Expression Of KLF4 Research Articles

MiR‑155 promotes an inflammatory response in HaCaT cells via the IRF2BP2/KLF2/NF‑κB pathway in psoriasis.

Psoriasis is a chronic inflammatory skin condition with numerous causes, including genetic, immunological and infectious factors. The course of psoriasis is long and recurrence is common; pathogenesis is not completely understood. However, there is an association between advancement of psoriasis and aberrant microRNA (miR or miRNA)‑155 expression. Through bioinformatics, the present study aimed to analyze the differentially expressed genes and miRNAs in psoriasis and its biological mechanism and function psoriatic inflammation. First of all, differentially expressed genes (DEGs) and miRNAs (DEMs) in patients with psoriasis were identified using GEO2R interactive web application. A psoriasis inflammatory model was established using lipopolysaccharide (LPS)‑treated HaCaT keratinocytes, which were transfected with miR‑155 mimic or inhibitor. Cell Counting Kit‑8 was used for the assessment of cell viability and proliferation, and changes in the cell cycle were examined using flow cytometry. ELISA and reverse transcription‑quantitative PCR (RT‑qPCR) were used to detect the expression levels of the inflammatory factors IL‑1β and IL‑6. The dual‑luciferase reporter assay was used to verify the targeting association between miR‑155‑5p and IFN regulatory factor 2 binding protein 2 (IRF2BP2). To verify the targeting association of miR‑155 and the IRF2BP2/kruppel‑like factor 2 (KLF2)/NF‑κB signaling pathway, expression levels of IRF2BP2, KLF2 and p65 were identified by RT‑qPCR and western blotting. IRF2BP2 levels were also confirmed by immunofluorescence, in conjunction with bioinformatics database analysis. Overexpression of miR‑155 inhibited proliferation of HaCaT cells and increased the number of cells in S phase and decreasing number of cells in G1 and G2 phase. In the LPS‑induced inflammatory state, miR‑155 overexpression heightened the inflammatory response of HaCaT cells while inhibition of miR‑155 lessened it. Suppression of inflammatory cytokine expression by miR‑155‑5p inhibitor was reversed by knockdown of IRF2BP2. miR‑155 was shown to interact with IRF2BP2 to negatively regulate its expression, leading to decreased KLF2 expression and increased p65 expression and secretion of inflammatory factors, intensifying the inflammatory response of HaCaT cells. Therefore, miR‑155 may contribute to development of psoriasis by inducing tissue and cell damage by increasing the inflammatory response of HaCaT cells via the IRF2BP2/KLF2/NF‑κB pathway. In conclusion, the results of the present study offer novel perspectives on the role of miR‑155 in the onset and progression of psoriasis.

Downregulation of Krüppel-like factor 15 expression delays endochondral bone ossification during fracture healing

ObjectiveThe role of Krüppel-like zinc finger transcription factor 15 (KLF15) in endochondral ossification during fracture healing remains unexplored. In this study, we aimed to elucidate the impact of KLF15 in a mouse model of tibial transverse fracture. MethodsWe created tamoxifen-inducible, cartilage-specific KLF15 knockout mice (KLF15 KO). KLF15 fl/fl Col2-CreERT mice from the same litters as the KLF15 KO mice, but not treated with 4-hydroxytamoxifen, were used as controls (CT). At 10 weeks, male KLF15 KO and CT mice underwent tibial fracture followed by intramedullary nailing. Both groups were administered tamoxifen at days 0, 3, and 7 after surgery. The tibiae were harvested on post-surgery days 7, 10, and 14 for radiological assessment using micro-computed tomography. Histological staining (Safranin-O) and immunohistochemistry for KLF15, SOX9, Indian hedgehog (IHH), RUNX2, and Osterix were performed. Additionally, cartilage from mouse fetus was cultured for qRT-PCR and western blot analyses of KLF15, SOX9, IHH, Col2, RUNX2, Osterix, TGF-β, SMAD3, and phosphor-SMAD3. ResultsThe radiological assessment revealed that immature callus formation was delayed in KLF15 KO, compared with that in CT, peaking on day 14 compared with that on day 10 in CT. KLF15 KO mice exhibited delayed fracture healing and reduced Safranin-O staining at days 7 and 10 post-surgery. The ratio of cells positive for KLF15 and SOX9 was significantly lower in KLF15 KO than in CT, whereas the ratios for IHH, RUNX2, and Osterix showed no significant difference. RT-PCR revealed reduced expression of KLF15, SOX9, and COL2, with no significant changes in IHH, Osterix, RUNX2, TGF-β, and SMAD3. Western blot analysis indicated decreased SMAD3 phosphorylation in KLF15 KO mice. ConclusionKLF15 regulates SOX9 via the TGF-β-SMAD3-SOX9 pathway, independent of IHH, in endochondral ossification. The KLF15 deficiency decreases SOX9 expression through reduced SMAD3 phosphorylation, subsequently delaying fracture healing.

HDAC6 promotes inflammation in lupus nephritis mice by regulating transcription factors MAFF and KLF5 in renal fibrosis

Aim This study explored the effect and mechanism of MAFF and HDAC6 on renal fibrosis and inflammation in lupus nephritis (LN). Methods IL-33 treated renal epithelial cells and MRL/lpr mice were respectively used for in vitro and in vivo experiments. The expressions of HDAC6, MAFF, and KLF5 were measured in cells and renal tissues. Before and after cell transfection, the morphological changes in renal tissues were observed using Hematoxylin and eosin (H&E) and Masson staining. The proteinuria, serum creatinine (SCr), blood urea nitrogen (BUN), and double-stranded DNA (dsDNA) levels were detected by biochemical analysis. The expressions of fibrosis and inflammation related proteins (including α-SMA, Vimentin, IL-1β, IL-6, and TNF-α), p65, and iNOS were also detected. The relationship among MAFF, HDAC6, and KLF5 was determined by chromatin immunoprecipitation and dual luciferase reporter gene assay. Results Renal tissues and cell models had elevated expressions of HDAC6 and KLF5, and decreased MAFF expression. HDAC6 suppression or MAFF overexpression led to suppression of proteinuria, SCr, BUN, and dsDNA levels, as well as attenuation of inflammatory infiltration and collagen deposition. HDAC6 can suppress MAFF expression via deacetylation to abolish its suppression of KLF5 expression, thus increasing KLF5 expression. In vivo and in vitro experiments showed the suppressive effect of HDAC6 suppression on renal fibrosis and inflammation can be abolished by KLF5 overexpression. Conclusion HDAC6 suppresses MAFF expression via deacetylation to elevate KLF5 expression, which consequently enhances fibrosis and inflammatory response in LN.

Renoprotective effect of liraglutide on diabetic nephropathy by modulation of Krüppel-like transcription factor 5 expression in rats.

Diabetic nephropathy (DN) is a serious consequence of diabetes that can develop through the lysophosphatidic acid axis. The purpose of this study was to determine whether the antidiabetic drug liraglutide can slow the development of diabetic kidney damage by altering the lysophosphatidic acid axis via KLF5. Wistar albino rats were divided into nondiabetic and diabetic rats (resulting from an intraperitoneal streptozotocin dose of 30mg/kg and a high-fat diet). These rats were further divided into four groups: nondiabetic control, liraglutide-treated nondiabetic, diabetic control, and liraglutide-treated diabetic. The nondiabetic and diabetic control groups received normal saline for 42 days, while the liraglutide-treated nondiabetic and diabetic groups received normal saline for 21 days, followed by a subcutaneous dose of liraglutide (200 μg/kg/day) for 21 days. Subsequently, serum levels of DN biomarkers were evaluated, and kidney tissues were histologically examined. The protein expression of PCNA, autotaxin, and KLF5 was detected. Liraglutide treatment in diabetic rats decreased DN biomarkers, histological abnormalities in kidney tissues, and the protein expression of PCNA, autotaxin, and KLF5. Liraglutide can slow the progression of DN by modulating KLF5-related lysophosphatidic acid axis. Thus, liraglutide may be an effective treatment for preventing or mitigating diabetes-related kidney damage.

The protective effects of annexin A1 against oxidized-LDL-induced monocytes adhesion to endothelial cells: implication in atherosclerosis.

