Cellular and molecular bioengineering最新文献

Introduction: Inflammatory activation of the vascular endothelium leads to overexpression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), contributing to the pro-thrombotic state underpinning atherogenesis. While the role of TEC family kinases (TFKs) in mediating inflammatory cell and platelet activation is well defined, the role of TFKs in vascular endothelial activation remains unclear. We investigated the role of TFKs in endothelial cell activation in vitro and in a nonhuman primate model of diet-induced atherosclerosis in vivo.

Methods and results: In vitro, we found that ibrutinib blocked activation of the TFK member, BMX, by vascular endothelial growth factors (VEGF)-A in human aortic endothelial cells (HAECs). Blockade of BMX activation with ibrutinib or pharmacologically distinct BMX inhibitors eliminated the ability of VEGF-A to stimulate VCAM-1 expression in HAECs. We validated that treatment with ibrutinib inhibited TFK-mediated platelet activation and aggregation in both human and primate samples as measured using flow cytometry and light transmission aggregometry. We utilized contrast-enhanced ultrasound molecular imaging to measure platelet GPIbα and endothelial VCAM-1 expression in atherosclerosis-prone carotid arteries of obese nonhuman primates. We observed that the TFK inhibitor, ibrutinib, inhibited platelet deposition and endothelial cell activation in vivo.

Conclusion: Herein we found that VEGF-A signals through BMX to induce VCAM-1 expression in endothelial cells, and that VCAM-1 expression is sensitive to ibrutinib in vitro and in atherosclerosis-prone carotid arteries in vivo. These findings suggest that TFKs may contribute to the pathogenesis of atherosclerosis and could represent a novel therapeutic target.

Introduction: Recent studies have revealed that several deubiquitinating enzymes (DUBs) play important roles in hepatocellular carcinoma (HCC) progression, but the roles of Otubain 2 (OTUB2) in HCC remain obscure.

Methods: In this study, we investigated the expression of OTUB2 in HCC based on clinical samples and a public online database (ENCORI), and its roles and working mechanisms were further explored by in vitro experiments.

Results: It was found that the expression of OTUB2 was significantly up-regulated in HCC tissues, and correlated with poor prognosis of HCC patients. Functionally, the overexpression of OTUB2 could promote malignant proliferation and metastasis of HCC cells, while knockdown of OTUB2 exerted the opposite results. Using two bioinformatics tools, PJA1 was identified as a potential gene regulated by OTUB2. Mechanistically, it was found that OTUB2 promoted the stabilization of PJA1 by deubiquitylation, based on immunoprecipitation (IP) and cycloheximide (CHX) assays. Moreover, the suppressive effects of OTUB2 depletion on the malignant phenotypes of HCC cells could be reversed by overexpressing PJA1.

Conclusion: In conclusion, our study indicated that OTUB2 could promote the malignant proliferation and migration of HCC cells by increasing the stability of PJA1 via deubiquitylation.

Introduction: Periodontitis is characterized by the destruction of tooth-supporting tissues including the alveolar bone. Barrier membranes are used in dentistry for tissue regenerative therapy. Nevertheless, conventional membranes have issues related to membrane stability and direct induction of bone mineralization. Amelotin (AMTN), an enamel matrix protein, regulates hydroxyapatite crystal nucleation and growth. To apply an AMTN membrane in clinical practice, we investigated the mineralizing and adhesive effects of recombinant human (rh) AMTN in vitro using a collagen-based system.

Methods: Collagen hydrogel incorporated with rhAMTN (AMTN gel) and rhAMTN-coated dentin slices were prepared. AMTN gel was then applied on a commercial membrane (AMTN membrane). Samples were incubated for up to 24 h in mineralization buffer, and the structures were observed. The peak adhesive tensile strength between the dentin and AMTN membrane was measured. Using an enzyme-linked immunosorbent assay, the release kinetics of rhAMTN from the membrane were investigated.

