Preparation and analysis of cutaneous primary myofibroblast three-dimensional culture: an experimental system for myofibroblast deactivation studies.

Myofibroblasts are activated fibroblasts involved in tissue repair and cancer that are characterized by de novo expression of alpha smooth muscle actin (α-SMA), increased secretion of growth factors and immunoregolatory fenotype. It is known that myofibroblasts form clusters during cancer, fibrotic tissue and wound healing. At the end of wound healing, myofibroblasts undergo apoptotic cell death, whereas in vitro fibroblasts undergo a necrosis-like programmed cell death, called nemosis, associated with inflammatory response. As primary myofibroblasts better resemble the tissue environment in vivo, in this work we generated spheroids from human primary cutaneous myofibroblasts to evaluate apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The study showed that spheroid formation does not trigger apoptosis, necrotic cell death and cyclooxygenase-2 (COX-2) protein induction associated with inflammatory response. The significant decrease of α-SMA in protein extracts of spheroids, compared to myofibroblasts monolayer, led to hypothesize that myofibroblasts have undergone a deactivation process within spheroids. This hypothesis is supported by cytostatic effect exerted by spheroid-conditioned medium on both normal and cancer cell lines, by anti-migratory effect on normal cells and also by the low number of nuclei positive for the proliferation marker Ki-67. The analysis of spheroid reversion to monolayer growth demonstrated that fibroblasts cultured as aggregates preserved their proliferation capability. Surprisingly, we obtained spontaneous clusters by seeding myofibroblasts on plastic and glass substrates: thus the cluster formation could be a physiological feature of cutaneous myofibroblasts. This work represents an experimental model to study myofibroblast deactivation and highlights the possible relevance of clusters as reservoir of myofibroblasts for regulating myofibroblast and tissue turnover

Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed

Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5?9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 ?M. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds

Generation of spheroids from human primary myofibroblasts: an experimental system to study myofibroblasts deactivation

Fibroblasts represent a heterogeneous cell population, that in adult body maintains the homeostasis of the extracellular matrix (ECM) and can acquire an immunoregulatory phenotype. Indeed, activated fibroblasts produce large amounts of cyclooxygenase-2 (COX-2) and proinflammatory cytokines (1). The activation of fibroblasts is represented by their differentiation into myofibroblasts. This process, either in wound healing or cancer tissue, is associated with the expression of alpha-smooth muscle actin (alpha-SMA), increased levels of growth factors and ECM-degrading proteases (2). Moreover, myofibroblasts form clusters in wound healing process and hypertrophic scars. In particular, cell clusters of hypertrophic scars are represented by nodules of myofibroblasts (3). It is known that human dermal fibroblasts established from neonatal foreskin, and forced in vitro to form clusters named spheroids, are activated to produce massive amounts of COX-2, prostaglandins and proinflammatory cytokines: this process leads to a programmed necrosis, designated “nemosis” (1). In the present study we generated spheroids from human primary myofibroblasts of skin, to evaluate necrotic, inflammation and activation markers during myofibroblasts clustering. Western blotting analysis, showing low levels of COX-2 and a significant decrease of alpha-SMA in protein extracts of spheroids, led to hypothesize that myofibroblasts have undergone a deactivation process within spheroids. This hypothesis is confirmed by cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines, by confocal immunofluorescence analysis of connexin 43 and immunohistochemical evaluation of proliferation marker Ki-67. This work could represent an experimental model to study myofibroblasts deactivation and highlights an alternative process regulating the turnover of myofibroblasts