Oxidized low-density lipoprotein (ox-LDL)-associated endothelial dysfunction is a critical factor in the initiation and progression of Atherosclerosis (AS). Annexin A1 is an important member of the annexin family. Despite its wide range of biological functions across various tissues and cells, the role of Annexin A1 in AS remains largely unexplored. In this study, we demonstrate that Annexin A1 treatment effectively reduced the expression of LOX-1 at both the mRNA and protein levels in HUVECs exposed to ox-LDL. Annexin A1 also ameliorated oxidative stress (OS) by decreasing mitochondrial ROS levels and restoring reduced GSH levels. Moreover, Annexin A1 decreased the expression of pro-inflammatory cytokines, including IL-6 and MCP-1. Importantly, Annexin A1 inhibited ox-LDL-induced expressions of the endothelial adhesion molecules, such as E-selectin and VCAM-1 in HUVECs, which leads to reduced attachment of THP-1 monocytes to HUVECs. Mechanically, we found that Annexin A1 reversed the expression of KLF2 against ox-LDL mediated by the PI3K/Akt axis. Notably, the silencing of KLF2 abrogated the protective effects of Annexin A1 on E-selectin and VCAM-1 expression and the attachment of THP-1 monocytes to HUVECs. Our findings suggest that Annexin A1 is a potential therapeutic agent for atherosclerosis, offering a novel approach to mitigate endothelial dysfunction and inflammation.

Pterosin B improves cognitive dysfunction by promoting microglia M1/M2 polarization through inhibiting Klf5/Parp14 pathway

BackgroundPterosin B (PB) exhibits strong neuroprotective effects in vitro, but its therapeutic effect and underlying mechanism on Alzheimer's disease (AD) remain elusive. PurposeThis study aimed to investigate the anti-AD effect and mechanism of PB. Study designThe therapeutic effect and mechanism of PB were investigated in APP/PS1 mice and lipopolysaccharide (LPS)-induced BV-2 cells. MethodsAfter 8 weeks of oral administration of PB or donepezil, the cognitive function was assessed using behavioral tests. Pathological damage was evaluated using histological analysis and immunohistochemical staining. Flow cytometry was applied to detect M1/M2 polarization. The expression levels of glycolysis- and oxidative phosphorylation-related proteins as well as enzyme activities were determined using Western blot and biochemical kits, respectively. The levels of inflammatory cytokines and Kruppel-like factor 5 (Klf5) were measured using enzyme-linked immunosorbent assay. AD biomarkers in serum were analyzed using single-molecular array. RNA sequencing identified the downstream molecules of Klf5, and interaction was evaluated using dual-luciferase reporter assay. ResultsOur findings demonstrated that PB effectively ameliorated cognitive impairment and reduced pathological damage in APP/PS1 mice. Furthermore, PB facilitated the transition of the phenotype of LPS-induced BV-2 cells from M1 to M2 by modulating metabolic reprogramming. Additionally, Klf5 had high expression levels in the serum of patients with AD, which strongly correlated with cognitive performance and AD biomarkers. PB downregulated Klf5 expression both in vitro and in vivo. Subsequently, poly-ADP ribosyl polymerase 14 (Parp14) was identified as a downstream molecule of Klf5 involved in regulating metabolic reprogramming, and PB regulated microglia M1/M2 polarization by inhibiting the Klf5/Parp14 pathway. ConclusionThe findings suggested that PB ameliorated cognitive dysfunction in AD by modulating microglia M1/M2 polarization via inhibiting Klf5/Parp14 pathway.

Low Intraocular Pressure Induces Fibrotic Changes in the Trabecular Meshwork and Schlemm's Canal of Sprague Dawley Rats.

