Artificial optical radiation, spanning from 100 nm to 1 mm, encompasses ultraviolet (UV) and infrared (IR) light. UV light is well known for its risks on the skin and eyes. Recently, there has been growing interest in light at 405 nm (violet-blue light, VBL) due to its antimicrobial properties and perceived safety for mammalian cells when administered in controlled amounts. This research delved into the impact of 405 nm VBL on corneal and retinal pigment epithelial cell cultures. ARPE-19 and corneal BCE C/D 1b cells were exposed to VBL for varying doses, according at different exposure times, to evaluate cell viability, oxidative stress levels and apoptotic indicators. A 3D printed prototype with 14 LEDs centred at 405 nm wavelength was used to ensure uniform distribution of light during exposure. Cell viability was assessed using the MTT assay, measurement of oxygen species (ROS) production was carried out, and Western blot analysis was employed to study catalase and SOD-1 expression and apoptotic marker activation. Exposure to 405 nm VBL for both term (3 h) and prolonged durations (9 h) led to a weak decrease in cell viability in ARPE-19 cells, whereas the effect on BCE C/D 1b cells was negligible. There was no increase in ROS production, with catalase and SOD-1 expression remaining stable, suggesting no pro-oxidative stress effects in these models. Moreover, no activation of caspase-3 and accumulation of cytochrome C were found. Based on our results, exposure to 405 nm light at regulated levels does not pose a threat to the viability of the tested cell lines and does not lead to oxidative stress and apoptosis under these conditions. These results suggest a favourable cytocompatibility profile for these specific ocular cell models, laying a foundation for further investigations into its ocular safety.

Antiangiogenic drugs (AADs) are an heterogenous group of molecules widely used in the treatment of cancers. Based on their mechanism of action, AADs are associated with an increased reporting of cardiovascular adverse events (AEs). However, pericarditis, pericardial effusion and myocarditis were not documented for most of AADs. The aim of this study was to determine whether AADs may be associated with pericarditis, pericardial effusion and myocarditis in a real-world setting. We performed an extensive descriptive and disproportionality analysis of pericarditis, pericardial effusion and myocarditis AEs related to AADs on the FDA Adverse Event Reporting System (FAERS) database, using the reporting odds ratio (ROR) as a measure of emerging safety signal. Statistically significant signals of pericardial effusion and myocarditis were found for AADs compared to all other drugs in the database. The RORs for the pooled AADs were 2.37 [(95

Future long-duration crewed space missions beyond Low Earth Orbit (LEO) will bring new healthcare challenges for astronauts for which pharmacological countermeasures (pharmacological countermeasures) are crucial. This paper highlights current pharmacological countermeasures challenges described in the ESA SciSpacE Roadmap, with a focus on the cardiovascular system as a model to demonstrate the potential implication of the challenges and recommendations. New pharmacological approaches and procedures need to be adapted to spaceflight (spaceflight) conditions, including ethical and reglementary considerations. Potential strategies include combining pharmacological biomarkers such as pharmacogenomics with therapeutic drug monitoring, advancing microsampling techniques, and implementing a pharmacovigilance system to gain deep insights into pharmacokinetics/pharmacodynamics (PK/PD) spaceflight alteration on drug exposure. Emerging therapeutic approaches (such as long-term regimens) or manufacturing drugs in the space environment, can address specific issues related to drug storage and stability. The integration of biobanks and innovative technologies like organoids and organ-on-a-chip, artificial intelligence (AI), including machine learning will further enhance PK modelling leading to personalized treatments. These innovative pharmaceutical tools will also enable reciprocal game-changing healthcare developments to be made on Earth as well as in space and are essential to ensure space explorers receive safe effective pharmaceutical care.

The field of biochemistry and pharmacology of nitric oxide (NO) has various clinical perspectives, such as cardiovascular and metabolic disorders; neurovascular and neurodegenerative diseases; muscular-skeletal disorders; ocular, respiratory, and a large series of inflammation-related pathologies; and, on top of these, infective and neoplastic diseases. In all these disorders, vascular endothelial cells (ECs) play a crucial role as both NO producers and NO responders. Depending on the pathology and pathophysiological mechanisms, different pharmacological approaches can be developed and used, either to stimulate NO synthases (NOS), improve NO availability, and activate downstream NO-related pathways or, on the opposite, to downregulate NOS, scavenge NO, and inhibit NO-related signaling. Introducing endothelial cell cultures, genetic manipulation, and molecular studies allows us to thoroughly investigate the role of NO and its signaling in EC functional responses and angiogenesis. The present chapter presents the cell types and the cellular assays used to study the functional aspects of NO-related strategies in vascular endothelium. By using selective biochemical inhibitors of signaling pathways, the involvement of intracellular messengers can be assessed and verified in functional responses associated with EC proliferation, migration, and permeability.

