Lentiviral Infection Research Articles

DEAD/H-box helicase 11 is transcriptionally activated by Yin Yang-1 and accelerates oral squamous cell carcinoma progression.

Oral squamous cell carcinoma (OSCC) is the most common oral malignancy. DEAD/H-box helicase 11 (DDX11), a DNA helicase, has been implicated in the progression of several cancers. Yet, the precise function of DDX11 in OSCC is poorly understood. The DDX11 expression in OSCC cells and normal oral keratinocytes was evaluated in the Gene Expression Omnibus database (GSE146483 and GSE31853). SCC-4 and CAL-27 cells expressing doxycycline-inducible DDX11 or DDX11 shRNA were generated by lentiviral infection. The role of DDX11 in OSCC cells was determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry assay, TUNEL staining, and western blot. The effects of DDX11 on tumor growth were explored in a xenograft nude mouse model. The relationship between DDX11 and transcription factor Yin Yang-1 (YY1) was researched using the dual luciferase report assay and chromatin immunoprecipitation assay. DDX11 expression was significantly upregulated in OSCC cells. Knockdown of DDX11 inhibited cell proliferation, induced cell cycle arrest, and suppressed PI3K-AKT pathway, while DDX11 overexpression showed opposite effects. The number of apoptotic cells was increased in DDX11 silenced cells. DDX11 upregulation or knockdown accelerated or suppressed tumor growth in vivo, respectively. Moreover, the YY1 bound and activated the DDX11 promoter, resulting in increasing DDX11 expression. Forced expression DDX11 reversed the anticancer effects of YY1 silencing on OSCC cells. DDX11 has tumor-promoting function in OSCC and is transcriptionally regulated by YY1, indicating that DDX11 may serve as a potential target for the OSCC treatment.

Targeting Mitochondrial Cholesterol Efflux via TCF21/ABCA10 Pathway to Enhance Cisplatin Efficacy in Ovarian Cancer.

Cisplatin (DDP) resistance is one of the causes of treatment failure for ovarian cancer (OV). Mitochondrial cholesterol level was reported to be associated with OV chemoresistance. We found that ABCA10, a potential cholesterol transport protein, was highly expressed in ovarian tissues and downregulated in OV tissues. Our study aimed to explore TCF21/ABCA10 axis resistance to DDP therapy in ovarian cancer based on regulating mitochondrial cholesterol efflux. Thirty epithelial ovarian cancer tumors and thirty ovarian tissues from non-cancer patients were collected. Western blot and RT-qPCR were used to measure ABCA10 and TCF21 expression levels in these tissues, as well as in a human ovarian epithelial cell line (IOSE-80), OV cells (A2780 and SKOV3), and DDP-resistant OV cell lines (A2780/DDP and SKOV3/DDP). IOSE-80 cells were also infected with ABCA10 knockdown lentivirus to identify the most effective ABCA10 knockdown plasmid. Lentiviral infection was used to create ABCA10 knockdown, ABCA10 overexpression, and TCF21 overexpression anti-DDP OV cell lines. Cell proliferation was detected by CCK-8 and EDU staining, flow cytometry for apoptosis, MTT for metabolic activity, calcium-induced Cytochrome C release, and mitochondrial matrix swelling for mitochondrial function and Oil Red O staining for lipid accumulation. Cholesterol metabolism was evaluated by measuring mitochondrial cholesterol and cholesterol efflux. Protein concentration was determined using the BCA method. A dual-luciferase reporter assay confirmed TCF21's interaction with ABCA10. ChIP also verified this interaction. The mRNA level (P < 0.01) and protein level (P < 0.001) of ABCA10 were downregulated in cancer tissues of OV patients relative to normal ovarian tissues. Relative to human ovarian epithelial cells, ABCA10 expression was significantly downregulated in OV cells (P < 0.01) and even more significantly downregulated in DDP-resistant OV cells (P < 0.001). Compared to the group treated solely with DDP, the overexpression of ABCA10 significantly inhibited the proliferation of DDP-resistant OV cells (P < 0.01), markedly reduced the staining intensity of EDU in these cells (P < 0.05), and substantially accelerated apoptosis in DDP-resistant OV cells (P < 0.01).Overexpression of ABCA10 further accelerated Cytochrome C expression and mitochondrial matrix swelling in DDP-resistant OV cells compared to the DDP-alone group (P < 0.01). The addition of cholesterol reversed the decrease in lipid accumulation, the decrease in mitochondrial cholesterol levels (P < 0.05), and the increase in cholesterol efflux (P < 0.01) in DDP-resistant OV cells caused by overexpression of ABCA10. The transcription factor TCF21 was bound to the promoter of ABCA10. Overexpression of TCF21 significantly increased ABCA10 expression in DDP-resistant OV cells (P < 0.01) and increased cytochrome C expression in A2780/DDP (P < 0.05) and SKOV3/DDP (P < 0.01) cells, with accelerated mitochondrial matrix swelling in A2780/DDP (P < 0.01) and SKOV3/DDP (P < 0.001) cells, while knockdown of ABCA10 reversed these effects. Our study found that TCF21 boosts ABCA10 expression, which in turn reduces DDP resistance in OV cells by enhancing mitochondrial cholesterol efflux. This mechanism increases the sensitivity of DDP-resistant OV cells to DDP. Our findings will provide new therapeutic targets for the treatment of ovarian cancer.