Results: The AMTN gel resulted in the formation of hydroxyapatite deposits both onto and within the collagen matrix. Furthermore, coating the dentin surface with rhAMTN promoted the precipitation of mineral deposits on the surface. Interestingly, site-specific mineralization was observed in the AMTN membrane. Only 1% of rhAMTN was released from the membrane. Hence, the AMTN membrane adhered to the dentin surface with more than twofold greater tensile strength than that detected for a rhAMTN-free barrier membrane.

Conclusions: RhAMTN can accelerate mineralization and adhesion in collagen-based systems. Furthermore, the AMTN membrane could inform the optimal design of calcified tissue regenerative materials.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-022-00722-2.

Introduction: The present study aimed to evaluate the effects of FTY720 as a neuromodulatory drug on the behaviors of neural stem/progenitor cells (NS/PCs) in two-dimensional (2-D) and three-dimensional (3-D) cultures and in spinal cord injury (SCI).

Methods: The NS/PCs isolated from the ganglionic eminence of the 13.5-day old embryos were cultured as free-floating spheres. The single cells obtained from the second passage were cultured in 96-well plates without any scaffold (2-D) or containing PuraMatrix (PM, 3-D) or were used for transplantation in a mouse model of compression SCI. After exposure to 0, 10, 50, and 100 nanomolar of FTY720, the survival, proliferation, and migration of the NS/PCs were evaluated in vitro using MTT assay, neurosphere assay, and migration assay, respectively. Moreover, the functional recovery, survival and migration capacity of transplanted cells exposure to 100 nanomolar FTY720 were investigated in SCI.

Results: Cell survival and migration capacity increased after exposure to 50 and 100 nanomolar FTY720. In addition, higher doses of FTY720 led to the formation of more extensive and more neurospheres. Although this phenomenon was similar in both 2-D and 3-D cultures, PM induced better distribution of the cells in a 3-D environment. Furthermore, co-administration of FTY720 and NS/PCs 7 days after SCI enhanced functional recovery and both survival and migration of transplanted cells in the lesion site.

Conclusions: Due to the positive effects of FTY720 on the behavior of NS/PCs, using them in combination therapies can be an appealing approach for stem cell therapy in CNS injury.

Introduction: The network of Interstitial Cells of Cajal (ICC) plays a plethora of key roles in maintaining, coordinating, and regulating the contractions of the gastrointestinal (GI) smooth muscles. Several GI functional motility disorders have been associated with ICC degradation. This study extended a previously reported 2D morphological analysis and applied it to 3D spatial quantification of three different types of ICC networks in the distal stomach guided by confocal imaging and machine learning methods. The characterization of the complex changes in spatial structure of the ICC network architecture contributes to our understanding of the roles that different types of ICC may play in post-prandial physiology, pathogenesis, and/or amelioration of GI dsymotility- bridging structure and function.

Methods: A validated classification method using Trainable Weka Segmentation was applied to segment the ICC from a confocal dataset of the gastric antrum of a transgenic mouse, followed by structural analysis of the segmented images.

Results: The machine learning model performance was compared to manually segmented subfields, achieving an area under the receiver-operating characteristic (AUROC) of 0.973 and 0.995 for myenteric ICC (ICC-MP; n = 6) and intramuscular ICC (ICC-IM; n = 17). The myenteric layer in the distal antrum increased in thickness (from 14.5 to 34 μm) towards the lesser curvature, whereas the thickness decreased towards the lesser curvature in the proximal antrum (17.7 to 9 μm). There was an increase in ICC-MP volume from proximal to distal antrum (406,960 ± 140,040 vs. 559,990 ± 281,000 μm³; p = 0.000145). The % of ICC volume was similar for ICC-LM and for ICC-CM between proximal (3.6 ± 2.3% vs. 3.1 ± 1.2%; p = 0.185) and distal antrum (3.2 ± 3.9% vs. 2.5 ± 2.8%; p = 0.309). The average % volume of ICC-MP was significantly higher than ICC-IM at all points throughout sample (p < 0.0001).

Conclusions: The segmentation and analysis methods provide a high-throughput framework of investigating the structural changes in extended ICC networks and their associated physiological functions in animal models.

Introduction: Sevoflurane (SEV) has been shown to inhibit the malignant progression in many cancers, including colorectal cancer (CRC). However, it is not clear whether SEV regulates the progression of CRC by mediating the circular RNA (circRNA) axis.