Continuous artificial aqueous humor drainage in the eyes of patients with glaucoma undergoing trabeculectomy likely exerts abnormal shear stress. However, it remains unknown how changes in intraocular pressure (IOP) can affect aqueous humor outflow (AHO). Here, we induced and maintained low intraocular pressure (L-IOP) in healthy Sprague Dawley (SD) rats by puncturing their eyes using a tube (200-µm diameter) for 2 weeks. After the rats were euthanized, their eyes were removed, fixed, embedded, stained, and scanned to analyze the physiological and pathological changes in the trabecular meshwork (TM) and Schlemm's canal (SC). We measured SC parameters using ImageJ software and assessed the expression of various markers related to flow shear stress (KLF4), fibrosis (TGF-β1, TGF-β2, α-SMA, pSmad1/5, pSmad2/3, and fibronectin), cytoskeleton (integrin β1 and F-actin), diastolic function (nitric oxide synthase and endothelial nitric oxide synthase [eNOS]), apoptosis (cleaved caspase-3), and proliferation (Ki-67) using immunofluorescence or immunohistochemistry. L-IOP eyes showed a larger SC area, higher eNOS expression, and lower KLF4 and F-actin expression in the TM and SC (both P < 0.05) than control eyes. The aqueous humor of L-IOP eyes had a higher abundance of fibrotic proteins and apoptotic cells than that of control eyes, with significantly higher TGF-β1, α-SMA, fibronectin, and cleaved caspase-3 expression (all P < 0.05). In conclusion, a persistence of L-IOP for 2 weeks may contribute to fibrosis in the TM and SC and might be detrimental to conventional AHO in SD rat eyes. Clinicians should consider that aberrant shear force induced by aqueous humor fluctuation may damage AHO outflow channel when treating patients.

TRβ activation confers AT2-to-AT1 cell differentiation and anti-fibrosis during lung repair via KLF2 and CEBPA

Aberrant repair underlies the pathogenesis of pulmonary fibrosis while effective strategies to convert fibrosis to normal regeneration are scarce. Here, we found that thyroid hormone is decreased in multiple models of lung injury but is essential for lung regeneration. Moreover, thyroid hormone receptor α (TRα) promotes cell proliferation, while TRβ fuels cell maturation in lung regeneration. Using a specific TRβ agonist, sobetirome, we demonstrate that the anti-fibrotic effects of thyroid hormone mainly rely on TRβ in mice. Cellularly, TRβ activation enhances alveolar type-2 (AT2) cell differentiation into AT1 cell and constrains AT2 cell hyperplasia. Molecularly, TRβ activation directly regulates the expression of KLF2 and CEBPA, both of which further synergistically drive the differentiation program of AT1 cells and benefit regeneration and anti-fibrosis. Our findings elucidate the modulation function of the TRβ-KLF2/CEBPA axis on AT2 cell fate and provide a potential treatment strategy to facilitate lung regeneration and anti-fibrosis.

KLF15 suppresses stemness of pancreatic cancer by decreasing USP21-mediated Nanog stability

The existence of cancer stem cells (CSCs) in pancreatic ductal adenocarcinoma (PDAC) is considered to be the key factor for metastasis and chemoresistance. Thus, novel therapeutic strategies for eradicating CSCs are urgently needed. Here we aimed to explore the role of KLF15 in stemness and the feasibility of using KLF15 to inhibit CSCs and improve chemotherapy sensitivity in PDAC. In this study, we report that KLF15 is negatively associated with poor survival and advanced pathological staging of PDAC. Moreover, tumorous KLF15 suppresses the stemness of PDAC by promoting the degradation of Nanog, and KLF15 directly interacts with Nanog, inhibiting interaction between Nanog with USP21. We also demonstrate that the KLF15/Nanog complex inhibit the stemness in vivo and in PDX cells. Tazemetostat suppresses stemness and sensitizes PDAC cells to gemcitabine by promoting KLF15 expression in PDAC. In summary, the findings of our study confirm the value of KLF15 level in diagnosis and prognosis of PDAC, it is the first time to explore the inhibition role of KLF15 in stemness of PDAC and the regulation mechanism of Nanog, contributing to provide a new therapeutic strategy that using Tazemetostat sensitizes PDAC cells to gemcitabine by promoting KLF15 expression for PDAC.Graphical

Role of KLF4 and SIAT7A interaction accelerates myocardial hypertrophy induced by Ang II.