EPO modified MSCs protects SH-SY5Y cells against ischemia/hypoxia-induced apoptosis via REST-dependent epigenetic remodeling

Hypoxic-ischemic encephalopathy (HIE) is a diffuse brain tissue injury caused by acute ischemia and hypoxia, and it is most commonly found in newborn infants but can also occur in adults. Mesenchymal stem cell (MSC) therapies have showed improved outcomes for treating HIE-induced neuronal defects. However, many key issues associated with poor cell viability and tolerance of grafted MSCs after HIE remain to be resolved. Genetic engineering could endow MSCs with more robust regenerative capacities. Our research, along with that of other scientists, has found that the expression of intracellular erythropoietin (EPO) in human umbilical cord MSCs (hUC-MSCs) increases proportionally with the duration of hypoxia exposure. Furthermore, we observed that EPO, when introduced into the EPO gene-modified hUC-MSCs, can be secreted into the extracellular space. However, the underlying mechanisms that support the neuroprotective effects of EPO-MSCs remain unclear. EPO-MSCs, hUC-MSCs, and NC-MSCs were identified by flow cytometry, osteogenic, and adipogenic differentiation assays. The oxygen-glucose deprivation (OGD)-induced SH-SY5Y cell-line was established, and five groups were set up: control, 24-h ischemia-hypoxia, co-cultured with hUC-MSCs, NC-MSCs, and EPO-MSCs after hypoxia. LEGENDplex™ multi-factor flow cytometry was used to detect the secretion of inflammatory factors in cell supernatants and cerebrospinal fluid. Chromosome-targeted excision and tagging (CUT&Tag) sequencing was applied to detect genomic H3K4me2 modifications, and conjoint analysis with transcriptome sequencing (RNA-seq) was performed. Lentiviral vector infection was used to construct SH-SY5Y cells with stable knockdown of RE1-silencing transcription factor (REST), and flow cytometry was used to detect alterations in apoptosis. Finally, the molecular mechanism underlying the neuroprotective and anti-apoptotic effects of EPO-MSCs was investigated using RNA sequencing, qRT-PCR, and western blot assays. Our results suggest that EPO-MSCs are genetically engineered to secrete significantly more EPO. EPO-MSCs treatment has anti-apoptotic properties and offers neuronal protection during ischemic-hypoxic injury. Furthermore, RNA-seq results suggest that multiple inflammation-related genes were down-regulated after EPO-MSCs treatment. Application of RNA-seq and CUT&Tag combined analysis found that the expressions of REST were significantly up-regulated. Lentiviral vector infection to construct REST knockdown SH-SY5Y failed to rescue apoptosis after hypoxia and co-culture with EPO-MSCs, and SETD2-mediated H3K36me3 protein level expression was reduced. EPO-MSCs may promote neuronal survival by affecting H3K4me2 and thus activating the expression of REST and TET3. EPO-MSCs also upregulated the modification level of SETD2-mediated H3K36me3 and regulated the expression of inflammation-related genes such as PLCG2, as well as apoptosis genes BCL2A1. To investigate the neuroprotective effects of EPO-modified hUC-MSCs and the underlying epigenetic regulatory mechanisms, this study aims to provide a theoretical foundation for the potential application of EPO gene-modified hUC-MSCs in the treatment of HIE.

Artemisitene induces apoptosis of breast cancer cells by targeting FDFT1 and inhibits the growth of breast cancer patient-derived organoids

Artemisitene (ATT) is a natural bioactive compound with anti-breast cancer activity. However, the direct target and clinical efficacy of ATT on breast cancer are still unclear. The current study aimed to identify the target protein and underlying mechanism of ATT in anti-breast cancer. Moreover, patient-derived organoids (PDOs) were employed to assess the clinical efficacy of ATT on breast cancer. Herein, molecular docking, molecular dynamics simulation, cellular thermal shift assay (CETSA) combined with Western blot, surface plasmon resonance (SPR) were applied to confirm the interactional target of ATT in breast cancer cells. Bioinformatics analysis, Western blot, flow cytometry, plasmid construction and lentivirus infection, chromatin immunoprecipitation (ChIP) assay, and quantitative real-time PCR (RT-qPCR) were performed to reveal the potential mechanism of ATT in treating breast cancer. PDOs were established to evaluate the clinical therapeutic efficiency of ATT on breast cancer. We found that ATT interacted with Farnesyl-diphosphate farnesyltransferase 1 (FDFT1) in breast cancer cells. Knockdown of FDFT1 induced NEDD4 expression and apoptosis in breast cancer cells. Overexpression of FDFT1 could rescue ATT-induced apoptosis, while interfering with FDFT1 expression decreased the level of RelA (NF-κB p65 subunit) in the nucleus in breast cancer cells. Knockdown of FDFT1 induced NEDD4 expression by regulating TNFR1/NF-κB pathway. Overexpression of FDFT1 could reverse the activation of ATT-induced TNFR1/NF-κB/NEDD4 pathway in breast cancer cells. Interestingly, the ChIP assay revealed that p65 could regulate NEDD4 transcription. Furthermore, ATT exhibited a broad-spectrum inhibitory effect on the growth of breast cancer PDOs with different pathological subtypes, and showed an excellent safety profile in comparison with that of conventional chemotherapy drugs. In summary, this work demonstrated that ATT targets FDFT1 to induce apoptosis of breast cancer cells through regulating TNFR1/NF-κB/NEDD4 pathway and suppresses breast cancer PDOs growth, which supported ATT as an effective and potential drug candidate for breast cancer treatment.

Plate centrifugation enhances the efficiency of polyethylenimine-based transfection and lentiviral infection

PurposeTo propose an efficient, reproducible, and consistent transgenic technology based on plate centrifugation, which is particularly useful for polyethylenimine (PEI) transfection and lentiviral infection. MethodsWe optimized multiple factors that could contribute to transfection efficiency, such as the dosage of the PEI or DNA, the working solution buffer used for diluting the PEI or DNA, the incubation time for the PEI/DNA complexes, and the transfection time. ResultsPlate centrifugation led to a 5.46-fold increase in the transfection efficiency of PEI-based transfection while maintaining the cell survival rate. Moreover, the average copy number of viral genes in each genome increased 4.96-fold with plate centrifugation. Plate centrifugation alters the spatial arrangement of the PEI/DNA complexes or lentiviruses, increasing the chances of these complexes or viruses coming into contact with target cells, ultimately resulting in improved transfection or infection efficiency. ConclusionsWe present a protocol based on plate centrifugation for transfection or lentiviral infection that is suitable for genetic modification of primary cells or stem cells.

C-CBL/LCK/c-JUN/ETS1/CD28 axis restrains childhood asthma by suppressing Th2 differentiation

BackgroundAsthma is a common immune disease with high morbidity in children. Type 2 inflammation is the center of asthma development, and mainly mediated by a subset of CD4 + T cells, T helper 2 (Th2) cells. Excess Th2 differentiation was generally associated with asthmatic attack. Casitas B-lineage lymphoma (c-CBL) was reported to involved in T cell development and databank showed its decreased expression in CD4 + T cells from peripheral blood of asthmatic children. This study aims to investigate the role of c-CBL in childhood asthma and Th2 differentiation, and explore the underlying mechanism.MethodsWe collected peripheral blood samples from clinical childhood asthma cases and healthy controls, and determined c-CBL expression in CD4 + T cells. Asthma was induced in neonatal mice by ovalbumin (OVA) intraperitoneal injection and aerosol inhalation, and c-CBL expression in CD4 + T cells from peripheral blood and spleen was measured. Gain-of-function experiments was performed to confirm the effects of c-CBL on Th2 differentiation in vitro. Finally, c-CBL was delivered into asthmatic mice via lentivirus infection to verify its effects on experimental asthma.Resultsc-CBL was lowly expressed in CD4 + T cells from asthmatic children than those of healthy controls. Similarly, it was downregulated in CD4 + T cells from peripheral blood and spleen of asthma mice. Overexpression of c-CBL restrained lung pathological injury and type 2 inflammation in experimental asthmatic mice. Gain-of-function experiments demonstrated that c-CBL inhibited Th2 differentiation of CD4 + T cells from healthy children, and mediated the ubiquitination of lymphocyte cell-specific protein-tyrosine kinase (LCK). LCK acted as a kinase to phosphorylate and activate c-JUN, which was predicted to bind promoter sequence of CD28 by bioinformatic analysis. Dual-luciferase reporter assay verified that c-JUN and ETS1 synergically enhanced transcription of CD28, and this transcription activation was aggravated by LCK overexpression.Conclusionc-CBL alleviated asthma and suppressed Th2 differentiation by facilitating LCK ubiquitination, interrupting c-JUN activation and CD28 expression in vivo and in vitro. c-CBL/LCK/c-JUN/ETS1/CD28 axis was partially involved in childhood asthma, and may provide novel insights for clinical treatment for asthma.

T-Box Transcription Factor 2 Mediates Chemoresistance of Endometrial Cancer via Regulating FSP1-involved Ferroptosis.

Chemotherapy is increasingly being used in the first-line treatment of endometrial cancer (EC) patients. However, chemoresistance seriously affects its efficacy. Understanding the underlying molecular mechanisms is critical for EC treatment. We explored the regulatory role of T-Box transcription factor 2 (TBX2)-ferroptosis suppressor protein 1 (FSP1) axis in ferroptosis and chemoresistance of EC. Cisplatin-resistant cell line Ishikawa/DDP cells were utilized to generate TBX2 and FSP1 overexpression and knockdown stable cell lines by using lentivirus infection and puromycin selection. Cell viability and ferroptosis status were evaluated in EC cells with or without Cisplatin and/or FSP1 inhibitor (iFSP1) using CKK-8, lipid peroxidation, malondialdehyde, and lactate dehydrogenase release assays. Endometrial carcinoma xenograft mouse model was established to further explore the function of TBX2-FSP1 axis on ferroptosis and tumor progression in EC. TBX2 suppressed Cisplatin-induced ferroptosis through up-regulating FSP1 expression level in EC cells. On the contrary, knockdown of TBX2 reduced FSP1 expression and significantly promoted Cisplatin-induced ferroptosis. TBX2 or FSP1 overexpression and knockdown promote and inhibit EC tumor growth under Cisplatin treatment, respectively. Interestingly, silence FSP1 could reverse TBX2-mediated ferroptosis inhibition and tumor-promoting effect. TBX2-FSP1 axis inhibits ferroptosis and enhances the Cisplatin resistance, which will provide an important theoretical basis and potential solution for the clinical treatment of EC.

Identification of New Single Nucleotide Polymorphisms Potentially Related to Small Ruminant Lentivirus Infection Susceptibility in Goats Based on Data Selected from High-Throughput Sequencing.

Small ruminant lentivirus (SRLV) infections are spread in the flocks of sheep and goats all over the world, causing economic loss. Although only a fraction of infected animals develop disease symptoms, all of them may shed the virus, causing uncontrolled spread of the infection. Antibodies against the virus can be detected in the blood of infected animals and are the main marker of infection. Additionally, in most infected animals, proviral DNA can also be detected, but at different levels. Due to the lack of treatment or vaccines, the most effective strategy to prevent SRLV infections are control programmes introduced by several countries based on the elimination of seropositive individuals from the flock. An alternative approach, which has currently become the rationale, is an identification of host factors which may predispose certain individuals or breeds to resistance or susceptibility to small ruminant lentivirus infection. In our work, attention was paid to goats of the Carpathian breed infected with SRLV. Available RNA-seq results from the blood of 12 goats with a determined level of SRLV proviral load were used to analyse single nucleotide polymorphisms (SNPs) by the variant calling method. Six SNPs within five genes (POU2AF1, BCAT2, TMEM154, PARP14, UBASH3A) were selected for genotyping to determine their association with the level of small ruminant lentivirus proviral DNA in a group of 60 goats. Interestingly, in seronegative individuals, only the TT genotype of the PARP14 gene was observed, while the TMEM154 CC genotype was found only in seropositive goats. Both genes may be considered potential markers for resistance/susceptibility to SRLV infection. In contrast, polymorphisms identified in POU2AF1 and UBASH3A genes seemed to be deleterious for respective protein functions; therefore, these genes are less likely to be recognised as resistance/susceptibility markers of SRLV infection.

Remifentanil-induced inflammation in microglial cells: Activation of the PAK4-mediated NF-κB/NLRP3 pathway and onset of hyperalgesia

BackgroundThe perioperative use of remifentanil is associated with postoperative hyperalgesia, which can impair recovery and extend hospitalization. Recent studies have revealed that microglia-mediated activation of the NLRP3 inflammasome plays a critical role in opioid-induced hyperalgesia, with NF-κB acting as a pivotal activation point for NLRP3. Despite these findings, the specific molecular mechanisms underlying remifentanil-induced postoperative hyperalgesia remain unclear. This study aims to develop a model of remifentanil-induced hyperalgesia and investigate the molecular mechanisms, focusing on the NF-κB/NLRP3 pathway, using both in vitro and in vivo approaches. MethodWe established a remifentanil-induced hyperalgesia model and performed proteomic analysis to identify differential protein expression in the spinal cord tissue of rats. NLRP3 or PAK4 antagonists were administered intrathecally in vivo, and mechanical pain thresholds in the hind paws were measured using Von Frey testing. In vitro, we applied NLRP3 or PAK4 inhibitors or used lentivirus infection to silence PAK4, NF-κB, and NLRP3 genes. Protein expression was assessed through immunohistochemistry, immunofluorescence, and Western blotting. Additionally, ELISA was performed to measure IL-1β and IL-18 levels, and RT-qPCR was conducted to evaluate the transcription of target genes. ResultsProteomic analysis revealed that remifentanil upregulates PAK4 protein in spinal cord tissue two hours after the surgery. In addition, remifentanil induces morphological changes in the spinal cord dorsal horn, characterized by increased expression of PAK4, p-p65, NLRP3 and Iba-1 proteins, which in turn leads to elevated IL-1β and IL-18 levels and an inflammatory response. Intrathecal injection of NLRP3 or PAK4 inhibitors mitigates remifentanil-induced hyperalgesia and associated changes. In vitro, downregulation of PAK4 inhibits the increase in PAK4, p-p65, NLRP3 and Caspase-1 induced by LPS. Conversely, the downregulation of NLRP3 does not impact the levels of PAK4 and p-p65 proteins, aligning with the in vivo results and suggesting that PAK4 acts as an upstream signaling molecule of NLRP3. ConclusionRemifentanil can increase PAK4 expression in spinal cord dorsal horn cells by activating the NF-κB/NLRP3 pathway and mediating microglial activation, thereby contributing to postoperative hyperalgesia.