Analysis of SOD3 and Akt in ascending aortic aneurysm

Ascending aortic aneurysm (AsAA) is divided into three different forms: syndromic, familial non-syndromic, and degenerative. Bicuspid aortic valve (BAV), occuring in 2% of the population, is the most frequent cardiac congenital abnormality, associated to AsAA. All the different forms of AsAAs are a consequence of cystic medial necrosis (CMN), characterized by apoptotic loss of smooth muscle cells (SMCs), fragmentation of elastic and collagen fibers and increased accumulation of mucoid material. The extracellular superoxide dismutase (SOD3) is a Cu/Zn enzyme, affecting redox state and homeostasis of extracellular matrix (ECM) (1). Moreover, the outsidein signalling from ECM modulates intracellular pathways regulating many cellular functions. The multifunctional Akt pathway affects survival and cellular proliferation and has important effects on the cardiovascular function. In this study we examined the relevance of SOD3 and Akt in AsAA pathogenesis. To this aim, the SOD3 and Akt protein levels were evaluated in normal ascending aortic tissues (n=6) and in tissues from AsAAs associated both to tricuspid aortic valve (TAV) (n=6) and BAV (n=6); moreover, we measured SOD3 activity in sera from healthy donors and patients with AsAA. Our data showed a reduction of SOD3 and phospho-Akt (pAkt) protein levels in AsAAs from BAV patients compared to normal donors; on the other hand, no differences emerged in SOD3 activity. Furthermore, immunohistochemical analysis performed on normal and pathological ascending aortic tissues showed a SOD3 immunostaining in both extracellular space and tunica media cells from normal ascending aortic tissues; conversely, no SOD3 immunostaining was detected in AsAAs tissues from both TAV and BAV patients. Our data show that SOD3 and pAkt could be associated to AsAA pathogenesis and suggest a link between ECM homeostasis and Akt survival pathway

Antagonists of Growth Hormone-Releasing Hormone Inhibit the Growth of Pituitary Adenoma Cells by Hampering Oncogenic Pathways and Promoting Apoptotic Signaling

Pituitary adenomas (PAs) are intracranial tumors, often associated with excessive hormonal secretion and severe comorbidities. Some patients are resistant to medical therapies; therefore, novel treatment options are needed. Antagonists of growth hormone-releasing hormone (GHRH) exert potent anticancer effects, and early GHRH antagonists were found to inhibit GHRH-induced secretion of pituitary GH in vitro and in vivo. However, the antitumor role of GHRH antagonists in PAs is largely unknown. Here, we show that the GHRH antagonists of MIAMI class, MIA-602 and MIA-690, inhibited cell viability and growth and promoted apoptosis in GH/prolactin-secreting GH3 PA cells transfected with human GHRH receptor (GH3-GHRHR), and in adrenocorticotropic hormone ACTH-secreting AtT20 PA cells. GHRH antagonists also reduced the expression of proteins involved in tumorigenesis and cancer progression, upregulated proapoptotic molecules, and lowered GHRH receptor levels. The combination of MIA-690 with temozolomide synergistically blunted the viability of GH3-GHRHR and AtT20 cells. Moreover, MIA-690 reduced both basal and GHRH-induced secretion of GH and intracellular cAMP levels. Finally, GHRH antagonists inhibited cell viability in human primary GH- and ACTH-PA cell cultures. Overall, our results suggest that GHRH antagonists, either alone or in combination with pharmacological treatments, may be considered for further development as therapy for PAs

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1β, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1β secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities

Aorta and pulmonary trunk - comparison of wall structure in typical and atypical (Ross procedure) blood pressure conditions

The ascending aorta and pulmonary trunk develop from the common truncus arteriosus that later becomes symmetrically divided by the aorticopulmonary septum. Normally, the systolic pressure value and the diastolic pressure gradient in the aorta is much higher than that in the pulmonary artery. In the Ross procedure, patient’s diseased aortic valve is replaced with their own pulmonary valve and as a consequence the pulmonary trunk is placed in the aortic root position. Typically, a reinforcement of transposed pulmonary trunk is necessary to avoid vessel dilation. In order to investigate how the blood flow characteristics determine the vessel wall structure we examined by immunochemistry the wall of normal aorta (NA), normal pulmonary trunk (NPT), transposed pulmonary trunk (trPT) and transposed pulmonary trunk with reinforcement (trPT-R). Throughout the tunica media of NA, elastic fibers form numerous, conspicuous and orderly arranged wavy lamellae that parallel thin layers of smooth muscle cells between the internal and external elastic membrane. In the NPT, smooth muscle cells run amid collagen fibers and form layers that are thicker and irregular, with elastic fibers arranged accordingly. In the trPT, intima denudation and media disruptions were observed. In the tunica media, smooth muscle cells were abundant, but muscle fibers, with irregular profiles and no discernible alignment, were widely spaced with intervening collagen fibers. In the trPA-R, the endothelial lining was preserved and elastic fibers formed a thick and highly organized layer of concentric lamellae in the middle third of tunica media. The structure of normal aorta and pulmonary trunk, both elastic arteries, with common embryological origin, differs significantly in terms of smooth muscle and elastic sheets number and organization. The animal model of Ross procedure with pulmonary trunk in aorta position further underscores the role of blood pressure and mechanical stress in vessel wall modification

Positional memory of fibroblasts may affect efficiency of iPSC reprogramming

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming