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A major obstacle in inducing therapeutic angiogenesis in the heart is inefficient gene transfer to endothelial cells (ECs). Here, we identify compounds able to enhance the permissiveness of cardiac ECs to adeno-associated virus (AAV) vectors, which stand as ideal tools for in vivo gene delivery. We screened a library of >1,500 US Food and Drug Administration (FDA)-approved drugs, in combination with AAV vectors, in cardiac ECs. Among the top drugs increasing AAV-mediated transduction, we found vatalanib, an inhibitor of multiple tyrosine kinase receptors. The increased AAV transduction efficiency by vatalanib was paralleled by induction of the endothelial-to-mesenchymal transition, as documented by decreased endothelial and increased mesenchymal marker expression. Induction of the endothelial-to-mesenchymal transition by other strategies similarly increased EC permissiveness to AAV vectors. In vivo injection of AAV vectors in the heart after myocardial infarction resulted in the selective transduction of cells undergoing the endothelial-to-mesenchymal transition, which is known to happen transiently after cardiac ischemia. Collectively, these results point to the endothelial-to-mesenchymal transition as a mechanism for improving AAV transduction in cardiac ECs, with implications for both basic research and the induction of therapeutic angiogenesis in the heart., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo., Competing Interests: Declaration of interests D.J.B. has potential financial interests related to this study. He is a co-inventor of patents held by the Mayo Clinic, patent applications licensed to or filed by Unity Biotechnology, and a Unity Biotechnology shareholder. Research in the Baker laboratory has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic Conflict of Interest policies. J. Gil has acted as a consultant for Unity Biotechnology, Geras Bio, Myricx Pharma Ltd., and Merck KGaA; owns equity in Geras Bio and share options in Myricx Pharma Ltd.; and is a named inventor in MRC and Imperial College patents related to senolytic therapies. J. Gil currently receives funding from Pfizer. Unity Biotechnology funded research on senolytics in J. Gil’s laboratory in the past. SenTraGor and GLF16 senescence detection compounds are under patent applications: EP3475287B1, and 20240100309 (Greek patent application) along with GB2406749.8 (UK patent application), respectively. J.M.S. is a co-inventor on patents held by Brown University on methods to inhibit retrotransposon activation in age-related diseases. He is the scientific co-founder of Transposon Therapeutics, chair of their scientific advisory board, and a consultant and holds stock options. He is also a consultant and holds equity in Atropos Therapeutics. Research in the Sedivy laboratory has been reviewed by the Brown University Conflict of Interest Review Board and is being conducted in compliance with Brown University Conflict of Interest policies. F.d.d.F. is an inventor on the patent applications PCT/EP2013/059753 and PCT/EP2016/068162. M.D. is co-inventor on patents held by the Buck Institute for Research on Aging. He is the scientific co-founder of Cleara Biotech and consultant for Oisin Biotechnologies. M.D.’s laboratory currently receives research funding from Ono Pharmaceuticals. J. Grillari is co-inventor on patents held by BOKU and is a co-founder and scientific advisor to TAmiRNA and Rockfish Bio., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pulmonary fibrosis is a devastating disease, in which fibrotic tissue progressively replaces lung alveolar structure, resulting in chronic respiratory failure. Alveolar type II cells act as epithelial stem cells, being able to transdifferentiate into alveolar type I cells, which mediate gas exchange, thus contributing to lung homeostasis and repair after damage. Impaired epithelial transdifferentiation is emerging as a major pathogenetic mechanism driving both onset and progression of fibrosis in the lung. Here, we show that lung endothelial cells secrete angiocrine factors that regulate alveolar cell differentiation. Specifically, we build on our previous data on the anti-fibrotic microRNA-200c and identify the Vascular Endothelial Growth Factor receptor 1, also named Flt1, as its main functional target in endothelial cells. Endothelial-specific knockout of Flt1 reproduces the anti-fibrotic effect of microRNA-200c against pulmonary fibrosis and results in the secretion of a pool of soluble factors and matrix components able to promote epithelial transdifferentiation in a paracrine manner. Collectively, these data indicate the existence of a complex endothelial-epithelial paracrine crosstalk in vitro and in vivo and position lung endothelial cells as a relevant therapeutic target in the fight against pulmonary fibrosis., (© 2023. The Author(s).)

The spatial boundaries of tissue response to wounding are unknown. Here, we show that in mammals, the ribosomal protein S6 (rpS6) is phosphorylated in response to skin injury, forming a zone of activation surrounding the region of the initial insult. This p-rpS6-zone forms within minutes after wounding and is present until healing is complete. The zone is a robust marker of healing as it encapsulates features of the healing process, including proliferation, growth, cellular senescence, and angiogenesis. A mouse model that is unable to phosphorylate rpS6 shows an initial acceleration of wound closure, but results in impaired healing, identifying p-rpS6 as a modulator but not a driver of healing. Finally, the p-rpS6-zone accurately reports on the status of dermal vasculature and the effectiveness of healing, visually dividing an otherwise homogeneous tissue into regions with distinct properties., Competing Interests: Declaration of interests M.O., N.A.R.R., H.D., B.S., and H.R. have submitted a patent application based on the application of p-rpS6 as a clinical diagnostic tool in the assessment of wound healing., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds., (© 2023. The Author(s).)

Senescence is a cellular state which involves cell cycle arrest and a proinflammatory phenotype, and it has traditionally been associated with cellular and organismal aging. However, increasing evidence suggests key roles in tissue growth and regrowth, especially during development and regeneration. Conversely, cellular plasticity-the capacity of cells to undergo identity change, including differentiation and dedifferentiation-is associated with development and regeneration but is now being investigated in the context of age-related diseases such as Alzheimer disease. Here, we discuss the paradox of the role for cellular senescence in cellular plasticity: senescence can act as a cell-autonomous barrier and a paracrine driver of plasticity. We provide a conceptual framework for integrating recent data and use the interplay between cellular senescence and plasticity to provide insight into age-related diseases. Finally, we argue that age-related diseases can be better deciphered when senescence is recognized as a core mechanism of regeneration and development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Therapies halting the progression of fibrosis are ineffective and limited. Activated myofibroblasts are emerging as important targets in the progression of fibrotic diseases. Previously, we performed a high-throughput screen on lung fibroblasts and subsequently demonstrated that the inhibition of myofibroblast activation is able to prevent lung fibrosis in bleomycin-treated mice. High-throughput screens are an ideal method of repurposing drugs, yet they contain an intrinsic limitation, which is the size of the library itself. Here, we exploited the data from our "wet" screen and used "dry" machine learning analysis to virtually screen millions of compounds, identifying novel anti-fibrotic hits which target myofibroblast differentiation, many of which were structurally related to dopamine. We synthesized and validated several compounds ex vivo ("wet") and confirmed that both dopamine and its derivative TS1 are powerful inhibitors of myofibroblast activation. We further used RNAi-mediated knock-down and demonstrated that both molecules act through the dopamine receptor 3 and exert their anti-fibrotic effect by inhibiting the canonical transforming growth factor β pathway. Furthermore, molecular modelling confirmed the capability of TS1 to bind both human and mouse dopamine receptor 3. The anti-fibrotic effect on human cells was confirmed using primary fibroblasts from idiopathic pulmonary fibrosis patients. Finally, TS1 prevented and reversed disease progression in a murine model of lung fibrosis. Both our interdisciplinary approach and our novel compound TS1 are promising tools for understanding and combating lung fibrosis., (© 2021. The Author(s).)