Carsten Kuenne

Summary: Marfan syndrome (MFS) is an inherited connective tissue disorder caused by pathogenic variants in FBN1, encoding fibrillin-1, with life-threatening aortic complications arising in part from endothelial cell (EC) dysfunction. To study this in a human model, we generated hiPSC-derived ECs from three MFS patients (iMFS-ECs). We show that iMFS-ECs recapitulate known disease phenotypes, including impaired alignment in the direction of flow. Moreover, we found that iMFS-ECs do not recover from TNF-α-induced loss of barrier integrity, due to sustained EC contractility. iMFS-ECs exhibited TNF-α-induced ICAM1 upregulation and NF-κB activation comparable to healthy donor-derived hiPSC-ECs by bulk RNA-seq, while expression of genes linked to cytoskeletal arrangements, cell signaling and ECM remodeling were dysregulated. In conclusion, we show that hiPSC derived ECs can serve as a model to investigate MFS pathology. These findings establish a human iPSC platform for MFS endothelial research and suggest impaired inflammatory resolution as a novel therapeutic target.

Aims: Microproteins (miPs) translated from small open reading frames (smORFs) are crucial regulators of cell function. However, the expression and function of miPs in endothelial cells and alterations in miP expression linked with inflammation and cardiovascular disease, remain largely unexplored. Methods and results: An optimized proteogenomic approach combining RiboTag RNA-sequencing and mass spectrometry of the small molecular mass proteome was utilized to identify endothelial cell-specific miPs. Heart, lung, and blood vessels from endothelial cell-specific RiboTag mice and human endothelial cells were studied under homeostatic and inflammatory conditions. We identified 2739 murine as well as 1365 intracellular and 607 extracellular human endothelial cell miPs encoded from previously non-canonical (unannotated) smORFs. Vascular inflammation induced in vitro by interleukin-1β (IL-1β) and in vivo through PCSK9 overexpression, high-fat diet, and partial carotid artery ligation significantly altered smORF expression. An additional 347 miPs were detected in human serum, 23 decreasing and 31 increasing, after cardiac damage. The expression of an inflammation-induced miP encoded by an internal smORF within the proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) transcript, that is, miP-PSTPIP2, was assessed using a custom antibody. miP-PSTPIP2 expression was upregulated in IL-1β-treated human endothelial cells, in pre-atherosclerotic murine carotid arteries and detected in carotid arteries from patients with atherosclerosis. The relevance of 250 miPs for endothelial cell growth and viability was demonstrated using a high-throughput clustered regularly interspaced Short palindromic Repeats (CRISPR)/Cas9 screen. Conclusion: Taken together, we document the existence of a large number of human and murine miPs encoded by non-canonical smORFs and their altered expression in inflammatory conditions. The identification of secreted miPs suggests that they may also exert autocrine or paracrine functions. These novel small peptides modulate cell proliferation and survival in endothelial cells and may play a significant role in human cardiovascular disease.

BACKGROUND: The Cre/loxP recombination system is the main tool for cell type–specific lineage tracing and gene targeting. Currently available smooth muscle cell (SMC)–specific Cre mouse lines show off-target activity outside the SMC lineage and are unable to distinguish among arterial SMCs (ASMCs), venous SMCs, and nonvascular SMCs (NVSMCs). These limitations prevent ASMC- and NVSMC-specific gene targeting, which is required to characterize the role of SMCs in different organs and diseases. METHODS: To achieve precise manipulation of ASMCs in vivo, we combined alleles for Cspg4-Dre and rox-Stop–containing Acta2-CreER (Acta2-rox-CreER ), generating a mouse line with Cre activity exclusively in ASMCs. RNA sequencing of fluorescence-activated cell sorting–isolated ASMCs was used to reveal differences in ASMCs among multiple organs. To specifically target and characterize NVSMCs in different organs, a combination of Chrm2-Dre and Acta2-rox-CreER was used. Disease-specific transcriptional changes of pulmonary ASMCs and NVSMCs were determined in the Sugen 5416/hypoxia model of pulmonary arterial hypertension. Usefulness for functional studies was assessed by inactivation of the genes for the splicing factors RBPMS (RNA-binding protein with multiple splicing) and RBPMS2 (RNA-binding protein with multiple splicing 2) in ASMCs. RESULTS: Intersectional genetic approaches using Cspg4-Dre and Acta2-rox-CreER mouse lines specifically targeted ASMCs within various organs. A combination of Chrm2-Dre with Acta2-rox-CreER achieved specific targeting of NVSMCs. Transcriptomic profiling revealed distinct gene expression signatures in ASMCs of different organs, indicating organ-dependent transcriptional adaptation of ASMCs. Transcriptional differences among NVSMCs were found in the lung, intestine, and bladder. Activation of distinct pathways was uncovered in pulmonary ASMCs and bronchial SMCs after induction of pulmonary arterial hypertension. Inactivation of Rbpms and Rbpms2 in ASMCs increased thickness of the muscular layer in pulmonary arteries, whereas inactivation in all SMCs abolished the contractile phenotype of NVSMCs in the intestine. CONCLUSIONS: The successful generation of mouse lines specifically targeting different subtypes of SMCs enhances specificity, allowing distinction between vascular and nonvascular effects of diseased SMCs. The identification of vessel bed–specific gene signatures will pave the way for specific manipulation of SMCs in distinct diseased organs, such as the lung in pulmonary arterial hypertension.

A bstract: Recruitment and resolution of immune cell accumulation after myocardial infarction (MI) is critical for effective wound healing and prevention of maladaptive remodeling. Numerous signals are known to recruit neutrophils, critical drivers of inflammation, but knowledge about signals containing and resolving their accumulation is limited. We discovered that Regenerating-islet derived protein 3 beta (REG3β) limits the persistence of neutrophils after MI, thereby promoting resolution of inflammation. REG3β selectively binds to aged and hyperactive neutrophils and induces rapid cell death, enabling clearance via macrophage-mediated efferocytosis. Selective binding of REG3β is achieved by interaction with paucimannosylated proteins that translocate from azurophile granules to the plasma membrane in an activation- and age-dependent manner. Endocytotic uptake and accumulation of REG3β in lysosomes initiates programmed cell death of neutrophils via lyosomal membrane permeabilization and release of cathepsins. Our work establishes REG3β as a local immune checkpoint essential for neutrophil resolution and cardiac repair.

Scientific research relies on transparent dissemination of data and its associated interpretations. This task encompasses accessibility of raw data, its metadata, details concerning experimental design, along with parameters and tools employed for data interpretation. Production and handling of these data represents an ongoing challenge, extending beyond publication into individual facilities, institutes and research groups, often termed Research Data Management (RDM). It is foundational to scientific discovery and innovation, and can be paraphrased as Findability, Accessibility, Interoperability and Reusability (FAIR). Although the majority of peer-reviewed journals require the deposition of raw data in public repositories in alignment with FAIR principles, metadata frequently lacks full standardization. This critical gap in data management practices hinders effective utilization of research findings and complicates sharing of scientific knowledge. Here we present a flexible design of a machine-readable metadata format to store experimental metadata, along with an implementation of a generalized tool named FRED. It enables i) dialog based creation of metadata files, ii) structured semantic validation, iii) logical search, iv) an external programming interface (API), and v) a standalone web-front end. The tool is intended to be used by non-computational scientists as well as specialized facilities, and can be seamlessly integrated in existing RDM infrastructure.

BACKGROUND: ADAR1 (adenosine deaminase acting on RNA 1) catalyzes the conversion of adenosine to inosine in double stranded RNAs (dsRNAs), which is critical to prevent autoinflammatory responses mediated by activation of the type I IFN (interferon) signaling. We define the role of ADAR1-dependent RNA editing in IFNβ activation in pulmonary arterial smooth muscle cells (PASMCs) from idiopathic pulmonary arterial hypertension (IPAH), a devastating disease leading to right heart failure and death. METHODS: RNA editing levels were analyzed in PASMCs from IPAH patients versus healthy controls. A conditional transgenic mouse model, Adar1 ^{SMC - KO} , was generated by knocking out Adar1 selectively in Sma (smooth muscle actin)–expressing cells, followed by 3 weeks of hypoxic exposure to induce pulmonary hypertension (PH). RESULTS: PASMCs from patients with IPAH displayed decreased levels of ADAR1 mRNA and isoform p150 protein, accompanied by accumulated dsRNA compared with healthy PASMCs. ADAR1 knockdown in PASMCs upregulated MDA5 (melanoma differentiation-associated protein 5), PKR (protein kinase R), IFNβ, and IFN-stimulated genes. Compared with controls in vivo, hypoxic Adar1 ^{SMC -KO} mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles and increased endothelium leakage, resulting in elevated right ventricular systolic pressure and right ventricular hypertrophy. Mechanistically, Ifnβ signaling in Adar1 ^SMC-KO induced the recruitment of macrophages, enhancing pulmonary artery muscularization. Pharmacological treatment with Pkr-relevant inhibitor 2BAct decreased Ifnβ and macrophages, thus attenuating pulmonary hypertension (PH) in hypoxic Adar1 ^SMC-KO mice. CONCLUSIONS: Our study describes a fundamental molecular mechanism underlying the progression of PH. We highlight the detrimental role of innate immune responses, where smooth muscle cell and context-specific RNA editing, along with the sensing of dsRNA, mediate disease progression and excessive vascular remodeling. This finding suggests that targeting PKR could be the new therapeutic strategy for treating pulmonary arterial hypertension.