Methods: Different concentrations of SEV were used to treat CRC cells. Cell proliferation, migration and invasion were determined by cell counting kit 8 assay, colony formation assay and transwell assay. The expression of circ_0000423, microRNA (miR)-525-5p and sphingosine-1-phosphate phosphatase 1 (SGPP1) mRNA was measured by quantitative real-time PCR. Cell apoptosis was assessed using flow cytometry, and protein expression was measured by western blot analysis. Dual-luciferase reporter assay and RIP assay were performed to confirm the interactions among circ_0000423, miR-525-5p and SGPP1. Animal experiments were performed to explore the effect of SEV and circ_0000423 on CRC tumorigenesis.

Results: SEV could inhibit CRC cell proliferation, migration and invasion. Circ_0000423 was upregulated in CRC and its expression could be reduced by SEV. Overexpressed circ_0000423 reversed the inhibitory effect of SEV on CRC cell proliferation, migration and invasion and the promotion effect on cell apoptosis. MiR-525-5p could be sponged by circ_0000423, and its overexpression also abolished the regulation of circ_0000423 on the progression of SEV-treated CRC cells. In addition, SGPP1 was confirmed to be a target of miR-525-5p, and its expression was positively regulated by circ_0000423. MiR-525-5p inhibitor promoted CRC cell progression under the treatment of SEV, while these effects could be overturned by SGPP1 silencing. Furthermore, the inhibition effect of SEV on CRC tumorigenesis also could be abolished by overexpressing circ_0000423.

Conclusion: Our results showed that SEV inhibited CRC progression through the regulation of circ_0000423/miR-525-5p/SGPP1 axis.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-021-00717-5.

Introduction: Trophoblast invasion is a complex biological process necessary for establishment of pregnancy; however, much remains unknown regarding what signaling factors coordinate the extent of invasion. Pregnancy-specific glycoproteins (PSGs) are some of the most abundant circulating trophoblastic proteins in maternal blood during human pregnancy, with maternal serum concentrations rising to as high as 200-400 μg/mL at term.

Methods: Here, we employ three-dimensional (3D) trophoblast motility assays consisting of trophoblast spheroids encapsulated in 3D gelatin hydrogels to quantify trophoblast outgrowth area, viability, and cytotoxicity in the presence of PSG1 and PSG9 as well as epidermal growth factor and Nodal.

Results: We show PSG9 reduces trophoblast motility whereas PSG1 increases motility. Further, we assess bulk nascent protein production by encapsulated spheroids to highlight the potential of this approach to assess trophoblast response (motility, remodeling) to soluble factors and extracellular matrix cues.

Conclusions: Such models provide an important platform to develop a deeper understanding of early pregnancy.

Introduction: Short interfering RNAs (siRNAs) are potent nucleic acid-based drugs designed to target disease driving genes that may otherwise be undruggable with small molecules. However, therapeutic potential of siRNA in vivo is limited by poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. Backpacking on natural carriers such as albumin, which is present at high concentration and has a long half-life in serum, is an effective way to modify pharmacokinetics of biologic drugs that otherwise have poor bioavailability. In this work, we sought to develop albumin-binding aptamer-siRNA chimeras to improve the bioavailability of siRNA.

Methods: A Systematic Evolution of Ligands through Exponential Enrichment (SELEX) approach was used to obtain modified RNA-binding aptamers, which were then fused directly to siRNA via in vitro transcription. Molecular and pharmacokinetic properties of the aptamer-siRNA chimeras were subsequently measured in vitro and in vivo.

Results: In vitro assays show that albumin-binding aptamers are stable in serum while maintaining potent gene knockdown capabilities in the chimera format. In vivo, the absolute circulation half-life of the best-performing aptamer-siRNA chimera (Clone 1) was 1.6-fold higher than a scrambled aptamer chimera control.

Conclusions: Aptamer-siRNA chimeras exhibit improved bioavailability without compromising biological activity. Hence, this albumin-binding aptamer-siRNA chimera approach may be a promising strategy for drug delivery applications.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-022-00718-y.