Primary Cells Research Articles

Enhancer of zeste homolog 2 facilitates phenotypic transition of vascular smooth muscle cells leading to aortic aneurysm/dissection.

Thoracic aortic aneurysms (TAAs) are a major cause of death owing to weaker blood vessel walls and higher rupture rates in affected individuals. Vascular smooth muscle cells (VSMCs) are the predominant cell type within the aortic wall and their dysregulation may contribute to TAA progression. Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, is involved in several pathological processes; however, the biological functions and mechanisms underlying VSMC phenotype transition and vascular intimal hyperplasia remain unclear. The present study aimed to determine the involvement of EZH2 in mediating VSMC function in the development of TAAs. The expression of EZH2 was revealed to be elevated in patients with thoracic aortic dissection and TAA mouse model through western blotting and reverse transcription-quantitative PCR experiments. Subsequently, a mouse model was established using β-aminopropionitrile. In vitro, EdU labeling, Transwell assay, wound healing assay and hematoxylin-eosin staining revealed that knocking down the Ezh2 gene could reduce the proliferation, invasion, migration, and calcification of mouse primary aortic smooth muscle cells. Flow cytometry analysis found that EZH2 deficiency increased cell apoptosis. Depletion of Ezh2 in mouse primary aortic VSMCs promoted the transformation of VSMCs from a synthetic to a contractile phenotype. Using RNA-sequencing analysis, it was demonstrated that Ezh2 regulated a group of genes, including integrin β3 (Itgb3), which are critically involved in the extracellular matrix signaling pathway. qChIP found Ezh2 occupies the Itgb3 promoter, thereby suppressing the expression of Itgb3. Ezh2 promotes the invasion and calcification of VSMCs, and this promoting effect is partially reversed by co-knocking down Itgb3. In conclusion, the present study identified a previously unrecognized EZH2-ITGB3 regulatory axis and thus provides novel mechanistic insights into the pathophysiological function of EZH2. EZH2 may thus serve as a potential target for the management of TAAs.

Knockdown of Antisense Noncoding Mitochondrial RNA Reduces Tumorigenicity of Patient-Derived Clear Cell Renal Carcinoma Cells in an Orthotopic Xenograft Mouse Model.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of renal cancer and its treatment is hindered by a resistance to targeted therapies, immunotherapies and combinations of both. We have reported that the knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) with chemically modified antisense oligonucleotides induces proliferative arrest and apoptotic death in tumor cells from many human and mouse cancer types. These studies have been mostly performed in vitro and in vivo on commercially available cancer cell lines and have shown that in mouse models tumor growth is stunted by the treatment. The present work was performed on cells derived from primary and metastatic ccRCC tumors. We established primary cultures from primary and metastatic ccRCC tumors, which were subjected to knockdown of ASncmtRNAs in vitro and in vivo in an orthotopic xenograft model in NOD/SCID mice. We found that these primary ccRCC cells are affected in the same way as tumor cell lines and in the orthotopic model tumor growth was significantly reduced by the treatment. This study on patient-derived ccRCC tumor cells represents a model closer to actual patient ccRCC tumors and shows that knockdown of ASncmtRNAs poses a potential treatment option for these patients.

Identifying PLAUR as a Pivotal Gene of Tumor Microenvironment and Regulating Mesenchymal Phenotype of Glioblastoma.

The mesenchymal (MES) phenotype of glioblastoma (GBM) is the most aggressive and therapy-resistant subtype of GBM. The MES phenotype transition during tumor progression results from both tumor-intrinsic genetic alterations and tumor-extrinsic microenvironmental factors. In this study, we sought to identify genes that can modulate the MES phenotype via both mechanisms. By integrating weighted gene co-expression network analysis (WGCNA) and the differential expression analysis of hypoxia-immunosuppression-related genes, we identified the plasminogen activator, urokinase receptor (PLAUR) as the hub gene. Functional enrichment analysis and GSVA analysis demonstrated that PLAUR was associated with the MES phenotype of glioma and the hypoxia-immunosuppression-related microenvironmental components. Single-cell sequencing analysis revealed that PLAUR mediated the ligand-receptor interaction between tumor-associated macrophages (TAMs) and glioma cells. Functional experiments in vitro with cell lines or primary glioma cells and xenograft models using BALB/c nude mice confirmed the role of PLAUR in promoting the MES phenotype of GBM. Our findings indicate that PLAUR regulates both glioma cells and tumor cell-extrinsic factors that favor the MES phenotype and suggest that PLAUR might be a potential target for GBM therapy.

Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

A comparison of endothelial cell growth on commercial coronary stents with and without laser surface texturing

Complete endothelialisation of coronary stents is an important determinant of future thrombotic complications following coronary stenting. Stent surface texture is an important factor that influences endothelial cell growth. With the emergence of second and third generation coronary stents, is limited comparative data describing endothelial cell growth in contemporary stent platforms, and limited data available on approaches used to rapidly modify the surfaces of commercial coronary stents to improve endothelialisation. In this study we have determined the in vitro proliferation of the primary human coronary artery endothelial cells on the commonly used 4 types of commercial coronary stents and found that the inner surface of BioMatrix drug-eluting stents (DES), after eliminating of the polymer and drug coating, had significantly higher endothelial cell proliferation compared to that of other bare metal stents (BMS): Multi-Link8, Integrity and Omega. The surfaces of the 3 types of BMS which are smooth, displayed similar endothelial cell proliferation, suggesting the importance of surface features in manipulating endothelial cell growth. Laser surface texturing was used to create micro/nano patterns on the stents. The laser treatment has significantly increased endothelial proliferation on the inner surfaces of all 4 types of stents, and Multi-Link8 stents displayed the highest (>100%) improvement. The laser textured BioMatrix stents had the highest absolute number of endothelial cells growth. Our results provided useful information in the endothelialisation potential for the commonly used commercial coronary stents and suggested a potential future application of laser surface bioengineering to coronary stents for better biocompatibility of the device.

Pacritinib inhibits proliferation of primary effusion lymphoma cells and production of viral interleukin-6 induced cytokines

Primary effusion lymphoma (PEL) and a form of multicentric Castleman’s disease (MCD) are both caused by Kaposi sarcoma herpesvirus (KSHV). There is a critical need for improved therapies for these disorders. The IL-6/JAK/STAT3 pathway plays an important role in the pathogenesis of both PEL and KSHV-MCD. We explored the potential of JAK inhibitors for use in PEL and KSHV-MCD, and found that pacritinib was superior to others in inhibiting the growth of PEL cell lines. Pacritinib induced apoptosis in PEL cells and inhibited STAT3 and NF-κB activity as evidenced by reduced amount of phosphorylated moieties. Pacritinib also inhibits FLT3, IRAK1, and ROS1; studies utilizing other inhibitors of these targets revealed that only FLT3 inhibitors exhibited similar cell growth inhibitory effects. FLT3’s likely contribution to pacritinib’s cell growth inhibition was further demonstrated by siRNA knockdown of FLT3. RNA sequencing and RT-PCR showed that many key host genes including cyclins and IL-6 were downregulated by pacritinib, while KSHV genes were variably altered. Finally, pacritinib suppressed KSHV viral IL-6-induced human IL-6 and IL-10 production in peripheral blood mononuclear cells, which may model an important step in KSHV-MCD pathogenesis. These results suggest that pacritinib warrants testing for the treatment of KSHV-MCD and PEL.