Sialylation catalysed by sialyltransferase 7A (SIAT7A) plays a role in the development of cardiac hypertrophy. However, the regulatory mechanisms upstream of SIAT7A in this context remain poorly elucidated. Previous study demonstrated that KLF4 activates the SIAT7A gene in ischemic myocardium by binding to its promoter region. Nevertheless, the potential involvement of KLF4 in regulating SIAT7A expression in Ang II-induced hypertrophic cardiomyocytes remains uncertain. This study seeks to deepen the underlying mechanisms of the KLF4 and SIAT7A interaction in the progression of Ang II-induced cardiac hypertrophy. The results showed a concurrent increase in SIAT7A and KLF4 levels in hypertrophic myocardium of essential hypertension patients and in hypertrophic cardiomyocytes stimulated by Ang II. Invitro experiments revealed that reducing KLF4 levels led to a decrease in both SIAT7A synthesis and Sialyl-Tn antigen expression, consequently inhibiting Ang II-induced cardiomyocyte hypertrophy. Intriguingly, reducing SIAT7A levels also resulted in decreased KLF4 expression and suppression cardiomyocyte hypertrophy. Consistent with this, elevating SIAT7A levels increased KLF4 expression and exacerbated cardiomyocyte hypertrophy in both invivo and invitro experiments. Additionally, a time-course analysis indicated that KLF4 expression preceded that of SIAT7A. Luciferase reporter assays further confirmed that modulating SIAT7A levels directly influenced the transcriptional activity of KLF4 in cardiomyocytes. In summary, KLF4 expression is upregulated in cardiomyocytes treated with Ang II, which subsequently induces the expression of SIAT7A. The elevated levels of SIAT7A, in turn, enhance the transcription of KLF4. These findings suggest a positive feedback loop between KLF4 and SIAT7A-Sialyl-Tn, ultimately promoting Ang II-induced cardiac hypertrophy.

Single-cell and spatial transcriptomics reveal apelin/APJ pathway's role in microvessel formation and tumour progression in hepatocellular carcinoma.

The apelin receptor (APJ) is a key player in tumour angiogenesis, but its role in hepatocellular carcinoma (HCC) remains unclear. This study aims to elucidate the function of the apelin/APJ pathway in HCC using a multi-omics approach and identify potential therapeutic biomarkers. Differentially expressed genes related to the apelin/APJ axis were identified from bulk transcriptomics to reveal HCC-associated disparities. Single-cell and spatial transcriptomics were used to localize and analyse the function of these genes. Machine learning models were constructed to predict outcomes based on apelin/APJ expression, and experimental validation was conducted to explore the pathway's impact on HCC angiogenesis. Single cell analysis revealed an overexpression of APJ/Aplin in the endothelium. The stemness of endothelial cell (EC) with high apelin/APJ was enhanced, as well as the expression of TGFb, oxidative stresses and PI3K/AKT pathway genes. Spatial transcriptomics confirmed that EC populations with high APJ scores were enriched within the tumour. Machine learning models showed high prognostic accuracy. High APJ expression was linked to worse outcomes (p = 0.001), and AUC values were high (1 year, 3 year, 5 year) (0.95, 0.97, 0.98). Immune suppression and non-responsiveness of immune therapy were also seen in high-risk groups. The experimental validation showed that silencing apelin reduced angiogenesis (p < 0.05), endothelial proliferation, decreased expression of ANG2, KLF2, VEGFA and lower ERK1/2 phosphorylation. Apelin may serve as a potential therapeutic target in HCC, given its role in promoting tumour angiogenesis and poor patient outcomes.

A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer.

Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the three Klf4 enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in Klf4 enhancer E2 rescued enhancer function and Klf4 expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities of Klf4 enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.

Disturbed function of TBL1X has a differential effect on T3-regulated gene expression in two human liver cell models.

Mutations in TBL1X, part of the NCOR1/SMRT corepressor complex, were identified in patients with hereditary X-linked central congenital hypothyroidism and associated hearing loss. The role of TBL1X in thyroid hormone (TH) action, however, is incompletely understood. The aim of the present study was to investigate the role of TBL1X on T3-regulated gene expression in two human liver cell models. A human hepatoma cell line (HepG2) wherein TBL1X was downregulated using siRNAs, and human-induced pluripotent stem cell-derived hepatocytes (iHeps) generated from individuals with a TBL1X N365Y mutation. Both cell types were treated with increasing concentrations of T3. The expression of T3-regulated genes was measured by qPCR. KLF9, CPT1A, and PCK1 mRNA expression were higher upon T3 stimulation in the HepG2 cells with decreased TBL1X expression compared to controls, while DIO1 mRNA expression was lower. Hemizygous TBL1X N365Y iHeps exhibited decreased expression of CPT1A, G6PC1, PCK1, FBP1, and ELOVL2 compared to cells with the heterozygous TBL1X N365Y allele, but KLF9 and HMGCS2 expression was unaltered. Downregulation of TBL1X in HepG2 cells and the TBL1X N365Y variant in iHeps have differential effects on T3-regulated gene expression. This suggests that TBL1X may play a gene context role in TH action.

The mechanism of TGF-β mediating BRD4/STAT3 signaling pathway to promote fibroblast proliferation and thus promote keloid progression

ObjectiveThe purpose of this study was to investigate the effect of TGF-β on keloid and its molecular mechanism in fibroblasts. Methods: The difference between normal tissue and keloid tissue can be detected using HE staining. Fibroblasts were treated with TGF-β, and then treated with the BRD4 inhibitor JQ1 and the STAT3 activator Colivelin TFA. Western blot was used to measure the relative protein expression of TGF-β, BRD4, p-STAT3, p-EZH2, C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Immunofluorescence staining was used to measure the relative fluorescence intensity of BRD4, p-STAT3, α-SMA, and Collagen-I. Cell proliferation ability was evaluated by CCK-8 assay and colony formation assay. Results:The expression of TGF-β and BRD4 was significantly higher in keloid tissue compared to normal tissue. TGF-β mediated the BRD4/STAT3 signaling pathway to inhibit p-EZH2 and promote the expression of C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Additionally, TGF-β mediated the BRD4/STAT3 signaling pathway to enhance fibroblast proliferation. Conclusion:TGF-β mediates the BRD4/STAT3 signaling pathway to promote fibroblast proliferation and contribute to the progression of keloid.

Biochanin A suppresses Klf6-mediated Smad3 transcription to attenuate renal fibrosis in UUO mice

BackgroundRenal fibrosis is a hallmark of chronic kidney disease (CKD). Smad3 serves as the principal transcription factor mediating the pro-fibrosis effects of TGF-β signaling in renal fibrosis. Biochanin A (BCA), a natural isoflavone, has been shown to attenuate renal fibrosis by inhibiting TGF-β signaling but the detailed mechanisms remain unresolved. This study aimed to elucidate the specific mechanisms by which BCA modulates TGF-β signaling. MethodsRenal fibrosis models were established both in vitro, using TGF-β1-stimulated mouse renal tubular TCMK1 cells, and in vivo, employing mice with unilateral ureter obstruction (UUO). RNA-seq was conducted to identify BCA-regulated genes. The AnimalTFDB4.0 database was utilized to predict transcription factors with potential binding to Smad3 promoter. The activities of TGF-β signaling and the cloned mouse Smad3 promoter were assessed using luciferase reporter assays. Plasmid transfection was performed using polyethylenimine in TCMK1 cells or ultrasound microbubbles in UUO kidneys. Gene expression was analyzed by RT-PCR, western blot, and immunohistochemistry assays. ResultsBCA significantly inhibited TGF-β signaling activity and suppressed TGF-β1-induced fibrotic gene expression in TCMK1 cells. RNA-seq and in silico analyses identified Smad3 as the key gene downregulated by BCA, while leaving Smad2 unaffected. This selective transcriptional suppression of Smad3 by BCA was validated by luciferase reporter assays using the cloned Smad3 promoter. Furthermore, transcription factor binding prediction identified that Klf6, a transcription factor downregulated by BCA, has binding potential to the Smad3 promoter and promotes Smad3 transcription. Klf6 expression was induced in TGF-β1-stimulated TCMK1 cells and UUO kidneys, but this induction was abolished upon BCA treatment. Importantly, Klf6 overexpression restored Smad3 expression and counteracted the anti-fibrosis effects of BCA in both TGF-β1-stimulated TCMK1 cells and UUO kidneys. ConclusionTGF-β-responsive Klf6 transcriptionally transactivates Smad3 expression. BCA exerts anti-renal fibrosis effects by inhibiting the Klf6-Smad3 signaling axis, underscoring its therapeutic potential in the treatment of CKD.

