The endothelium promotes a non-adherent vascular surface that facilitates tissue perfusion, prevents clotting, and limits inflammation. Endothelial cells (ECs) execute these tissue-specific functions through the integration of signaling pathways promoted by growth factors, cytokines, extracellular matrix components, and signals from mechanosensory complexes. Furthermore, ECs secrete various molecular signals, leading to the establishment of a specific niche microenvironment. Importantly, ECs can serve as sentinels against invading viral pathogens, propagating anti-viral responses such as the secretion of type I interferons (IFNs). Identification of mechanisms that alter immunity and inflammation at this critical barrier is important to understanding endothelial dysfunctions and the endothelium’s overall role in disease. To investigate the regulation and function of IFN signaling in endothelial cells, we used a conditionally immortalized human cell line. We analyzed IFN gene expression by RT-qPCR and used an Mx2 promoter-dependent bioassay to quantify the levels of secreted IFN during homeostatic conditions. Multiple cell types were screened for the ability to enhance tonic IFN production by endothelial cells in a direct coculture model. The role of direct cell-cell interactions in this behavior was studied using cell culture insert settings and inhibitors specifically targeting gap junction communication. The antiviral effects of endothelial tonic IFN production were determined with SARS-CoV-2 and HCMV infections. We demonstrate that endothelial cells can generate a type I IFN response in absence of infection under homeostatic conditions. These tonic IFN levels rise dramatically when endothelial cells are in direct contact with epithelial cells, though not when cultured with other cell types. The transcriptional induction of type I IFN genes occurs only in endothelial cells and requires direct cell-cell contacts. We further show that IFN induction can be blocked by interfering with gap junction communication and is partially dependent on the cGAS/STING pathway. Notably, the IFN activity derived by this cell type-specific interaction efficiently protects neighboring lung epithelial cells against SARS-CoV-2 infection. We propose that the upregulation of tonic IFN production by the endothelial-epithelial cell axis can contribute directly to pathogen defense and/or strengthens the innate immune response by elevated priming. While the contributing molecular signaling pathways underlying this activation have not been fully identified, this newly described mechanism has potential relevance during acute or chronic lung injuries, as it enhances the level of tonic antiviral activity. Furthermore, excessive lung inflammation in nonviral pathologies may be dampened by elevated levels of tonic IFNs.

BACKGROUND: Peri-implant health depends on the complex interactions between the dental implant, surrounding soft/hard tissues and the oral microbial environment. However, existing 2D and monoculture models fail to replicate this complexity, limiting their clinical relevance. Therefore, this study aimed to develop a clinically relevant 3D in vitro model that integrates oral soft tissue, hard tissue and a titanium implant in a 3D setup to accurately replicate the peri-implant environment. In addition, the model was designed to integrate bacterial biofilms, in order to mimic incipient peri-implant infections. METHODS: As a hard tissue component, osteoblast-covered HA/TCP scaffold structures were developed and merged with peri-implant mucosa, resulting in a 3D in vitro peri-implant bone-mucosa composite model. The composite model was then cultivated for 2, 7 and 14 days. At each time point, histological analysis, live/dead staining and collagen immunofluorescence staining were performed to assess its structural integrity, osteoblast viability and bone ECM characteristics. To demonstrate proof-of-concept for suitability in simulating implant infection, an oral multispecies biofilm was integrated on top of the implant in the peri-implant bone-mucosa model. RESULTS: Cell viability and osteoblastic phenotype were maintained throughout the study period. Microscopic and histological analyses confirmed a homogenous structure, with a stratified epithelium overlying collagen-embedded human gingival fibroblasts closely connected to the underlying scaffold structure interspersed with bone cells. Combined with a living multispecies biofilm, this model represents several essential components observed in peri-implant interaction. CONCLUSIONS: By combining oral soft tissue, hard tissue and a titanium implant in a 3D setup, this model represents the first and most complex model for evaluating innovative implant materials and novel treatment strategies as well as studying the development of peri-implant diseases. Incorporating different biofilms could enhance the model’s clinical relevance, enabling the study of pro-inflammatory responses to bacterial infections in a setting that includes both soft and hard tissue.