Propyl gallate induces human pulmonary fibroblast cell death through the regulation of Bax and caspase-3

Propyl gallate (PG) has been found to exert an inhibitory effect on the growth of different cell types, including lung cancer cells. However, little is known about the cytotoxicological effects of PG specifically on normal primary lung cells. The current study examined the cellular effects and cell death resulting from PG treatment in human pulmonary fibroblast (HPF) cells. DNA flow cytometry results demonstrated that PG (100–1,600 μM) had a significant impact on the cell cycle, leading to G1 phase arrest. Notably, 1,600 μM PG slightly increased the number of sub-G1 cells. Additionally, PG (400–1,600 μM) resulted in the initiation of cell death, a process that coincided with a loss of mitochondrial membrane potential (MMP; ΔΨm). This loss of MMP (ΔΨm) was evaluated using a FACS cytometer. In PG-treated HPF cells, inhibitors targeting pan-caspase, caspase-3, caspase-8, and caspase-9 showed no significant impact on the quantity of annexin V-positive and MMP (ΔΨm) loss cells. The administration of siRNA targeting Bax or caspase-3 demonstrated a significant attenuation of PG-induced cell death in HPF cells. However, the use of siRNAs targeting p53, Bcl-2, or caspase-8 did not exhibit any notable effect on cell death. Furthermore, none of the tested MAPK inhibitors, including MEK, c-Jun N-terminal kinase (JNK), and p38, showed any impact on PG-induced cell death or the loss of MMP (ΔΨm) in HPF cells. In conclusion, PG induces G1 phase arrest of the cell cycle and cell death in HPF cells through apoptosis and/or necrosis. The observed HPF cell death is mediated by the modulation of Bax and caspase-3. These findings offer insights into the cytotoxic and molecular effects of PG on normal HPF cells.

NGF Signaling Exacerbates KOA Peripheral Hyperalgesia via the Increased TRPV1-Labeled Synovial Sensory Innervation in KOA Rats.

Knee osteoarthritis (KOA) pain is caused by nociceptors, which are actually sensory nerve fiber endings that can detect stimuli to produce and transmit pain signals, and high levels of NGF in synovial tissue led to peripheral hyperalgesia in KOA. The purpose of this study is to investigate how sensory nerve fibers respond to the NGF/TrKA signal pathway and mediate the peripheral hyperalgesia in KOA rats. Forty SD male rats were randomly divided into 4 groups: normal, KOA, KOA + NGF, and KOA + siRNA TrKA. KOA model rats were induced by anterior cruciate ligament transection (ACLT). Mechanical and cold withdrawal thresholds (MWT and CWT) were measured 4 times in each group. The synovial tissues were harvested on day 28, and the expressions of NGF, TrKA, TRPV1, IL-1β, and PGP9.5 were determined using western blot, qPCR, and immunofluorescence staining. The primary rat fibroblast-like synoviocytes (FLSs) and DRG cells were divided into 4 groups as in vivo. The expressions of NGF, TrKA, TRPV1, and CGRP in vitro were determined using western blot and qPCR. KOA and intra-articular injection with NGF protein increased both mRNA and protein levels, not only TRPV1, PGP 9.5, and IL-1β in the synovial tissue, but also TRPV1, PGP 9.5, and S100 in the DRG tissue, while above changes were partly reversed after siRNA TrKA intervention. Besides, siRNA TrKA could improve peripheral hyperalgesia and decreased the TRPV1 positive nerve fiber innervation in synovial tissue. The results in vitro were consistent with those in vivo. This study showed the activation of the NGF/TrKA signaling pathway in KOA promoted the release of pain mediators, increased the innervation of sensory nerve fibers in the synovium, and worsened peripheral hyperalgesia. It also showed increased TRPV1 positive sensory innervation in KOA was mediated by NGF/TrKA signaling and exacerbated peripheral hyperalgesia.

Establishment of immortalized Egyptian Rousettus bat cell lines.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin-dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung-derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart-derived SV40 cell lines had aberrant karyotypes and the young bat-derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

Novel Immortalized Human Multipotent Mesenchymal Stromal Cell Line for Studying Hormonal Signaling.