Idiopathic pulmonary fibrosis (IPF) is a progressive and debilitating interstitial lung disease characterized by limited therapeutic options, with only two FDA-approved palliative agents currently available. Given its poor prognosis and the high incidence of lung transplantation, there is a pressing need to develop innovative and effective therapeutics. Histone deacetylase 6 (HDAC6) has been identified as a key driver of fibrotic progression in IPF, and selective inhibitors of this isoform were able to revert the fibrotic phenotypes. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that can trigger the degradation of a specific target in cells, including HDAC6 enzyme. Therefore, the application of PROTAC technology may represent a novel therapeutic strategy for IPF. We showcase the design, synthesis, and biological evaluation of a library of first-in-class antifibrotic HDAC6-targeting PROTACs, incorporating our in-house inhibitor 1 as the HDAC6 ligand. Newly developed PROTACs 5a and 5c showed effective degradation of HDAC6 in A549 lung cells and IMR-90 lung fibroblasts. 5a and 5c exhibited significant antifibrotic effects against the TGF-beta 1 induced fibrotic phenotype on IMR-90 cells, a model that mimics IPF conditions. The generation of putative ternary complexes involving PROTAC molecules, the E3-ligase cereblon (CRBN) and the HDAC6 target protein was supported by molecular modelling techniques, including protein-protein docking and molecular dynamics simulations. Mechanistic investigations, based on pull-down experiments, confirmed that the newly synthetized compounds were able to reduce HDAC6 levels through a proteasome-and CRBN-dependent mechanism as confirmed by experiments with neddylation and proteasome inhibitors. This pioneering exploration of targeted protein degradation in IPF-like conditions provides compelling evidence of its therapeutic promise, paving the way for a broader application of PROTACs in treating rare diseases.

Cyclooxygenase-2 (COX-2) is overexpressed in various cancers and has emerged as a promising target in oncological pharmacotherapy. This study investigates the in vitro antitumor properties and mechanism of action of novel vicinal diaryl-substituted heterocyclic COX-2 inhibitors, with a focus on VA1213, in comparison to celecoxib, a widely marketed COX-2 inhibitor known for its off-target effects. We assessed cytotoxicity, apoptosis induction, cell-cycle distribution, antimetastatic activity, and alterations in key signaling pathways in HT-29 colorectal carcinoma and MDA-MB-231 breast carcinoma cell lines. Among the novel compounds, VA1213 exhibited the most potent growth-inhibitory activity, demonstrating time-dependent cytotoxicity with a lower IC50 after 48-72 h of treatment compared to VA692 and VA694, and consistent with that observed for celecoxib. Unlike celecoxib, which produced rapid cytotoxic effects, VA1213 required prolonged exposure, suggesting a distinct mechanism of action. VA1213 induced G₀/G₁ phase cell cycle arrest and apoptosis via caspase-3 activation. Furthermore, it impaired EGFR downstream signaling by reducing ERK1/2 and AKT phosphorylation, without directly inhibiting EGFR itself. At sub-cytotoxic concentrations, VA1213 was more effective than celecoxib in inhibiting cell migration and demonstrated a comparable reduction in clonogenic potential. These findings highlight VA1213 as a COX-2 inhibitor with noteworthy in vitro antitumor efficacy, comparable to that of celecoxib. Its ability to interfere with multiple cancer-associated signaling pathways and reduce tumor cell aggressiveness underscores its potential as a promising therapeutic candidate. Further in vivo studies are warranted to confirm its efficacy and assess potential off-target effects.

Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap “Biology in Space and Analogue Environments” focusing on “How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?” The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.

Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.

Tissue homeostasis, function recovery, and protection mechanisms are boosted by the balanced and timely control of inflammation and oxidative stress. Nowadays, many natural products and bio-derivates exhibit antioxidant and anti-inflammatory activity, supporting medical care and tissue wellness against inflammation, oxidative stress, and inflammaging. Castanea sativa wood distillate (WD) is a bio-derivative used as a corroborant and biofertilizer in agriculture. Based on the safety profile of low concentrations of WD on human cells, the present study aims to assess the anti-inflammatory and antioxidant activity of WD on different cell types in the integumentary system. Human keratinocytes, mucosal epithelium, dermal fibroblasts, and endothelial cells were exposed to WD, and the concentrations devoid of pro-apoptotic potential were profiled. Then, the effect of nontoxic doses of WD revealed an anti-inflammatory effect, observed through the immunodetection of prostanoid cascade markers in experimentally induced inflammation. A reduction in endothelial hyperpermeability was evidenced by the immunofluorescence analysis of cell-cell adhesion proteins, VE-cadherin and ZO-1. In addition, WD buffered the exogenously produced oxidative stress. On the whole, WD showed both anti-inflammatory and antioxidant activities on the various cell types, preserving endothelial barrier integrity. Overall, this study supports the involvement of this bio-derivative in novel exploitable fields, such as therapeutic dermatological applications for human and animal medical care.

The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations.Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors.

Insufficient vessel maintenance adversely impacts patients in terms of tissue reperfusion following stroke or myocardial infarction, as well as during wound healing. Angiogenesis impairment is a feature typical of metabolic disorders acting at the cardiovascular level, such as diabetes. Therapeutic angiogenesis regulation offers promising clinical implications, and natural compounds as pro-angiogenic nutraceuticals hold valuable applications in regenerative medicine. By using cultured endothelial cells from human umbilical veins (HUVEC) we studied functional and molecular responses following exposure to erucin, a natural isothiocyanate derived from Brassicaceae plants and extracted from the seeds of rocket. Erucin (at nanomolar concentrations) promotes cell migration and tube formation, similar to vascular endothelial growth factor (VEGF), through mobilizing paxillin at endothelial edges. At the molecular level, erucin induces signaling pathways typical of angiogenesis activation, namely Ras, PI3K/AKT, and ERK1/2, leading to VEGF expression and triggering its autocrine production, as pharmacological inhibition of soluble VEGF and VEGFR2 dampens endothelial functions. Furthermore, erucin, alone and together with VEGF, preserves endothelial angiogenic functions under pathological conditions, such as those induced in HUVEC by high glucose (HG) exposure. Erucin emerges as a compelling candidate for therapeutic revascularization applications, showcasing promising prospects for natural compounds in regenerative medicine, particularly in addressing angiogenesis-related disorders.

The present white paper concerns the indications and recommendations of the SciSpacE Science Community to make progress in filling the gaps of knowledge that prevent us from answering the question: “How Do Gravity Alterations Affect Animal and Human Systems at a Cellular/Tissue Level?” This is one of the five major scientific issues of the ESA roadmap “Biology in Space and Analogue Environments”. Despite the many studies conducted so far on spaceflight adaptation mechanisms and related pathophysiological alterations observed in astronauts, we are not yet able to elaborate a synthetic integrated model of the many changes occurring at different system and functional levels. Consequently, it is difficult to develop credible models for predicting long-term consequences of human adaptation to the space environment, as well as to implement medical support plans for long-term missions and a strategy for preventing the possible health risks due to prolonged exposure to spaceflight beyond the low Earth orbit (LEO). The research activities suggested by the scientific community have the aim to overcome these problems by striving to connect biological and physiological aspects in a more holistic view of space adaptation effects.

As human spaceflight progresses with extended mission durations, the demand for effective and safe drugs will necessarily increase. To date, the accepted medications used during missions (for space motion sickness, sleep disturbances, allergies, pain, and sinus congestion) are administered under the assumption that they act as safely and efficaciously as on Earth. However, physiological changes have been documented in human subjects in spaceflight involving fluid shifts, muscle and bone loss, immune system dysregulation, and adjustments in the gastrointestinal tract and metabolism. These alterations may change the pharmacokinetics (PK) and pharmacodynamics of commonly used medications. Frustratingly, the information gained from bed rest studies and from in‐flight observations is incomplete and also demonstrates a high variability in drug PK. Therefore, the objectives of this review are to report (i) the impact of the space environmental stressors on human physiology in relation to PK; (ii) the state‐of‐the‐art on experimental data in space and/or in ground‐based models; (iii) the validation of ground‐based models for PK studies; and ( iv) the identification of research gaps.

The normalization of the tumor ecosystem, consisting in many different cell types and components, is an important new tool for cancer treatment. This chapter discusses the potential targets within the tumor microenvironment (TME) with pharmacological perspective on tumor vascularization and optimization of cancer treatments: hypoxic, acidic and oxidative environment responsible for angiogenic switch and chaotic neovascularization; switch of normal ECs (NECs) to tumor ECs (TECs) in term of cell behavior, metabolism and signaling; cancer associated fibroblasts (CAFs) mainly responsible for angiogenic factor release and processing of the extracellular matrix (ECM); macrophage recruitment in the TME to become tumor associated macrophages (TAMs) and differentiation toward M2 phenotype; normalization of the tumor vasculature; activation of the antitumoral activity by immune cells; miRNAs and epigenetic determinants. The combination of therapies targeting different stromal components, together with traditional antitumor agents, represents the key element to definitely impair cancer progression. Ongoing studies in the field which focus on evaluating the TME with an integrative approach bear the potential to significantly control tumor angiogenesis and broaden the spectrum of current anticancer treatments.