A nanobody interaction with SARS-COV-2 Spike allows the versatile targeting of lentivirus vectors.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

Senegenin suppresses hepatocellular carcinoma by regulating O-GlcNAcylation.

Based on current knowledge, hepatocellular carcinoma (HCC) is a condition with numerous etiologies and risk factors. However, the pathogenesis of HCC remains unclear. To investigate the roles of senegenin and O-GlcNAcylation in the growth and metastasis of HCC. The levels of O-linked N-acetylglucosamine transferase (OGT) and O-GlcNAcylation in HCC cells and tissues were detected using western blot analysis. The effects of senegenin and O-GlcNAcylation on the proliferation of HCC cells were investigated in vitro using cell counting kit-8 and clonogenic assays. The potential effects of senegenin and O-GlcNAcylation on HCC metastasis were examined using the transwell migration assay. O-GlcNAcylation levels were altered via drug treatment and lentiviral infection, and western blot analysis was used to detect proteins involved in various pathways. Western blot analysis revealed that OGT and O-GlcNAcylation levels were significantly elevated in HCC tissues and cells. O-GlcNAcylation levels in HCC cells were significantly altered by drug treatment and lentiviral infection. An increase in the glycosylation level was linked to enhanced proliferation, invasiveness, clonogenicity, and metastatic potential of cancer cells. O-GlcNAcylation induced by senegenin was found to slow the proliferation and migration of HCC cells. The levels of proteins involved in nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, which are associated with endoplasmic reticulum stress, were altered. Senegenin lowers O-GlcNAcylation levels, decreases OGT expression, and inhibits cancer cell growth and metastasis by regulating proteins involved in NF-κB and JNK pathways.

Abstract P125: In an Autocrine Mechanism, Endothelial Progranulin Regulates Vascular Tone and Blood Pressure via AMPK Activation

Dysfunctional endothelial cells (ECs) contribute to the development of hypertension (HTN). Progranulin (PGRN) is a secreted protein that plays a major role in the inflammatory response. We recently demonstrated that HTN is associated with increased circulating PGRN, whereas the global lack of PGRN elicits vascular dysfunction and raises blood pressure (BP). However, the cellular origin of PGRN and the molecular mechanisms by which PGRN regulates vascular tone and BP are still to be elucidated. We hypothesized that ECs secrete PGRN, which regulates the vascular phenotype and BP in an autocrine manner. We used mice with tamoxifen-inducible, Cdh5-CreERT2-mediated deletion of PGRN in ECs (PGRN EC-/-) and their counterparts as controls (PGRN EC+/+). The effectiveness of endothelial PGRN depletion was validated using en face immunostaining in the aorta and qPCR in ECs from mesenteric arteries (MA). PGRN EC-/- mice exhibited a 2.5-fold reduction in circulating PGRN levels followed by EC dysfunction in MA characterized by reduced maximal response (Emax) and sensitivity (pD2) to acetylcholine [Emax (%): PGRN EC+/+: 99.3 ± 0.7 vs PGRN EC-/-: 95.9 ± 0.5* and pD2: PGRN EC+/+: 8.1 ± 0.2 vs PGRN EC-/-: 7.0 ± 0.3*, *P&lt;0.05]. Supplementation with recombinant PGRN (20 μg/day) normalized endothelial function. Moreover, under baseline conditions, BP (analyzed by telemetry) was slightly higher in PGRN EC-/- mice [MAP (mmHg): PGRN+/+: 110.6 ± 1.6 vs PGRN-/-: 114.3 ± 1.2]. However, treatment with a subpressor dose of angiotensin II (120 ng/kg/day for 2 weeks) only increased BP in PGRN EC-/- mice. Mechanistically, we found that knockdown of PGRN (via siRNA) in cultured mesenteric ECs (MECs) suppressed PGRN secretion, AMPK phosphorylation, and nitric oxide (NO) formation. Conversely, MECs overexpressing PGRN (via lentivirus infection) led to exacerbated PGRN secretion followed by AMPK activation and NO formation, which were blunted by blocking AMPK or EphrinA2 (PGRN receptor) with dorsomorphin (10 μM) or ALWII4127 (5 μM), respectively. Finally, endothelial-specific AMPK-deficient mice exhibited increased BP and endothelial dysfunction in MA; such effects were not affected by PGRN treatment, suggesting that AMPK is a downstream target for PGRN. In summary, our data indicate that ECs are important sources of PGRN, which supports the maintenance of the EC phenotype and BP via EphrinA2, AMPK, and NO formation. This pathway occurs in an autocrine-dependent manner.