Multipotent mesenchymal stromal cells (MSCs) integrate hormone and neuromediator signaling to coordinate tissue homeostasis, tissue renewal and regeneration. To facilitate the investigation of MSC biology, stable immortalized cell lines are created (e.g., commercially available ASC52telo). However, the ASC52telo cell line has an impaired adipogenic ability and a depressed response to hormones, including 5-HT, GABA, glutamate, noradrenaline, PTH and insulin compared to primary cells. This markedly reduces the potential of the ASC52telo cell line in studying the mechanisms of hormonal control of MSC's physiology. Here, we have established a novel immortalized culture of adipose tissue-derived MSCs via forced telomerase expression after lentiviral transduction. These immortalized cell cultures demonstrate high proliferative potential (up to 40 passages), delayed senescence, as well as preserved primary culture-like functional activity (sensitivity to hormones, ability to hormonal sensitization and differentiation) and immunophenotype up to 17-26 passages. Meanwhile, primary adipose tissue-derived MSCs usually irreversibly lose their properties by 8-10 passages. Observed characteristics of reported immortalized human MSC cultures make them a feasible model for studying molecular mechanisms, which regulate the functional activities of these cells, especially when primary cultures or commercially available cell lines are not appropriate.

TET1 inhibits liver fibrosis by blocking hepatic stellate cell activation.

Hepatic stellate cells (HSCs) are critical regulator contributing to the onset and progression of liver fibrosis. Chronic liver injury triggers HSCs to undergo vast changes and trans-differentiation into a myofibroblast HSCs, the mechanism remains to be elucidated. This study investigated that the involvement of hydroxymethylase TET1 (ten-eleven translocation 1) in HSC activation and liver fibrosis. It is revealed that TET1 levels were downregulated in the livers in mouse models of liver fibrosis and patients with cirrhosis, as well as activated HSCs in comparison to quiescent HSCs. In vitro data showed that the inhibition of TET1 promoted the activation HSC, whereas TET1 overexpression inhibited HSC activation. Moreover, TET1 could regulate KLF2 (Kruppel-like transcription factors) transcription by promoting hydroxymethylation of its promoter, which in turn suppressed the activation of HSCs. In vivo, it is confirmed that liver fibrosis was aggravated in Tet1 knockout mice after CCl4 injection, accompanied by excessive activation of primary stellate cells, in contrast to wild-type mice. In conclusion, we suggested that TET1 plays a significant role in HSC activation and liver fibrosis, which provides a promising target for anti-fibrotic therapies.

Generation of IPi001-A/B/C human induced pluripotent stem cell lines from healthy amniotic fluid cells

Human induced Pluripotent Stem Cells (hiPSCs) represent an invaluable source of primary cells to investigate development, establish cell and disease models, provide material for regenerative medicine and allow more physiological high-content screenings. Here, we generated three healthy hiPSC control lines - IPi001-A/B/C - from primary amniotic fluid cells (AFCs), an infrequently used source of cells, which can be readily obtained from amniocentesis for the prenatal diagnosis of numerous genetic disorders. These AFCs were reprogrammed by non-integrative viral transduction. The resulting hiPSCs displayed normal karyotype and expressed classic pluripotency hallmarks.

Liver X receptor activation mitigates oxysterol-induced dysfunction in fetoplacental endothelial cells

Maintaining the homeostasis of the placental vasculature is of paramount importance for ensuring normal fetal growth and development. Any disruption in this balance can lead to perinatal morbidity. Several studies have uncovered an association between high levels of oxidized cholesterol (oxysterols), and complications during pregnancy, including gestational diabetes mellitus (GDM) and preeclampsia (PE). These complications often coincide with disturbances in placental vascular function. Here, we investigate the role of two oxysterols (7-ketocholesterol, 7β-hydroxycholesterol) in (dys)function of primary fetoplacental endothelial cells (fpEC). Our findings reveal that oxysterols exert a disruptive influence on fpEC function by elevating the production of reactive oxygen species (ROS) and interfering with mitochondrial transmembrane potential, leading to its depolarization. Moreover, oxysterol-treated fpEC exhibited alterations in intracellular calcium (Ca2+) levels, resulting in the reorganization of cell junctions and a corresponding increase in membrane stiffness and vascular permeability. Additionally, we observed an enhanced adhesion of THP-1 monocytes to fpEC following oxysterol treatment. We explored the influence of activating the Liver X Receptor (LXR) with the synthetic agonist T0901317 (TO) on oxysterol-induced endothelial dysfunction in fpEC. Our results demonstrate that LXR activation effectively reversed oxysterol-induced ROS generation, monocyte adhesion, and cell junction permeability in fpEC. Although the effects on mitochondrial depolarization and calcium mobilization did not reach statistical significance, a strong trend towards stabilization of calcium mobilization was evident in LXR-activated cells. Taken together, our results suggest that high levels of systemic oxysterols link to placental vascular dysfunction and LXR agonists may alleviate their impact on fetoplacental vasculature.