This study is preliminary to an experiment to be performed onboard the International Space Station (ISS) and on Earth to investigate how low gravity influences the healing of sutured human skin and vein wounds. Its objective was to ascertain whether these tissue explants could be maintained to be viable ex vivo for long periods of time, mimicking the experimental conditions onboard the ISS. We developed an automated tissue culture chamber, reproducing and monitoring the physiological tensile forces over time, and a culture medium enriched with serelaxin (60 ng/mL) and (Zn(PipNONO)Cl) (28 ng/mL), known to extend viability of explanted organs for transplantation. The results show that the human skin and vein specimens remained viable for more than 4 weeks, with no substantial signs of damage in their tissues and cells. As a further clue about cell viability, some typical events associated with wound repair were observed in the tissue areas close to the wound, namely remodeling of collagen fibers in the papillary dermis and of elastic fibers in the vein wall, proliferation of keratinocyte stem cells, and expression of the endothelial functional markers eNOS and FGF-2. These findings validate the suitability of this new ex vivo organ culture system for wound healing studies, not only for the scheduled space experiment but also for applications on Earth, such as drug discovery purposes.

ALDH1A1 is a cytosolic enzyme upregulated in tumor cells, involved in detoxifying cells from reactive aldehydes and in acquiring resistance to chemotherapeutic drugs. Its expression correlates with poor clinical outcomes in a number of cancers, including melanoma. The present study hypothesized that the increased ALDH1A1 expression and activity upregulated the release of proangiogenic factors from melanoma cells, which regulate angiogenic features in endothelial cells (ECs) through a rearrangement of the Notch pathway. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing melanoma cells displayed a higher microvessel density. In a 3D multicellular system, obtained co-culturing melanoma cancer cells with stromal cells, including ECs, melanoma ALDH1A1 overexpression induced the recruitment of ECs into the core of the tumorspheres. By using a genes array, overexpression of ALDH1A1 in tumor cells also promoted modulation of Notch cascade gene expression in ECs, suggesting an interaction between tumor cells and ECs mediated by enrichment of angiogenic factors in the tumor microenvironment. To confirm this hypothesis, inactivation of ALDH1A1 by the pharmacological inhibitor CM037 significantly affected the release of angiogenic factors, including IL-8, from melanoma cells. High levels of ALDH1A1, through the retinoic acid pathway, regulated the activation of NF-kappa B-p65 and IL-8. Further, in a 2D co-culture system, the addition of an IL-8 neutralizing antibody to ECs co-cultured with melanoma cells forced to express ALDH1A1 dampened endothelial angiogenic features, both at the molecular (in terms of gene and protein expression of mediators of the Notch pathway) and at the functional level (proliferation, scratch assay, tube formation and permeability). In conclusion, these findings demonstrated the existence of a link between melanoma ALDH1A1 expression and EC Notch signaling modification that results in a pro-angiogenic phenotype. Based on the crucial role of ALDH1A1 in melanoma control of the tumor microenvironment, the enzyme seems a promising target for the development of novel drugs able to interrupt the cross-talk between cancer (stem) cells and endothelial cells.

Exogenous nitric oxide appears a promising therapeutic approach to control cancer progression. Previously, a nickel-based nonoate, [Ni(SalPipNONO)], inhibited lung cancer cells, along with impairment of angiogenesis. The Zn(II) containing derivatives [Zn(PipNONO)Cl] exhibited a protective effect on vascular endothelium. Here, we have evaluated the antitumor properties of [Zn(PipNONO)Cl] in human lung cancer (A549) and melanoma (A375) cells. Metastasis initiates with the epithelial–mesenchymal transition (EMT) process, consisting of the acquisition of invasive and migratory properties by tumor cells. At not cytotoxic levels, the nonoate significantly impaired A549 and A375 EMT induced by transforming growth factor-β1 (TGF-β1). Reduction of the mesenchymal marker vimentin, upregulated by TGF-β1, and restoration of the epithelial marker E-cadherin, reduced by TGF-β1, were detected in both tumor cell lines in the presence of Zn-nonoate. Further, the endothelial–mesenchymal transition achieved in a tumor-endothelial cell co-culture was assessed. Endothelial cells co-cultured with A549 or A375 acquired a mesenchymal phenotype with increased vimentin, alpha smooth muscle actin and Smad2/3, and reduced VE-cadherin. The presence of [Zn(PipNONO)Cl] maintained a typical endothelial phenotype. In conclusion, [Zn(PipNONO)Cl] appears a promising therapeutic tool to control tumor growth and metastasis, by acting on both tumor and endothelial cells, reprogramming the cells toward their physiologic phenotypes.