Search

Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency., (© 2024. The Author(s).)

Tumor cells subvert immune surveillance or lytic stress by harnessing inhibitory signals. Hence, bispecific antibodies have been developed to direct CTLs to the tumor site and foster immune-dependent cytotoxicity. Although applied with success, T cell-based immunotherapies are not universally effective partially because of the expression of pro-survival factors by tumor cells protecting them from apoptosis. Here, we report a CRISPR/Cas9 screen in human non-small cell lung cancer cells designed to identify genes that confer tumors with the ability to evade the cytotoxic effects of CD8 + T lymphocytes engaged by bispecific antibodies. We show that the gene C22orf46 facilitates pro-survival signals and that tumor cells devoid of C22orf46 expression exhibit increased susceptibility to T cell-induced apoptosis and stress by genotoxic agents. Although annotated as a non-coding gene, we demonstrate that C22orf46 encodes a nucleolar protein, hereafter referred to as "Tumor Apoptosis Associated Protein 1," up-regulated in lung cancer, which displays remote homologies to the BH domain containing Bcl-2 family of apoptosis regulators. Collectively, the findings establish TAAP1/ C22orf46 as a pro-survival oncogene with implications to therapy., (© 2024 Döring et al.)

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE., (© 2023. The Author(s).)

The programmable CRISPR/Cas9 DNA nuclease is a versatile genome editing tool, but it requires the host cell DNA repair machinery to alter genomic sequences. This fact leads to unpredictable changes of the genome at the cut sites. Genome editing tools that can alter the genome without causing DNA double-strand breaks are therefore in high demand. Here, we show that expression of promoter-associated short guide (sg)RNAs together with dead Cas9 (dCas9) fused to a Krüppel-associated box domains (KRABd) in combination with the transcription repression domain of methyl CpG-binding protein 2 (MeCP2) can lead to persistent gene silencing in mouse embryonic stem cells and in human embryonic kidney (HEK) 293 cells. Surprisingly, this effect is achievable and even enhanced in DNA (cytosine-5)-methyltransferase 3A and 3B (Dnmt3A -/- , Dnmt3b -/- ) depleted cells. Our results suggest that dCas9-KRABd-MeCP2 fusions are useful for long-term epigenetic gene silencing with utility in cell biology and potentially in therapeutical settings., (© 2022 Ding et al.)