Natural product Kaji-ichigoside F1 exhibits rapid antidepression via activating the AMPA–BDNF–mTOR pathway and inhibiting the NMDAR–CaMKIIα pathway

BackgroundDepression is a common and recurrent neuropsychiatric disorder. Recent studies have shown that the N-methyl-d-aspartate (NMDA) receptor (NMDAR) is involved in the pathophysiology of depression. Previous studies have found that Kaji-ichigoside F1 (KF1) has a protective effect against NMDA-induced neurotoxicity. However, the antidepressant mechanism of KF1 has not been confirmed yet. PurposeIn the present study, we aimed to evaluate the rapid antidepressant activity of KF1 and explore the underlying mechanism. Study designFirst, we explored the effect of KF1 on NMDA-induced hippocampal neurons and the underlying mechanism. Second, depression was induced in C57BL/6 mice via chronic unpredictable mild stress (CUMS), and the immediate and persistent depression-like behavior was evaluated using the forced swimming test (FST) after a single administration of KF1. Third, the contributions of NMDA signaling to the antidepressant effect of KF1 were investigated using pharmacological interventions. Fourth, CUMS mice were treated with KF1 for 21 days, and then their depression-like behaviors and the underlying mechanism were further explored. MethodsThe FST was used to evaluate immediate and persistent depression-like behavior after a single administration of KF1 with or without NMDA pretreatment. The effect of KF1 on depressive-like behavior was investigated in CUMS mice by treating them with KF1 once daily for 21 days through the sucrose preference test, FST, open field test, and tail suspension test. Then, the effects of KF1 on the morphology and molecular and functional phenotypes of primary neuronal cells and hippocampus of mice were investigated by hematoxylin–eosin staining, Nissl staining, propidium iodide staining, TUNEL staining, Ca2+ imaging, JC-1 staining, ELISA, immunofluorescence analysis, RT-PCR, and Western blot. ResultsKF1 could effectively improve cellular viability, reduce apoptosis, inhibit the release of LDH and Ca2+, and increase the mitochondrial membrane potential and the number of dendritic spines numbers in hippocampal neurons. Moreover, behavioral tests showed that KF1 exerted acute and sustained antidepressant-like effects by reducing Glu-levels and ameliorating neuronal damage in the hippocampus. Additionally, in vivo and in vitro experiments revealed that PSD95, Syn1, α-amino-3‑hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and brain-derived neurotrophic factor (BDNF) were upregulated at the protein level, and BDNF and AMPA were upregulated at the mRNA level. NR1 and NR2A showed the opposite trend. ConclusionThese results confirm that KF1 exerts rapid antidepressant effects mainly by activating the AMPA–BDNF–mTOR pathway and inhibiting the NMDAR–CaMKIIα pathway. This study serves as a new reference for discovering rapid antidepressants.

CD24 negativity reprograms mitochondrial metabolism to PPARα and NF-κB-driven fatty acid β-oxidation in triple-negative breast cancer

CD24 is a well-characterized breast cancer (BC) stem cell (BCSC) marker. Primary breast tumor cells having CD24-negativity together with CD44-positivity is known to maintain high metastatic potential. However, the functional role of CD24 gene in triple-negative BC (TNBC), an aggressive subtype of BC, is not well understood. While the significance of CD24 in regulating immune pathways is well recognized in previous studies, the significance of CD24 low expression in onco-signaling and metabolic rewiring is largely unknown. Using CD24 knock-down and over-expression TNBC models, our in vitro and in vivo analysis suggest that CD24 is a tumor suppressor in metastatic TNBC. Comprehensive in silico gene expression analysis of breast tumors followed by lipidomic and metabolomic analyses of CD24-modulated cells revealed that CD24 negativity induces mitochondrial oxidative phosphorylation and reprograms TNBC metabolism toward the fatty acid beta-oxidation (FAO) pathway. CD24 silencing activates PPARα-mediated regulation of FAO in TNBC cells. Further analysis using reverse-phase protein array and its validation using CD24-modulated TNBC cells and xenograft models nominated CD24-NF-κB-CPT1A signaling pathway as the central regulatory mechanism of CD24-mediated FAO activity. Overall, our study proposes a novel role of CD24 in metabolic reprogramming that can open new avenues for the treatment strategies for patients with metastatic TNBC.

Lipid nanoparticles containing labile PEG-lipids transfect primary human skin cells more efficiently in the presence of apoE

Nucleic acid-based therapeutics encapsulated into lipid nanoparticles (LNPs) can potentially target the root cause of genetic skin diseases. Although nanoparticles are considered impermeable to skin, research and clinical studies have shown that nanoparticles can penetrate into skin with reduced skin barrier function when administered topically.Studies have shown that epidermal keratinocytes express the low-density lipoprotein receptor (LDLR) that mediates endocytosis of apolipoprotein E (apoE)-associated nanoparticles and that dermal fibroblasts express mannose receptors. Here we prepared LNPs designed to exploit these different endocytic pathways for intracellular mRNA delivery to the two most abundant skin cell types, containing: (i) labile PEG-lipids (DMG-PEG2000) prone to dissociate and facilitate apoE-binding to LNPs, enabling apoE-LDLR mediated uptake in keratinocytes, (ii) non-labile PEG-lipids (DSPE-PEG2000) to impose stealth-like properties to LNPs to enable targeting of distant cells, and (iii) mannose-conjugated PEG-lipids (DSPE-PEG2000-Mannose) to target fibroblasts or potentially immune cells containing mannose receptors. All types of LNPs were prepared by vortex mixing and formed monodisperse (PDI ∼ 0.1) LNP samples with sizes of 130 nm (±25%) and high mRNA encapsulation efficiencies (≥90%). The LNP-mediated transfection potency in keratinocytes and fibroblasts was highest for LNPs containing labile PEG-lipids, with the addition of apoE greatly enhancing transfection via LDLR. Coating LNPs with mannose did not improve transfection, and stealth-like LNPs show limited to no transfection.Taken together, our studies suggest using labile PEG-lipids and co-administration of apoE when exploring LNPs for skin delivery.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

TBX3 reciprocally controls key trophoblast lineage decisions in villi during human placenta development in the first trimester

Human trophoblastic lineage development is intertwined with placental development and pregnancy outcomes, but the regulatory mechanisms underpinning this process remain inadequately understood. In this study, based on single-nuclei RNA sequencing (snRNA-seq) analysis of the human early maternal-fetal interface, we compared the gene expression pattern of trophoblast at different developmental stages. Our findings reveal a predominant upregulation of TBX3 during the transition from villous cytotrophoblast (VCT) to syncytiotrophoblast (SCT), but downregulation of TBX3 as VCT progresses into extravillous trophoblast cells (EVT). Immunofluorescence analysis verified the primary expression of TBX3 in SCT, partial expression in MKi67-positive VCT, and absence in HLA-G-positive EVT, consistent with our snRNA-seq results. Using immortalized trophoblastic cell lines (BeWo and HTR8/SVneo) and human primary trophoblast stem cells (hTSCs), we observed that TBX3 knockdown impedes SCT formation through RAS-MAPK signaling, while TBX3 overexpression disrupts the cytoskeleton structure of EVT and hinders EVT differentiation by suppressing FAK signaling.In conclusion, our study suggests that the spatiotemporal expression of TBX3 plays a critical role in regulating trophoblastic lineage development via distinct signaling pathways. This underscores TBX3 as a key determinant during hemochorial placental development.