Interleukin-6 (IL-6) is a central mediator of endothelial activation in inflammatory diseases. Synthetic glucocorticoids such as dexamethasone (Dex) are commonly used to treat vascular inflammation. Despite their clinical relevance, the molecular mechanisms underlying the crosstalk between IL-6 and glucocorticoid signalling in endothelial cells remain poorly defined.The interaction between glucocorticoids and IL-6-induced JAK/STAT signalling was analysed in human endothelial cells by examining successive steps from receptor expression to STAT3 activation and downstream gene regulation. The expression and stability of SOCS3, CCL2, ICAM1, and VEGF mRNA were monitored at transcriptional and post-transcriptional level. The functional effects of the interaction between IL-6 and glucocorticoids were investigated by tube formation assays. In endothelial cells, Dex enhances IL-6 trans-signalling-induced STAT3 phosphorylation without affecting the expression of IL-6 receptor subunits. Instead, Dex destabilizes IL-6–induced SOCS3 mRNA, thereby weakening SOCS3 negative feedback on STAT3 signalling. Despite increased STAT3 activation, Dex suppresses expression of IL-6-induced STAT3 target genes, including CCL2, ICAM1, and VEGF, through accelerated mRNA degradation. Functionally, this suppression leads to reduced IL-6-driven angiogenesis.In summary, Dex regulates IL-6 signalling in endothelial cells at multiple levels. By destabilizing SOCS3 mRNA, glucocorticoids increase STAT3 activation. At the same time, glucocorticoids suppress pro-inflammatory and pro-angiogenic gene expression by increasing mRNA turnover, thereby attenuating endothelial activation and angiogenesis. This mechanism effectively decouples STAT3 phosphorylation from downstream gene induction. Together, these findings reveal an endothelial cell–specific crosstalk between glucocorticoid and IL-6 signalling that is relevant to vascular inflammation and cytokine release syndrome.

IntroductionUnderstanding the biology of implant-associated infections is essential in order to provide adequate detection, prevention and therapeutic strategies. Advanced 3D in vitro models offer valuable insights into the complex interactions between cells and bacteria in the presence of implant materials. This review aims to give a comprehensive overview of current 3D in vitro models that mimic implant-associated infections.MethodsThe structured literature search initially identified 258 publications, seven of which fitted the inclusion criteria.ResultsThe included 3D models were established either to mimic the in vivo situation (organotypic model) or to investigate future implant materials. In three studies, organotypic models for dental implants were created and one study described an organotypic model containing immune cells. In the remaining three studies, biomaterials for constructing future orthopedic implants were developed and tested. All authors included specific cells and bacteria suitable for the respective implants. The dental implant models used fibroblasts and keratinocytes; the orthopedic implant models used stem cells and fibroblast-like cells; the model containing immune cells incorporated co-cultivation of fibroblasts and THP-1 derived macrophages. For bacterial challenge, most authors used Gram positive bacteria, but three studies employed Gram negative bacterial species. A wide variety of analytical methods of different complexity were applied after co-culture of cells and bacteria and between one and five different methods were used.DiscussionAll models could be employed to provide answers to specific scientific questions regarding implant-associated infections. Nonetheless, this review reveals the limitations of current 3D models for the investigation of implant-associated infections and highlights the opportunities for further development in this scientific field.

Creation of a biological seal and efficient antibacterial qualities in the peri-implant environment is essential for the success of dental implants. Therefore, novel multifunctional strategies are being developed to address these issues, aiming at the simultaneous improvement of tissue integration and hindering pathological biofilm formation. In this study, we investigated the effect of tissue-promotive strontium acetate (SrAc), antibacterial silver nitrate (AgNO3), and their combination on oral soft tissue cells and an oral multispecies biofilm not only in monoculture setups but also in a three-dimensional (3D) implant-tissue-oral bacterial-biofilm model (INTERbACT model) that takes the naturally occurring interactions into account. Application of SrAc led to improved fibroblast migration in the monoculture setting, without impairment of metabolic activity, even upon additional AgNO3 administration. Notably, the combined treatment of SrAc and AgNO3 resulted in a synergistic antibacterial effect during biofilm formation as well as on early matured biofilms. Most interestingly, the antibacterial effect of the combined treatment was even further enhanced within the coculture setup leading to increased bacterial death and decreased biofilm volume. The 3D tissue in the coculture setup underwent the combined treatment with a notable rise in CCL20 and IL-1β levels. Histologically, only the AgNO3-treated groups exhibited damage to the integrity of the epithelial barrier. Therefore, the results of this study demonstrated promising dual antibacterial and tissue-integrative characteristics of combined AgNO3 and SrAc in the dental implant environment. Additionally, the study emphasizes the importance of considering naturally occurring tissue-bacteria interactions for reliable in vitro testing of novel implant materials.

Macrophages are pivotal in regulating inflammation and facilitating pathogen clearance, particularly in periimplant environments prone to biofilm infections. For a better understanding of the role of these immune cells in host-biofilm-implant interactions, we developed a complex three-dimensional (3D) implant-tissue-oralbacterial-biofilm model (INTERbACT) with integrated macrophages to mimic the intricate triangular interactions between implant materials, biofilms, and the host, in conditions simulating the in vivo environment. The INTERbACT model includes a peri-implant mucosa with collagen-embedded fibroblasts and macrophages, overlaid with a multi-layered oral epithelium, as well as a multi-species biofilm. Histological analysis demonstrated a preserved peri-implantarchitecture with high cell viability. Co-cultivation with multi-species biofilms provided valuable insights into macrophage roles in pathogen defence and inflammation regulation. In tissues lacking macrophages, biofilm exposure led to compromised epithelial integrity, whereas macrophage-containing tissues maintained their epithelial structure. Macrophages significantly reduced the biofilm biomass while promoting bacterial death. Inflammatory cytokine levels elevated under sterile conditions decreased upon biofilm exposure, suggesting a biofilm-mediated immune suppression. Cytokine expression levels differentially reacted to biofilm exposure and macrophage concentration, with IL-1 beta and IL-17 levels notably elevated in biofilm-exposed tissues, aligning with clinical observations of peri-implant inflammation. Our findings demonstrate that the INTERbACT model, after incorporation of macrophages, serves as a physiologically relevant platform for studying host-pathogen dynamics and allows to identify key inflammatory markers associated with biofilm-related complications. Furthermore, this model holds significant potential for evaluating novel therapeutic strategies, such as sensor-actuator systems to detect and combat pathogenic biofilms, improving clinical outcomes in peri-implantitis management.

Background & Aims: Hepatitis C virus (HCV) has a narrow species tropism and cannot infect mice. To understand HCV species tropism and to develop better animal models, we adapted HCV to infect mouse cells deficient in innate immunity and with minimal human HCV host factors. Methods: HCV was adapted via passaging an HCV infectious virus clone several times in human hepatoma cells, mouse liver cells, and eventually primary mouse hepatocytes deficient in innate immunity and ectopically expressing human occludin and human CD81. Using RNAseq the sequence of the adapted virus was analyzed, and several clones were generated to study replication and infection kinetics as well as neutralization assays in several human/mouse cell lines and primary hepatocytes from human, mouse, and macaques. Results: Accumulation of 35 non-synonymous and 66 synonymous mutations correlated with >1,000-fold enhanced production of infectious progeny from primary mouse hepatocytes. These mutations did not confer drug resistance or evasion from innate immunity. They did not enhance fitness in human or macaque hepatocytes. We show that non-synonymous mutations are necessary and sufficient for adaptation, and that changes to the glycoproteins are not essential. Mutations outside of viral envelope proteins enhanced specific infectivity and facilitated viral spread in murine cells. Conclusions: This study reveals key viral factors governing HCV species tropism. The mouse-adapted HCV opens up possibilities for the development of animal models to analyze HCV pathogenesis, immune control, and vaccine development. (c) 2025 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Two features of macrophages make them attractive for targeted transport of drugs: they efficiently take up a broad spectrum of nanoparticles (NPs) and, by sensing cytokine gradients, they are attracted to the sites of infection and inflammation. To expand the potential of macrophages as drug carriers, we investigated whether macrophages could be simultaneously coloaded with different types of nanoparticles, thus equipping individual cells with different functionalities. We used superparamagnetic iron oxide NPs (SPIONs), which produce apoptosis-inducing hyperthermia when exposed to an alternating magnetic field (AMF), and co-loaded them on macrophages together with drug-containing NPs (inorganic–organic nanoparticles (IOH-NPs) or mesoporous silica NPs (MSNs)). We show that individual macrophages can take up both SPIONs and drug-loaded NPs efficiently, thereby generating drug-loaded cells susceptible to AMF-induced cell death. Macrophages co-loaded with SPIONs and drug-containing IOH-NPs spontaneously released the drugs at similar rates irrespective of the application of an AMF. Notably, while the spontaneous drug release from macrophages co-loaded with SPIONs and drug-associated MSNs was low, AMF exposure accelerated the drug release. Thus, AMF exposure of SPION/drug-MSN coloaded macrophages provides a simple strategy for trigger-controlled drug release since it does not require any chemical modification of NPs or drugs. Thus, we assume that the coloading of different types of NPs will expand the potential of macrophages for drug delivery.

