Ferric uptake regulator Fur is conditionally essential in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the ferric uptake regulator (Fur) protein controls both metabolism and virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion mutants of P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this bacterium. By investigating a P. aeruginosa conditional fur mutant, we demonstrate that Fur is not essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are severely impaired in colony formation. Transposon-mediated random mutagenesis experiments identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the conditional fur mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do not accumulate higher levels of reactive oxygen species (ROS) and are not rescued by antioxidant agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that pyochelin production has no inhibitory effects on a fur deletion mutant of Pseudomonas syringae pv. tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves a P. aeruginosa-specific pathway(s)

Pharmacological modulation of endothelial function during tissue remodeling in physiopathological conditions

Vascular endothelium plays a pivotal role in the maintenance of many biological functions, including angiogenesis, defined as the formation of new capillaries from pre-existing vasculature. The process is aimed at supplying nutrients and oxygenation to growing or healing tissues following injury and, in the context of wound healing, endothelial cells lining the inner surface of blood vessels, are continuously engaged in a crosstalk with other cell types, immunity cells or fibroblasts, to assure a correct progression in the process of regeneration. Disruption of endothelial functions, which can occur for several reasons, including systemic administration of certain dugs or hyperglycemia, leads to a variety of pathological cardiovascular consequences which are commonly featured by inflammation and a dysregulation in pro-angiogenic factors release. Despite many efforts have been made to address this issue, endothelial dysfunction continues to be one of the main causes of morbidity and mortality all over the world, for which new therapeutical approaches are needed to prevent the damage or revert cardiovascular disorders. The first aim of this thesis was to investigate the pro-angiogenic effect of erucin, a natural isothiocyanate with “smart” H2S-releasing properties, particularly abundant in the edible cruciferous plant Eruca sativa. In this study we characterized the pro-angiogenic effect of erucin on endothelial cells by using different functional in vitro assays aimed at evaluating cell migration and ability to organize in a capillary network. A special focus was paid to the molecular mechanisms involved in endothelial cell response to the compound by investigating the early activation of enzymes involved in angiogenesis, such as eNOS, ERK1/2, Akt. Secondly, we demonstrated erucin ability, alone or in combination with vascular endothelial growth factor (VEGF), to protect endothelial cells from high glucose-induced damage and recover impaired functional responses to physiological levels. In the second part of this topic we started to analyze the activity of the isothiocyanate in the context of wound healing by assessing in vitro its pro-migratory, pro- survival effect on dermal fibroblasts (NHDF) and keratinocytes (HaCaT). Lastly, a preliminary study using indirect co-cultures on NHDF and HUVEC, was carried out in order to investigate erucin ability to promote and sustain endothelial-stromal crosstalk, a fundamental step in wound healing. The second part of my thesis addressed the role of activated fibroblasts in the context of impaired wound healing characterized by eccessive inflammation, which underlies several pathologic conditions ranging from healing delay to fibrosis. The aim was to characterize the cellular and molecular events associated with the anti-inflammatory activity of photobiomodulation therapy on human dermal fibroblasts exposed to a mix of inflammatory cytokines followed by laser treatment. Results demonstrated laser ability to revert fibroblast inflammatory phenotype by reducing to basal levels pro-angiogenic factors, as VEGF, and inducible inflammatory key enzymatic pathways, as iNOS and COX-2/mPGES-1/PGE2, by retaining NF-kB transcription factor in a cytoplasmic localization. These molecular changes are accompanied by a shift in cell morphology attributed to a re-distribution of fundamental cytoskeletal proteins (Tubulin, F- actin, and α-SMA) to basal localization following laser treatments. In the third and final topic of this dissertation we discussed the importance of assuring endothelial safety during drug development. The cardiovascular system has proven to be particularly sensitive to a large variety of drugs, especially chemotherapeutic agents, which can promote or accelerate the onset of relevant cardiovascular diseases by impairing vascular integrity and tone. Recently, carbonic anhydrase IX (CA-IX), emerged as a promising new anticancer target for the treatment of solid hypoxic tumors and many efforts have been made to develop selective inhibitors for biomedical applications. In the last project presented, the safety profile of two CA-IX inhibitors, SLC-0111 and AA-06-05 on human endothelial cells was assessed

Effect of Microgravity on Endothelial Cell Function, Angiogenesis, and Vessel Remodeling During Wound Healing

Wound healing is a complex phenomenon that involves different cell types with various functions, i.e., keratinocytes, fibroblasts, and endothelial cells, all influenced by the action of soluble mediators and rearrangement of the extracellular matrix (ECM). Physiological angiogenesis occurs in the granulation tissue during wound healing to allow oxygen and nutrient supply and waste product removal. Angiogenesis output comes from a balance between pro- and antiangiogenic factors, which is finely regulated in a spatial and time-dependent manner, in order to avoid insufficient or excessive nonreparative neovascularization. The understanding of the factors and mechanisms that control angiogenesis and their change following unloading conditions (in a real or simulated space environment) will allow to optimize the tissue response in case of traumatic injury or medical intervention. The potential countermeasures under development to optimize the reparative angiogenesis that contributes to tissue healing on Earth will be discussed in relation to their exploitability in space

The Effect of Space Travel on Bone Metabolism: Considerations on Today’s Major Challenges and Advances in Pharmacology