BACKGROUND: Nitric oxide (NO), generated by the endothelial NO synthase (eNOS), regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. Most eNOS is localized at the cell membrane or in the Golgi apparatus, but the enzyme is also present in the endothelial cell nucleus. Here, we assessed the relevance of nuclear eNOS and NO signaling for endothelial cell function. METHODS: eNOS loss-of-function approaches were combined with confocal microscopy and biochemical, histological, and multiomics analyses. The pathophysiological relevance of the findings was assessed in murine models of atherogenesis (hypercholesterolemia and partial carotid ligation) as well as in samples from patients with atherosclerosis. RESULTS: eNOS was present in the nucleus of unstimulated human and murine endothelial cells (in vitro and ex vivo) and stimulation with VEGF (vascular endothelial growth factor) enhanced its nuclear localization. Coimmunoprecipitation studies coupled with proteomics revealed the association of nuclear eNOS with 81 proteins involved in RNA binding and processing. Among the latter was ADAR1 (double-stranded RNA-specific adenosine deaminase), an enzyme involved in editing double-stranded RNA via the deamination of A to I. ADAR1 was S -nitrosated in human endothelial cells, and the knockdown of eNOS resulted in altered ADAR1-mediated A-to-I editing and an increase in double-stranded RNA. The latter phenomenon elicited aggregation of MAVS (mitochondrial antiviral signaling protein), activation of the type I IFN (interferon) signaling pathway and a marked downregulation of cell cycle–related genes. ADAR1 depletion elicited similar effects on the activation of type I IFN signaling. As a result, growth factor–stimulated cell proliferation was abrogated, and basal as well as stimulated cell death were increased in endothelial cells lacking eNOS. Endothelial dysfunction in mice as well as in subjects with atherosclerosis was accompanied by accumulation of double-stranded RNA and activation of type I IFN signaling. Preserving NO bioavailability in vivo (Tyr657Phe eNOS mice) prevented these effects. CONCLUSIONS: Our findings uncovered a novel mechanism linking nuclear eNOS-derived NO with the activity of ADAR1 to maintain vascular homeostasis. Reduced NO bioavailability results in previously unrecognized activation of a type I IFN response in the endothelium, which contributes to atherogenesis.

Background: Human endothelial cells express numerous microproteins (miPs) encoded by small open reading frames (smORFs) distributed throughout the genome, yet the biological functions of most remain unknown. This study set out to characterize a novel 69 amino acid miP encoded by a smORF located within the coding sequence of the FERM domain containing kindlin-3 transcript (miP-FERMT3). Methods: Confocal microscopy was used to determine the subcellular localization of miP-FERMT3 in endothelial cells and its interaction partners were determined by mass spectrometry and immunoblotting. RNA sequencing identified transcriptional alterations induced by miP-FERMT3 overexpression. Cell proliferation and cell cycle stages were assessed by live cell imaging, EdU incorporation and flow cytometry, while senescence was examined by senescence-associated β-galactosidase staining, live cell imaging and RT-qPCR-based measurement of telomere length. Results: In endothelial cells miP-FERMT3 localized mainly to centriole subdistal appendages, where it interacted with proteins involved in ubiquitin- and proteasome-dependent protein catabolism, including PSMD9, CUL2 and TRIM8. Consistent with these interactions, cells expressing miP-FERMT3 exhibited increased global protein ubiquitination, enhanced centrosomal neddylation and elevated proteasomal activity. MiP-FERMT3 also promoted the nuclear accumulation of p53, which subsequently repressed FOXM1 expression, leading to the downregulation of genes required for cell-cycle progression and upregulation of genes involved in cell cycle inhibition, resulting in cell-cycle arrest. Cells expressing the miP also demonstrated multiple hallmarks of cellular senescence, including enlarged size, DNA damage, increased senescence-associated β-galactosidase activity, telomere shortening and paracrine pro-inflammatory activation of naïve endothelial cells. Analyses of independent murine and human transcriptomic and proteomic aging datasets further revealed that FERMT3 expression and protein abundance increase with age. Conclusions: These findings identify miP-FERMT3 as a novel regulator of protein catabolism and p53-dependent cell cycle arrest and cellular senescence in endothelial cells. Given the aging-associated upregulation of FERMT3 in mouse and human endothelial cells, increased miP-FERMT3 expression may contribute to the onset of vascular senescence as a hallmark of aging.

Supplementary Data with figires_tables

The accelerated development of single-cell technologies has profoundly impacted the field of biological research, facilitating unparalleled insights into cellular heterogeneity. However, this progress has also produced new computational challenges in the field of bioinformatics: single-cell datasets are increasingly high-dimensional, multimodal, and large-scale, while analysis workflows often remain fragmented, data type specific, ad hoc, and difficult to reproduce. The prevailing methodologies are dependent on a combination of public tools, which hinders the reproducibility of results, limits scalability, and complicates the efforts to establish benchmarks. The necessity for a higher-level, unified framework for single-cell data analysis is paramount to address these inherent limitations. Here, we introduce the SC-Framework, providing the integration of standardized data structures, declarative workflows and standardized computational backends in a containerized environment, enabling analysts to focus on biological interpretation rather than technical overhead. SC-Framework is available at GitHub ( https://github.com/loosolab/SC-Framework ).