Nanoparticles have proven to be attractive carriers in therapeutic drug delivery since they can encapsulate, protect and stabilize a plethora of different drugs, thereby improving therapeutic efficacy and reducing side effects. However, specific targeting of drug-loaded nanoparticles to the tissue of interest and a timely and spatially controlled release of drugs on demand still represent a challenge. Recently, gas-filled microparticles, so-called antibubbles, have been developed which can efficiently encapsulate liquid drug droplets. Here, we show that antibubbles are efficiently taken up by macrophages in vitro and are stably maintained for more than 48 h without compromising antibubble integrity and macrophage viability. We show that application of diagnostic ultrasound induces the disintegration of both antibubbles and carrier cells while not affecting non-loaded macrophages. Using 4-hydroxytamoxifen as a model drug, we show ultrasound-mediated drug release upon adoptive transfer of antibubble-loaded macrophages in mice. Together with the ability of macrophages to accumulate in inflamed tissues, antibubble-loaded macrophages represent an attractive tool for targeted delivery of drugs and its ultrasound-mediated spatial and temporal drug release, highlighting the therapeutic perspective of this strategy.

Bacterial infection is a major challenge to human health. Although various potent antibiotics have emerged in recent decades, current challenges arise from the increasing number of multi-drug-resistant species. Infections associated with implants represent a particular challenge because they are usually diagnosed at an advanced stage and are difficult to treat with antibiotics owing to the formation of protective biofilms. In this study, we designed and explored a synthetic biology-inspired cell-based biosensor/actor for the detection and counteraction of bacterial infections. The system is generic, as it senses diverse types of infections and acts by enhancing the endogenous immune system. This strategy is based on genetically engineered sensor/actor cells that can sense type I interferons (IFNs), which are released by immune cells at the early stages of infection. IFN signalling activates a synthetic circuit to induce reporter genes with a sensitivity of only 5 pg ml(-1) of IFN and leads to a therapeutic protein output of 100 ng ml(-1), resulting in theranostic cells that can visualize and fight infections. Robustness and resilience were achieved by implementing a positive feedback loop. We showed that diverse gram-positive and gram-negative implant-associated pathogenic bacteria activate the cascade in co-culture systems in a dose-dependent manner. Finally, we showed that this system can be used to secrete chemoattractants that facilitate the infiltration of immune cells in response to bacterial triggers. Together, the system is not only universal to bacterial infections, but also hypersensitive, allowing the sensing of infections at initial stages.

Lentiviral gene transfer represents a versatile and powerful method for genetic transduction of many cell lines and primary cells including "hard-to-transfect" cells. As a consequence of the integration of the recombinant lentiviral vector into the cellular genome the transgene is stably maintained and long term producing cells are established. Here, we describe the current state of the art and give details for lab scale production of lentiviral vectors as well as for infection and titration of the viral vectors.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKƴ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