Abstract 12: Deficiency of Progranulin Decreases Mitochondrial Function and Induces a Whitening Phenotype in Perivascular Adipose Tissue, Suppressing Its Anti-Contractile Effects

Perivascular adipose tissue (PVAT) is recognized as an endocrine organ that secretes a variety of adipokines, which negatively regulate vascular contraction. Progranulin (PGRN), a glycoprotein encoded by the GRN gene, is highly expressed in adipocytes, and is implicated in inflammation, cellular proliferation, and repair mechanisms. The relationship between PGRN and PVAT remains unclear. We hypothesized that the lack of PGRN negatively affects PVAT quality. We used male C57BL/6J wild-type (WT) and global PGRN knockout (KO) mice, aged 10-12 weeks. Energy expenditure was analyzed via the Comprehensive Lab Animal Monitoring System (CLAMS). Vascular function was studied in endothelium-intact aortae with PVAT [PVAT(+)] and without PVAT [PVAT(-)]. Additionally, 3T3-L1 cells were used to evaluate the molecular mechanisms. The KO mice showed a decreased respiratory exchange ratio (RER) and a whitening phenotype of PVAT, which was accompanied by a loss of anti-contractile effects [(mN) WT_PVAT(-): 5.2±0.3 vs PVAT(+): 3.4±0.5*, *P&lt;0.05 and KO_PVAT(-): 6.1±0.7 vs KO_PVAT(+): 5.7±0.4]. Additionally, PVAT from KO displayed a significant reduction in nitric oxide production [(RFU) WT_PVAT: 223.1± 12.3 vs KO_PVAT: 67.3±9.1*, *P&lt;0.05] and endothelial NO synthase phosphorylation. Additionally, the lack of PGRN attenuated the expression of mitochondrial complexes [(AU) WT_PVAT: 1.3± 0.03 vs KO_PVAT: 0.6±0.06*, *P&lt;0.05] measured by the OXPHOS cocktail antibody. Via high-resolution respirometry, we found that isolated mitochondria from KO_PVAT showed impaired succinate-driven respiration or complex II activity. To understand if the whitening phenotype of PVAT was leading to its dysfunction, we induced a browning phenotype of PVAT by treating the mice with a β3-agonist for 7 days (CL 316243, 1mg/kg/day). This treatment increased RER restored the PVAT phenotype, and improved PVAT anti-contractile effect and mitochondrial respiration. Using 3T3-L1 cells, we engineered the overexpression and knockdown of PGRN via lentivirus (LV) infection and siRNA, respectively. Suppression of PGRN increased mRNA of whitening markers (1.5-fold increase vs scramble siRNA), while overexpression elevated mRNA of browning and mitochondrial markers (1.3-fold increase vs scramble LV). In summary, our findings reveal that PGRN is crucial for maintaining the phenotype and anti-contractile function of PVAT and highlight PGRN as a pharmacological target for vascular outcomes in cardiometabolic conditions.

Restriction of lentivirus infection by proteins encoded by the equine genome

Restriction of lentivirus infection by proteins encoded by the equine genome

Abstract P170: Angiotensin II Receptor Associated Protein Expression Within the PVN of Spontaneously Hypertensive Rats

Increased levels of circulating Angiotensin II (Ang II) have been linked to the development of neurogenic hypertension and elevated sympathetic activity, primarily through the Angiotensin II Receptor 1 (AT1R). Angiotensin II Receptor Associated Protein (ATRAP) is a transmembrane protein that regulates Ang II signaling by promoting the internalization of AT1R. Studies have demonstrated that in a model of Ang II-induced hypertension, enhancing ATRAP expression in the paraventricular nucleus of the hypothalamus (PVN) via Lentivirus infection reduced high blood pressure. However, whether ATRAP is differentially expressed in Spontaneously Hypertensive Rats (SHR) is unknown. We hypothesized that ATRAP gene and protein expression decreases during the development of hypertension in SHRs, leading to increased Ang II signaling. To test this, we used male SHRs at 4 (pre-hypertensive) and 12-weeks-old (wo, hypertensive; mean arterial pressure (MAP) 128.0±2.7 vs. 149.2±5.30 mmHg, respectively, measured by tail-cuff recording [Kent Scientific]) and age-matched normotensive Wistar rats (WIS, 92.6±3.3 vs. 123.6±4.6 mmHg, respectively). We measured ATRAP and AT1R mRNA expression in PVN samples using real-time PCR. Protein expression density of ATRAP was assessed using immunofluorescence (IF) and confocal imaging. We found no differences in ATRAP mRNA expression in the PVN between groups ( p &gt;0.05). On the other hand, we found a significant increase in AT1R mRNA expression after hypertension is developed (WIS 12wo: 1.12 ± 0.35; vs. SHR 12wo: 4.17 ± 1.07, p =0.02). While no differences were found in ATRAP, using ATRAP/AT1R ratio as an indicator of AT1R overactivity and decrease in ratio after the development of hypertension (SHR 12wo: 0.23 ± 0.06 vs. WIS 12wo:1.05 ± 0.25) further indicate increased AngII signaling. As observed with ATRAP mRNA expression, no statistical differences were observed in ATRAP protein density within the SHR at 12wo as measured in a separate cohort of animals (MAP SHR 12wo:149.0 ± 3.7 vs. WIS 12wo:115.8 ± 4.0; p &gt;0.05). These results suggest that the increased AngII signaling within the PVN of SHR are most likely due to increased AT1r expression rather than a differential expression of ATRAP. This work was partially funded by AHA 953524 to VCB.

Eupafolin hinders cross-talk between gastric cancer cells and cancer-associated fibroblasts by abrogating the IL18/IL18RAP signaling axis

BackgroundCancer-associated fibroblasts (CAFs) are involved in the progression of gastric cancer (GC) as a critical component of the tumor microenvironment (TME), yet specific interventions remain limited. Natural products hold a promising application prospect in the field of anti-tumor in view of their high activity and ease of binding with biological macromolecules. However, the role of natural products in modulating the cross-talk between CAFs and GC cells has not been fully investigated. PurposeThe aim of this study was to identify a potential therapeutic target in CAFs and then screen for natural small molecule drugs with anti-tumor activity against this target. MethodsIntegrating bioinformatics analysis of public databases and experimental validation of human samples and cell lines to identify a candidate target in CAFs. Molecular docking and biolayer interferometry technique were utilized for screening potential natural small molecule drugs. The efficacy and underlying mechanisms of the candidates were explored in vitro and in vivo through techniques such as lentiviral infection, cell spheroids culture, immunoprecipitation and cells-derived xenografts. ResultsIL18 receptor accessory protein (IL18RAP) was found to be overexpressed in CAFs derived from GC tissues and facilitated the protumor function of CAFs on GC. Based on virtual screening and experimental validation, we identified a natural product, eupafolin, that interfered with IL18 signaling. Phenotyping studies confirmed that the proliferation, spheroids formation and tumorigenesis of GC cells facilitated by CAFs were greatly attenuated by eupafolin both in vitro and in vivo. Mechanistically, eupafolin impeded the formation of IL18 receptor (IL18R) complex by directly binding to IL18RAP, thus blocking IL18-mediated nuclear factor kappa B (NF-κB) activation and reduced the synthesis and secretion of IL6 in CAFs. As a consequence, it inactivated signal transducer and activator of transcription 3 (STAT3) in GC cells. ConclusionThis study provides new evidence that IL18 signaling regulates the cross-talk between GC cells and CAFs. And it highlights a novel pharmacological role of eupafolin in inhibiting IL18 signaling, thereby curbing the development of GC via modulating CAFs.

Investigation of seroprevalence of small ruminant lentivirus infections in Erzurum province of Türkiye and determination of individual and environmental variables

Small ruminant lentiviruses (SRLVs) are chronic, incurable, and vaccine-free viral diseases that cause respiratory problems and nervous disorders and yield losses in sheep and goats. Caprine arthritis encephalitis virus in goats and maedi-visna virus in sheep have been named as SRLVs. This study aimed to determine the epidemiological status of SRLV infection in Erzurum province and to evaluate the risk factors of the disease based on breed, age, and sex. For this purpose, 204 animals including 184 sheep (Akkaraman, Morkaraman, and Hemşin breeds) and 20 goats (Anatolian Black goats) from 8 districts of Erzurum province (Aşkale, Hınıs, Horasan, Karaçoban, Palandöken, Pasinler, Pazaryolu, and Tekman) were included in the sample. Commercial antibody-ELISA kit was used to determine the seroprevalence of SRLV and 15.12% seropositivity was detected. In terms of SRLV, 14.67% of females and 20% of males were positive. In terms of breed, 20%, 13.76%, 0%, and 15% seropositivity was determined in Akkaraman, Morkaraman and Hemşin breed sheep and Anatolian Black goats, respectively. Although there was no statistically significant difference in terms of breed groups and sex, the detection rates in the districts were significant. In conclusion, the prevalence of SRLV infection was investigated in 8 locations of Erzurum province, which is one of the important centers of animal breeding and where small ruminant breeding is at a high level, and significant findings were obtained at the district level. With this study, updated data on seroprevalence of SRLV in the region were obtained and a broader perspective was tried to be provided by comparing with other SRLV studies in Türkiye and the world. These findings are important in terms of evaluating the prevalence and transmission risks of SRLV infections in the region and will shed light on future control and prevention strategies.