Microgravity-induced bone loss is currently a significant and unresolved health risk for space travelers, as it raises the likelihood for irreversible changes that weaken skeletal integrity and the incremental onset of fracture injuries and renal stone formation. Another issue related to bone tissue homeostasis in microgravity is its capacity to regenerate following fractures due to weakening of the tissue and accidental events during the accomplishment of particularly dangerous tasks. Today, several pharmacological and non-pharmacological countermeasures to this problem have been proposed, including physical exercise, diet supplements and administration of antiresorptive or anabolic drugs. However, each class of pharmacological agents presents several limitations as their prolonged and repeated employment is not exempt from the onset of serious side effects, which limit their use within a well-defined range of time. In this review, we will focus on the various countermeasures currently in place or proposed to address bone loss in conditions of microgravity, analyzing in detail the advantages and disadvantages of each option from a pharmacological point of view. Finally, we take stock of the situation in the currently available literature concerning bone loss and fracture healing processes. We try to understand which are the critical points and challenges that need to be addressed to reach innovative and targeted therapies to be used both in space missions and on Earth

Endothelium as a Source and Target of H2S to Improve Its Trophism and Function

The vascular endothelium consists of a single layer of squamous endothelial cells (ECs) lining the inner surface of blood vessels. Nowadays, it is no longer considered as a simple barrier between the blood and vessel wall, but a central hub to control blood flow homeostasis and fulfill tissue metabolic demands by furnishing oxygen and nutrients. The endothelium regulates the proper functioning of vessels and microcirculation, in terms of tone control, blood fluidity, and fine tuning of inflammatory and redox reactions within the vessel wall and in surrounding tissues. This multiplicity of effects is due to the ability of ECs to produce, process, and release key modulators. Among these, gasotransmitters such as nitric oxide (NO) and hydrogen sulfide (H2S) are very active molecules constitutively produced by endotheliocytes for the maintenance and control of vascular physiological functions, while their impairment is responsible for endothelial dysfunction and cardiovascular disorders such as hypertension, atherosclerosis, and impaired wound healing and vascularization due to diabetes, infections, and ischemia. Upregulation of H2S producing enzymes and administration of H2S donors can be considered as innovative therapeutic approaches to improve EC biology and function, to revert endothelial dysfunction or to prevent cardiovascular disease progression. This review will focus on the beneficial autocrine/paracrine properties of H2S on ECs and the state of the art on H2S potentiating drugs and tools

Effect of Carbonic Anhydrase IX inhibitors on human endothelial cell survival

The vascular endothelium is one of the first barriers encountered by drugs and xenobiotics, which, once administered, enter the blood stream and diffuse to all organs through blood vessels. The continuous exposure of endothelial cells to drugs and chemical compounds turns out to be a huge risk for the cardiovascular system, as these substances could compromise endothelial vitality and function and create irreparable, localized or systemic damages. For this reason, a special attention should be paid to the safety of developing drugs on the cardiovascular system. In this study we focused our attention on carbonic anhydrase (CA)-IX inhibitors. CA-IX is an enzyme overexpressed in tumor cells in response to hypoxia, which is involved in pH control of the neoplastic mass microenvironment and in tumor progression. Specifically, we evaluated the safety on human umbilical vein endothelial cells (HUVEC) of CA-IX inhibitor AA-06-05, compared to its lead compound SLC-0111, for which the efficacy on tumor cells has already been proven. In this analysis we detected an impairment in viability and mitochondrial metabolism of HUVECs treated with AA-06-05 (but not with SLC-0111) in the concentration range 1–10 μM. These data were accompanied by an increase in the expression of the cell cycle negative regulator, p21, and a down-regulation of the pro-survival proteins ERK1/2 and AKT, both in their phosphorylated and total forms. The data obtained document the likelihood for CA-IX inhibitor AA-06-05 to be developed as new anticancer drug, but a particular attention should be paid to its potential side effects on endothelial cells due to its targeting on other CA isoforms as CA-I, with ubiquitous localization and physiological significance

SARS-CoV-2 infection of thymus induces loss of function that correlates with disease severity

Background: Lymphopenia, particularly when restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in patients with coronavirus disease 2019 (COVID-19) and proposed as an indicator of disease severity. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood. Objective: Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on thymic function. Methods: We performed molecular quantification of T-cell receptor excision circles and κ-deleting recombination excision circles to assess, respectively, T- and B-cell neogenesis in SARS-CoV-2-infected patients. We developed a system for in vitro culture of primary human thymic epithelial cells (TECs) to mechanistically investigate the impact of SARS-CoV-2 on TEC function. Results: We showed that patients with COVID-19 had reduced thymic function that was inversely associated with the severity of the disease. We found that angiotensin-converting enzyme 2, through which SARS-CoV-2 enters the host cells, was expressed by thymic epithelium, and in particular by medullary TECs. We also demonstrated that SARS-CoV-2 can target TECs and downregulate critical genes and pathways associated with epithelial cell adhesion and survival. Conclusions: Our data demonstrate that the human thymus is a target of SARS-CoV-2 and thymic function is altered following infection. These findings expand our current knowledge of the effects of SARS-CoV-2 infection on T-cell homeostasis and suggest that monitoring thymic activity may be a useful marker to predict disease severity and progression