Pathological cardiac remodeling predisposes individuals to developing heart failure. Here, we investigated two co-regulated long non-coding RNAs (lncRNAs), termed Gadlor1 and Gadlor2, which are upregulated in failing hearts of patients and mice. Cardiac overexpression of Gadlor1 and Gadlor2 aggravated myocardial dysfunction and enhanced hypertrophic and fibrotic remodeling in mice exposed to pressure overload. Compound Gadlor1/2 knockout (KO) mice showed markedly reduced myocardial hypertrophy, fibrosis, and dysfunction, while exhibiting increased angiogenesis during short and prolonged periods of pressure overload. Paradoxically, Gadlor1/2 KO mice suffered from sudden death during prolonged overload, possibly due to cardiac arrhythmia. Gadlor1 and Gadlor2, which are mainly expressed in endothelial cells (ECs) in the heart, where they inhibit pro-angiogenic gene expression, are strongly secreted within extracellular vesicles (EVs). These EVs transfer Gadlor lncRNAs to cardiomyocytes, where they bind and activate calmodulin-dependent kinase II, and impact pro-hypertrophic gene expression and calcium homeostasis. Therefore, we reveal a crucial lncRNA-based mechanism of EC-cardiomyocyte crosstalk during heart failure, which could be specifically modified in the future for therapeutic purposes.

The aging process is characterized by cellular functional decline and increased susceptibility to infections. Understanding the association between virus infection and aging is crucial for developing effective strategies against viral infections in older individuals. However, the relationship between Kaposi's sarcoma-associated herpesvirus (KSHV) infection, a cause of increased Kaposi's sarcoma prevalence among the elderly without HIV infection, and cellular senescence remains enigmatic. This study uncovered a link between cellular senescence and enhanced KSHV infectivity in human endothelial cells. Through a comprehensive proteomic analysis, we identified caveolin-1 and CD109 as host factors significantly upregulated in senescent cells that promote KSHV infection. Remarkably, CRISPR/Cas9-mediated KO of these factors reduced KSHV binding and entry, leading to decreased viral infectivity. Furthermore, surface plasmon resonance analysis and confocal microscopy revealed a direct interaction between KSHV virions and CD109 on the cell surface during entry, with recombinant CD109 protein exhibiting inhibitory activity of KSHV infection by blocking virion binding. These findings uncover a previously unrecognized role of cellular senescence in enhancing KSHV infection through upregulation of specific host factors and provide insights into the complex interplay between aging and viral pathogenesis.

Many cytokines produced by Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells have been shown to participate in the pathogenesis of KSHV. Determination of the exact role of cytokines in Kaposi's sarcoma (KS) pathogenesis is limited, however, by the difficulty to manipulate the target genes in human endothelial cells. In this study, we sought to elucidate the role of cytokines in KSHV-infected human immortalized endothelial cell line (HuARLT cells) by knockout (KO) of the corresponding target genes using the CRISPR/Cas9 system. The cytokine production profile of KSHV-infected HuARLT cells was analyzed using a protein array, and several cytokines were found to be highly upregulated following KSHV infection. This study focused on CXCL1, which was investigated by knocked out in HuARLT cells. KSHV-infected CXCL1 KO cells underwent increased cell death compared to KSHV-infected wild-type (WT) cells and mock-infected CXCL1 KO cells. Lytic replication was not observed in KSHV-infected WT nor CXCL1 KO cells. Phosphorylation of STAT3 was significantly suppressed in KSHV-infected CXCL1 KO cells. Additionally, inhibitors of STAT3 and CXCL1 induced cell death in KSHV-infected endothelial cells. Our results show that CXCL1 production is required for the survival of KSHV-infected endothelial cells, and the CXCL1 to STAT3 phosphorylation signaling pathway may be a therapeutic target for KS.

More than one hundred herpesviruses have been isolated from different species so far, with nine infecting humans. Infections with herpesviruses are characterized by life-long latency and represent a significant challenge for human health. To investigate the consequences of infections and identify novel treatment options, in vivo models are of particular relevance. The mouse has emerged as an economical small animal model to investigate herpesvirus infections. However, except for herpes simplex viruses (HSV-1, HSV-2), human herpesviruses cannot infect mice. Three natural herpesviruses have been identified in mice: mouse-derived cytomegalovirus (MCMV), mouse herpesvirus 68 (MHV-68), and mouse roseolovirus (MRV). These orthologues are broadly used to investigate herpesvirus infections within the natural host. In the last few decades, immunocompromised mouse models have been developed, allowing the functional engraftment of various human cells and tissues. These xenograft mice represent valuable model systems to investigate human-restricted viruses, making them particularly relevant for herpesvirus research. In this review, we describe the various mouse models used to study human herpesviruses, thereby highlighting their potential and limitations. Emphasis is laid on xenograft mouse models, covering the development and refinement of immune-compromised mice and their application in herpesvirus research.