Tobacco smoking and hemodynamic forces are key stimuli for the development of endothelial dysfunction. As an alternative to smoking, next generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their potential pro-inflammatory and atherogenic effects on the endothelium. In this study, we analyzed key parameters of endothelial function after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine. All experiments were performed under atheroprotective high laminar or atherogenic low flow with primary human endothelial cells. Treatment with 3R4F, but not alternative smoking products, reduced endothelial cell viability and wound healing capability via the PI3K/AKT/eNOS(NOS3) pathway. Laminar flow delayed detrimental effects on cell viability by 3R4F treatment. 3R4F stimulation led to activation of NRF2 antioxidant defense system at nicotine concentrations ≥0.56 μg/ml and increased expression of its target genes HMOX1 and NQO1. Treatment with HTP revealed an induction of HMOX1 and NQO1 at dosages with ≥1.68 μg/ml nicotine, whereas e-cig and nicotine exposure had no impact. Analyses of pro-inflammatory genes revealed an increased ICAM1 expression under 3R4F treatment. 3R4F reduced VCAM1 expression in a dose-dependent manner; HTP treatment had similar but milder effects; e-cig and nicotine treatment had no impact. SELE expression was induced by 3R4F under static conditions. High laminar flow prevented this upregulation. Stimulation with laminar flow led to downregulation of CCL2 (MCP-1). From this downregulated level, only 3R4F increased CCL2 expression at higher concentrations. Finally, under static conditions, all components increased adhesion of monocytes to endothelial cells. Interestingly, only stimulation with 3R4F revealed increased monocyte adhesion under atherosclerosis-prone low flow. In conclusion, all product categories activated anti-oxidative or pro-inflammatory patterns. NGP responses were typically lower than in 3R4F exposed cells. Also, 3R4F stimulation led to an impaired endothelial wound healing and induced a pro-inflammatory phenotype compared to NGP treatment., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumor cells subvert immune surveillance by harnessing signals from immune checkpoints to acquire immune resistance. The protein PD-L1 is an important component in this process, and inhibition of PD-L1 elicits durable anti-tumor responses in a broad spectrum of cancers. However, immune checkpoint inhibition that target known pathways is not universally effective. A better understanding of the genetic repertoire underlying these processes is necessary to expand our knowledge in tumor immunity and to facilitate identification of alternative targets. Here, we present a CRISPR/Cas9 screen in human cancer cells to identify genes that confer tumors with the ability to evade the cytotoxic effects of the immune system. We show that the transcriptional regulator MLLT6 (AF17) is required for efficient PD-L1 protein expression and cell surface presentation in cancer cells. MLLT6 depletion alleviates suppression of CD8 + cytotoxic T cell-mediated cytolysis. Furthermore, cancer cells lacking MLLT6 exhibit impaired STAT1 signaling and are insensitive to interferon-γ-induced stimulation of IDO1, GBP5, CD74, and MHC class II genes. Collectively, our findings establish MLLT6 as a regulator of oncogenic and interferon-γ-associated immune resistance., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Tobacco smoking and hemodynamic forces are key stimuli in the development of endothelial dysfunction and atherosclerosis. High laminar flow has an atheroprotective effect on the endothelium and leads to a reduced response of endothelial cells to cardiovascular risk factors compared to regions with disturbed or low laminar flow. We hypothesize that the atheroprotective effect of high laminar flow could delay the development of endothelial dysfunction caused by cigarette smoking. Primary human endothelial cells were stimulated with increasing dosages of aqueous cigarette smoke extract (CSEaq). CSEaq reduced cell viability in a dose-dependent manner. The main mediator of cellular adaption to oxidative stress, nuclear factor erythroid 2-related factor 2 (NRF2) and its target genes heme oxygenase (decycling) 1 (HMOX1) or NAD(P)H quinone dehydrogenase 1 (NQO1) were strongly increased by CSEaq in a dose-dependent manner. High laminar flow induced elongation of endothelial cells in the direction of flow, activated the AKT/eNOS pathway, increased eNOS expression, phosphorylation and NO release. These increases were inhibited by CSEaq. Pro-inflammatory adhesion molecules intercellular adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1), selectin E (SELE) and chemokine (C-C motif) ligand 2 (CCL2/MCP-1) were increased by CSEaq. Low laminar flow induced VCAM1 and SELE compared to high laminar flow. High laminar flow improved endothelial wound healing. This protective effect was inhibited by CSEaq in a dose-dependent manner through the AKT/eNOS pathway. Low as well as high laminar flow decreased adhesion of monocytes to endothelial cells. Whereas, monocyte adhesion was increased by CSEaq under low laminar flow, this was not evident under high laminar flow. This study shows the activation of major atherosclerotic key parameters by CSEaq. Within this process, high laminar flow is likely to reduce the harmful effects of CSEaq to a certain degree. The identified molecular mechanisms might be useful for development of alternative therapy concepts., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Endothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2, and 5 reduces nitric oxide availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H2O2 produced by main endothelial isoform Nox4. Therefore, we analysed the impact of genetic deletion of Nox4 on endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model., Methods and Results: Ex vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4(-/-)/Ldlr(-/-) mice. Further progression of endothelial dysfunction due to high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4(-/-)/Ldlr(-/-) mice compared with Ldlr(-/-) mice. Under physiological conditions, loss of Nox4 does not influence aortic vascular function. In this setting, loss of Nox4-derived H2O2 production could be partially compensated for by nNOS upregulation. Using an innovative optical coherence tomography approach, we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach revealed an altered flow-mediated dilation in Nox4(-/-) mice, indicating a role for Nox4 under physiological conditions in peripheral arteries in vivo., Conclusions: Nox4 plays an important role in maintaining endothelial function under physiological and pathological conditions. Loss of Nox4-derived H2O2 could be partially compensated for by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Arteries and veins show marked differences in their anatomy, physiology and genetic expression pattern. In this study, we analyzed impact of overexpression or downregulation of arterial marker gene Hey2 and venous marker gene COUP-TFII in human venous and arterial endothelial cells on genes involved in arteriovenous differentiation. Lentiviral overexpression of venous marker gene COUP-TFII in arterial endothelial cells led to downregulation of NICD4, arterial marker gene Hey2 and EphrinB2. Downregulation of Hey2 could be mediated by direct binding of COUP-TFII to Hey2 promoter as shown by ChIP, EMSA and promoter analysis. Downregulation of Hey2 by shRNA causes downregulation of EphrinB2 expression. Overexpression of arterial marker Hey2 in venous endothelial cells did not change expression pattern of COUP-TFII. Downregulation of venous marker gene COUP-TFII in venous endothelial cells resulted in upregulation of VEGF-A, Dll4 and EphrinB2 expression. Our data support an important role of Hey2 and COUP-TFII in arteriovenous differentiation of human endothelial cells.