LncRNAPVT1 is Associated with Cancer-Associated Fibroblasts Proliferation Through Regulating TGF-βin Oral Squamous Cell Carcinoma

ABSTRACT Introduction Human oral squamous cell carcinoma (OSCC) is the most common type of oral cancer and has a poor survival rate. Cell-cell communication between OSCC cells and cancer-associated fibroblasts (CAFs) plays important roles in OSCC progression. We previously demonstrated that CAFs promote OSCC cell migration and invasion. However, how OSCC cells influence CAFs proliferation is unknown. Methods Knockdown of PVT1 was confirmed using lentivirus infection technique. CAFs in tissues were identified by staining the cells with α-SMA using immunohistochemical technique. CCK-8 assay was used to evaluate cell proliferation. The mRNA level of a gene was measured by qRT-PCR. Secreted TGF-β were detected using ELISA assay. Results We found that knockdown of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was associated with a low density of CAFs in xenograft tumors in mice; further analysis revealed that PVT1 in OSCC cells induced CAF proliferation through transforming growth factor (TGF)-β. Discussion Our results demonstrate that lncRNA PVT1 in tumor cells participates in CAF development in OSCC by regulating TGF-β. This study revealed a new mechanism by which PVT1 regulates OSCC progression and PVT1 is a potential therapeutic target in OSCC

Engineered mitochondria exert potent antitumor immunity as a cancer vaccine platform.

The preferable antigen delivery profile accompanied by sufficient adjuvants favors vaccine efficiency. Mitochondria, which feature prokaryotic characteristics and contain various damage-associated molecular patterns (DAMPs), are easily taken up by phagocytes and simultaneously activate innate immunity. In the current study, we established a mitochondria engineering platform for generating antigen-enriched mitochondria as cancer vaccine. Ovalbumin (OVA) and tyrosinase-related protein 2 (TRP2) were used as model antigens to synthesize fusion proteins with mitochondria-localized signal peptides. The lentiviral infection system was then employed to produce mitochondrial vaccines containing either OVA or TRP2. Engineered OVA- and TRP2-containing mitochondria (OVA-MITO and TRP2-MITO) were extracted and evaluated as potential cancer vaccines. Impressively, the engineered mitochondria vaccine demonstrated efficient antitumor effects when used as both prophylactic and therapeutic vaccines in murine tumor models. Mechanistically, OVA-MITO and TRP2-MITO potently recruited and activated dendritic cells (DCs) and induced a tumor-specific cell-mediated immunity. Moreover, DC activation by mitochondria vaccine critically involves TLR2 pathway and its lipid agonist, namely, cardiolipin derived from the mitochondrial membrane. The results demonstrated that engineered mitochondria are natively well-orchestrated carriers full of immune stimulants for antigen delivery, which could preferably target local dendritic cells and exert strong adaptive cellular immunity. This proof-of-concept study established a universal platform for vaccine construction with engineered mitochondria bearing alterable antigens for cancers as well as other diseases.

Abstract Tu021: Genome-wide CRISPR Screen In Vivo Decodes Monocyte Infiltration Associated With Abdominal Aortic Aneurysm

Introduction: Aortic aneurysm is the second most common aortic disease following atherosclerosis and accounts for the ninth-leading cause of death overall. The inflammatory response has a crucial role in the Abdominal Aortic Aneurysm (AAA) development. The infiltration of monocyte into aortic wall is a hallmark of aortic inflammation and it still requires further understanding of this complicated process. CRISPR screening is a powerful tool to investigate biological process, but it requires large pool of mutant cells and reliable selection procedure, which can usually only be performed in vitro or in xenograft models. To overcome this problem, we developed an unbiased genome-wide CRISPR-Cas Screen for Monocyte Infiltration in vivo (CRISPR-MI) and employed this new method in mouse AAA model to systematically study key genes regulating monocyte infiltration into aortic wall. Hypothesis: Monocyte infiltration is an important process in AAA development and CRISPR-MI could be utilized to study this process in vivo. Methods: To acquire enough monocytes for screen, we used the adoptive transfer method to transplant in vivo differentiated monocytes (bone marrow derived macrophage, BMDM) back into mouse circulation via tail vein injection. We optimized the lentivirus infection protocol, so that sgRNA library delivered by lentivirus can effectively knock out target genes in BMDMs. The BMDMs were infected with sgRNA library before adoptive transfer into AAA-model mice. After transfer, by comparing the sgRNA enrichment in different organs of receipt mice, we could identify key genes inhibiting monocyte infiltration. Results: CRISPR-MI identified novels genes regulating monocyte infiltration into aorta in AAA model, including Trem2 gene. In vitro experiments confirmed that Trem2 knockout increased adhesion molecules and chemokine receptors expression. Conclusions: CRISPR-MI can be used to identify new therapeutical targets for cardiovascular diseases.