Synthetic expression cassettes provide the ability to control transgene expression in experimental animal models through external triggers, enabling the study of gene function and the modulation of endogenous regulatory networks in vivo. The performance of synthetic expression cassettes in transgenic animals critically depends on the regulatory properties of the respective chromosomal integration sites, which are affected by the remodeling of the chromatin structure during development. The epigenetic status may affect the transcriptional activity of the synthetic cassettes and even lead to transcriptional silencing, depending on the chromosomal sites and the tissue. In this study, we investigated the influence of the ubiquitous chromosome opening element (UCOE) HNRPA2B1-CBX3 and its subfragments A2UCOE and CBX3 on doxycycline-controlled expression modules within the chromosomal Rosa26 locus. While HNRPA2B1-CBX3 and A2UCOE reduced the expression of the synthetic cassettes in mouse embryonic stem cells, CBX3 stabilized the expression and facilitated doxycycline-controlled expression after in vitro differentiation. In transgenic mice, the CBX3 element protected the cassettes from overt silencing although the expression was moderate and only partially controlled by doxycycline. We demonstrate that CBX3-flanked synthetic cassettes can be activated by decitabine-mediated blockade of DNA methylation or by specific recruitment of the catalytic demethylation domain of the ten–eleven translocation protein TET1 to the synthetic promoter. This suggests that CBX3 renders the synthetic cassettes permissive for subsequent epigenetic activation, thereby supporting doxycycline-controlled expression. Together, this study reveals a strategy for overcoming epigenetic constraints of synthetic expression cassettes, facilitating externally controlled transgene expression in mice.

Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.

Background Pathological overload triggers maladaptive myocardial remodeling that leads to heart failure. Recent studies have shown that long non-coding RNAs (lncRNAs) regulate cardiac remodeling. This study investigates two recently discovered, secreted lncRNAs, Gadlor1 and Gadlor2 ( Gadlor 1/2 ).Methods We generated compound Gadlor 1/2 knock-out (KO) mice and compared their response to pressure overload by transverse aortic constriction (TAC) to that of wild-type (WT) littermates. Endothelial cells, fibroblasts and cardiomyocytes were isolated from the hearts of both genotypes after TAC and their transcriptome was investigated by RNA sequencing. Gadlor target proteins were identified by RNA antisense purification coupled with mass spectrometry (RAP-MS) in cardiomyocytes. In addition, we investigated the effects of cardiac overexpression of Gadlor1 / 2 .Results Gadlor1/2 are jointly upregulated in failing mouse hearts as well as in the myocardium of heart failure patients. Cardiac overexpression of Gadlor1 and Gadlor2 aggravated myocardial dysfunction and enhanced hypertrophic and fibrotic remodeling in mice exposed to pressure overload. Compound Gadlor1/2 KO mice, in turn, exerted markedly reduced myocardial hypertrophy, fibrosis and dysfunction, but more angiogenesis during short and long-standing pressure overload. Paradoxically, Gadlor1/2 KO mice suffered from sudden death during prolonged overload, possibly due to cardiac arrhythmia. Gadlor1 and Gadlor2 , which are mainly expressed in endothelial cells (ECs) in the heart, where they inhibit pro-angiogenic gene-expression, are strongly secreted within extracellular vesicles (EVs). These EVs transfer Gadlor lncRNAs to cardiomyocytes, where they bind and activate calmodulin-dependent kinase II, induce pro-hypertrophic gene-expression and enhance calcium re-uptake into the sarcoplasmic reticulum.Conclusion Gadlor1 and Gadlor2 are lncRNAs that are mainly enriched in EC-derived EVs and are jointly upregulated in mouse and human hearts during pathological overload. We reveal a crucial endothelial cell-cardiomyocyte crosstalk, which aims at restoring calcium homeostasis in cardiomyocytes during overload at the cost of aggravated hypertrophy and fibrosis.### Competing Interest StatementCompeting Interest The following patent has been granted to N.F., J.B. and J.H.: LncRNAs Gadlor1 and Gadlor2 for use in treating and preventing cardiac remodeling. US11208656 (granted 28.01.2021). The other authors declare that not no conflict of interest exists.