Sustainability of shear stress conditioning in endothelial colony-forming cells compared to human aortic endothelial cells to underline suitability for tissue-engineered vascular grafts

The endothelialization of cardiovascular implants is supposed to improve the long-term patency of these implants. In addition, in previous studies, it has been shown, that the conditioning of endothelial cells by dynamic cultivation leads to the expression of an anti-thrombogenic phenotype. For the creation of a tissue-engineered vascular graft (TEVG), these two strategies were combined to achieve optimal hemocompatibility. In a clinical setup, this would require the transfer of the already endothelialized construct from the conditioning bioreactor to the patient. Therefore, the reversibility of the dynamic conditioning of the endothelial cells with arterial-like high shear stress (20 dyn/cm2) was investigated to define the timeframe (tested in a range of up to 24 h) for the perseverance of dynamically induced phenotypical changes. Two types of endothelial cells were compared: endothelial colony-forming cells (ECFCs) and human aortic endothelial cells (HAECs). The results showed that ECFCs respond far more sensitively and rapidly to flow than HAECs. The resulting cell alignment and increased protein expression of KLF-2, Notch-4, Thrombomodulin, Tie2 and eNOS monomer was paralleled by increased eNOS and unaltered KLF-2 mRNA levels even under stopped-flow conditions. VCAM-1 mRNA and protein expression was downregulated under flow and did not recover under stopped flow. From these time kinetic results, we concluded, that the maximum time gap between the TEVG cultivated with autologous ECFCs in future reactor cultivations and the transfer to the potential TEVG recipient should be limited to ∼6 h.

Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis.

Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells. Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) induced expression of multiple EndoMT marker genes. However, the detailed molecular mechanisms underlying CD45 that drive EndoMT and the therapeutic potential of manipulation of CD45 expression in atherosclerosis are entirely unknown. We generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-iCD45KO) in an ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA sequencing. Biomedical, cellular, and molecular approaches were utilized to investigate the role of endothelial CD45-specific deletion in the prevention of EndoMT in ApoE-/- model of atherosclerosis. Single-cell RNA sequencing revealed that loss of endothelial CD45 inhibits EndoMT marker expression and transforming growth factor-β signaling in atherosclerotic mice. which is associated with the reductions of lesions in the ApoE-/- mouse model. Mechanistically, the loss of endothelial cell CD45 results in increased KLF2 expression, which inhibits transforming growth factor-β signaling and EndoMT. Consistently, endothelial CD45 deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls. These findings demonstrate that the loss of endothelial CD45 protects against EndoMT-driven atherosclerosis, promoting KLF2 expression while inhibiting TGFβ signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.

KLF3 impacts insulin sensitivity and glucose uptake in skeletal muscle.

Skeletal muscle is one of the predominant sites involved in glucose disposal, accounting for approximately 80% of postprandial glucose uptake, and plays a critical role in maintaining glycemic homeostasis. Dysregulation of energy metabolism in skeletal muscle is involved in developing insulin resistance and type 2 diabetes (T2D). Transcriptomic responses of skeletal muscle to exercise found that the expression of Klf3 was increased in T2D Goto-Kakizaki (GK) rats and decreased after exercise with improved hyperglycemia and insulin resistance, implying that Klf3 might be associated with insulin sensitivity and glucose metabolism. We also found that knockdown of Klf3 promoted basal and insulin-stimulated glucose uptake in L6 myotubes, while overexpression of Klf3 resulted in the opposite. Through pairwise comparisons of L6 myotubes transcriptome, we identified 2256 and 1988 differentially expressed genes in Klf3 knockdown and overexpression groups, respectively. In insulin signaling, the expression of Slc2a4, Akt2, Insr and Sorbs1 was significantly increased by Klf3 knockdown and decreased with klf3 overexpression; Ptprf and Fasn were markedly downregulated in klf3 reduced group and upregulated in klf3 overexpressed group. Moreover, downregulation of Klf3 promoted the expression of GLUT4 and AKT proteins, as well as the translocation of GLUT4 to the cell membrane in the basal situation, and enhanced insulin sensitivity, characterized by increased insulin-stimulated GLUT4 translocation and AKT, TBC1D1 and TBC1D4 phosphorylation, while overexpression of Klf3 showed contrary results. These results suggest that Klf3 affects glucose uptake and insulin sensitivity via insulin signal transduction and intracellular metabolism, offering a novel potential treatment strategy for T2D.