Objective: Hypercholesterolemia is an important risk factor of cardiovascular diseases. Lipoprotein apheresis is an efficient strategy to reduce the serum low-density lipoprotein (LDL)-cholesterol and lipoprotein(a) levels and cardiovascular complications in patients with severe hypercholesterolemia. The underlying molecular mechanisms are not well-understood. In this study, we analyzed the impact of lipoprotein apheresis on gene expression in human endothelial cells., Methods: Human endothelial cells were stimulated with serum of hypercholesterolemic patients before and after lipoprotein apheresis. The expression of endothelial lipoprotein receptors, nitric oxide (NO) synthase and adhesion molecules was quantified by real-time PCR and Western blot., Results: Lipoprotein apheresis reduced the expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells. Low-density lipoprotein (LDL) receptor expression remained unchanged. The mRNA expression of the endothelial nitric oxide synthase (eNOS) was increased with serum of hypercholesterolemic patients after lipoprotein apheresis. In contrast, endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) was reduced in response to serum after lipoprotein apheresis., Conclusion: Lipoprotein apheresis reduced the expression of the proatherosclerotic oxLDL receptor LOX-1 and adhesion molecule VCAM-1 and increased the expression of vasoprotective and NO generating eNOS in human endothelial cells in response to serum of hypercholesterolemic patients. These novel molecular mechanisms may account for the antiatherosclerotic and vasoprotective potential of lipoprotein apheresis in patients with hypercholesterolemia., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduces endothelial superoxide anion formation and Nox4 expression. In primary human endothelial cells, we identified a 47 bp 5'-untranslated region of Nox4 mRNA by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Cloning and functional analysis of human Nox4 promoter revealed a range between -1,490 and -1,310 bp responsible for flow-dependent downregulation. Mutation of an overlapping antioxidative response element (ARE)-like and Oct-1 binding site at -1,376 bp eliminated shear stress-dependent Nox4 downregulation. Consistent with these observations, electrophoretic mobility shift assays (EMSA) demonstrated an enhanced shear stress-dependent binding of Nox4 oligonucleotide containing the ARE-like/Oct-1 binding site, which could be inhibited by specific antibodies against the transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and octamer transcription factor 1 (Oct-1). Furthermore, shear stress caused the translocation of Nrf2 and Oct-1 from the cytoplasm to the nucleus. Knockdown of Nrf2 by short hairpin RNA (shRNA) increased Nox4 expression twofold, indicating a direct cross-talk between Nrf2 and Nox4. In conclusion, an ARE-like/Oct-1 binding site was noticed to be essential for shear stress-dependent downregulation of Nox4. This novel mechanism may be involved in the flow-dependent downregulation of endothelial superoxide anion formation.

Diabetes mellitus is an important risk factor for cardiovascular diseases. Clinical evidence supports a link between hyperglycemia, endothelial dysfunction, and vascular disorders. However, the precise molecular mechanisms causing endothelial dysfunction in diabetic patients remain unclear. An interesting novel mediator could be chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), which plays an essential role in glucose metabolism. COUP-TFII is known to be expressed in venous endothelial cells. In this study, we show COUP-TFII expression in human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells. HUVECs express glucose transporters 1, 3, 6, and 10, and the insulin receptor. Insulin in combination with glucose activates protein kinase B (PKB or Akt) phosphorylation via phosphoinositide 3-kinase (PI3-kinase). Short-term (60-240 min) stimulation of HUVECs with high glucose increased COUP-TFII expression independent of insulin. Long-term (48 h) stimulation of HUVECs with high glucose augmented expression of the insulin receptor and E-selectin, but downregulated COUP-TFII protein expression. Downregulation of COUP-TFII by shRNA leads to downregulation of E-selectin and upregulation of eNOS and glucose transporters. Our data suggest that COUP-TFII is regulated by glucose in a time- and dose-dependent manner in endothelial cells. COUP-TFII might affect endothelial function in a diabetic background., (Georg Thieme Verlag KG Stuttgart * New York.)