NEDD4L affects KLF5 stability through ubiquitination to control ferroptosis and radiotherapy resistance in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) contributes to high mortality. Modulating ferroptosis may reverse resistance to radiotherapy. This article was to explore the ubiquitination modification of KLF5 and its effect on ferroptosis in ESCC. KLF5 was under-expressed by shRNA plasmids in the cells and ROS levels were analysed by flow cytometry, ferroptotic gene expression was detected by qRT-PCR, MDA and GSH levels were determined by ELISA, cell morphology was observed by transmission electron microscopy, and Fe ion levels were analysed by immunofluorescence. Cells were treated with Ferrostatin-1 and NAC and analysed for cell proliferation by colony formation assay, cell migration and invasion by Transwell assays, and apoptosis by flow cytometry. DNA damage in cells was also analysed using comet assay, EdU doping assay, γH2AX fluorescence, DNA-PKcs and PCR. NEDD4L and KLF5 binding was analysed by immunoprecipitation. Changes in ferroptosis, DNA damage and resistance were analysed in cells with both silencing NEDD4L and KLF5. Changes in tumour resistance to radiation were analysed in mice underexpressing NEDD4L and KLF5. Low expression of KLF5 significantly promotes cellular lipid peroxidation levels, with decreased expression of SOD and GPX4, and increased expression of ACSL4. Concurrently, MDA levels deplete GSH, and cells exhibit typical ferroptotic morphology with increased Fe2+ content. KLF5 inhibition results in enhanced cellular clonogenicity, migration and invasion activities, reduced apoptosis, increased tail DNA, nuclear EdU incorporation, nuclear γH2AX foci and elevated expression of DNA-PKcs, LIG4, RAD9B and BMI1. Ferrostatin-1 and NAC reverse these effects. NEDD4L ubiquitination modifies and degrades KLF5, with NEDD4L/KLF5 inhibition mitigating cellular ferroptosis and DNA damage, thereby promoting radiosensitivity both invitro and invivo. NEDD4L increases radiosensitivity by accelerating cellular ferroptosis via ubiquitination modification of KLF5.

Metformin Improves the Function of Abdominal Aortic Aneurysm Patient-Derived Aortic Smooth Muscle Cells

Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor. Paradoxically, a decreased risk of abdominal aortic aneurysm (AAA) presence and growth rate is described among patients with T2DM, associated with metformin use. This study aimed to investigate the effect of metformin on AAA patient derived aortic smooth muscle cell (SMC) function. Aortic biopsies were obtained from patients with AAA (n = 21) and controls (n = 17) during surgery. The SMCs of non-pathological aortic controls, non-diabetic patients with AAA, and diabetic patients with AAA were cultured from explants and treated with or without metformin. The SMC contractility was measured upon ionomycin stimulation, as well as metabolic activity, proliferation, and migration. Then, mRNA and protein expression of markers for contraction, metabolic activity, proliferation, and inflammation were measured. The mRNA expression of KLF4 and GYS1, genes involved in metabolic activity, differed between the SMCs from non-diabetic and diabetic patients with AAA before metformin stimulation (p < .041). However, the effect of metformin on the various SMC functions was similar between non-diabetic and diabetic patients with AAA. Upon stimulation, metformin increased the contractility of AAA patient SMCs (p = .001). The mRNA expression of smoothelin, a marker for the contractile phenotype, increased in the SMCs of patients with AAA after treatment with metformin (p = .006). An increase in metabolic activity (p < .001) and a decrease in proliferation (p < .001) and migration were found in the SMCs of controls and patients with AAA with metformin. Increased mRNA expression of PPARγ, a nuclear receptor involved in mitochondrial biogenesis (p < .009), and a decrease in gene expression of Ki-67, a marker for proliferation (p < .005), were observed. Gene expression of inflammation markers MCP-1 and IL-6, and protein expression of NF-κB p65 decreased after treatment with metformin in patients with AAA. This study found that metformin increases contractility and metabolic activity, and reduces proliferation, migration, and inflammation in aortic SMCs in vitro.