In NIH3T3 cells, 0.001 nM of the synthetic androgen R1881 induces and stimulates association of androgen receptor (AR) with Src and phosphatidylinositol 3-kinase (Pl3-kinase), respectively, thereby triggering S-phase entry. 10 nM R1881 stimulates Rac activity and membrane ruffling in the absence of the receptor-Src-PI3-kinase complex assembly. The antiandrogen Casodex and specific inhibitors of Src and PI3-kinase prevent both hormonal effects, DNA synthesis and cytoskeletal changes. Neither low nor high R1881 concentration allows receptor nuclear translocation and receptor-dependent transcriptional activity in fibroblasts, although they harbor the classical murine AR. The very low amount of AR in NIH3T3 cells (7% of that present in LNCaP cells) activates the signaling pathways, but apparently is not sufficient to stimulate gene transcription. This view is supported by the appearance of receptor nuclear translocation as well as receptor-mediated transcriptional activity after overexpression of AR in fibroblasts. In addition, AR-negative Cos cells transiently transfected with a very low amount of hAR cDNA respond to low and high R1881 concentrations with signaling activation. Interestingly, they do not show significant transcriptional activation under the same experimental conditions. Fibroblasts are the first example of cells that respond to steroid hormones with activation of signaling pathways in the absence of endogenous receptor transcriptional activity. The data reported also show that hormone concentration can be crucial in determining the type of cell responsiveness.

The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.

Prostate cancer represents the major cause of cancer-related death in men and patients frequently develop drug-resistance and metastatic disease. Most studies focus on hormone-resistance mechanisms related to androgen receptor mutations or to the acquired property of prostate cancer cells to over-activate signaling pathways. Tumor microenvironment plays a critical role in prostate cancer progression. However, the mechanism involving androgen/androgen receptor signaling in cancer associated fibroblasts and consequences for prostate cancer progression still remains elusive. We now report that prostate cancer associated fibroblasts express a transcriptional-incompetent androgen receptor. Upon androgen challenging, the receptor co-localizes with the scaffold protein filamin A in the extra-nuclear compartment of fibroblasts, thus mediating their migration and invasiveness. Cancer-associated fibroblasts move towards epithelial prostate cancer cells in 2D and 3D cultures, thereby inducing an increase of the prostate cancer organoid size. Androgen enhances both these effects through androgen receptor/filamin A complex assembly in cancer-associated fibroblasts. An androgen receptor-derived stapled peptide, which disrupts the androgen receptor/filamin A complex assembly, abolishes the androgen-dependent migration and invasiveness of cancer associated fibroblasts. Notably, the peptide impairs the androgen-induced invasiveness of CAFs in 2D models and reduces the overall tumor area in androgen-treated 3D co-culture. The androgen receptor in association with β1 integrin and membrane type-matrix metalloproteinase 1 activates a protease cascade triggering extracellular matrix remodeling. The peptide also impairs the androgen activation of this cascade. This study offers a potential new marker, the androgen receptor/filamin A complex, and a new therapeutic approach targeting intracellular pathways activated by the androgen/androgen receptor axis in prostate cancer-associated fibroblasts. Such a strategy, alone or in combination with conventional therapies, may allow a more efficient treatment of prostate cancer.

Three-dimensional printing of poly(ε-caprolactone) (PCL) is a consolidated scaffold manufacturing technique for bone regenerative medicine. Simultaneously, the mesenchymal stem/stromal cell (MSC) secretome is osteoinductive, promoting scaffold colonization by cells, proliferation, and differentiation. The present paper combines 3D-printed PCL scaffolds with lyosecretome, a freeze-dried formulation of MSC secretome, containing proteins and extracellular vesicles (EVs). We designed a lyosecretome 3D-printed scaffold by two loading strategies: (i) MSC secretome adsorption on 3D-printed scaffold and (ii) coprinting of PCL with an alginate-based hydrogel containing MSC secretome (at two alginate concentrations, i.e., 6% or 10% w/v). A fast release of proteins and EVs (a burst of 75% after 30 min) was observed from scaffolds obtained by absorption loading, while coprinting of PCL and hydrogel, encapsulating lyosecretome, allowed a homogeneous loading of protein and EVs and a controlled slow release. For both loading modes, protein and EV release was governed by diffusion as revealed by the kinetic release study. The secretome's diffusion is influenced by alginate, its concentration, or its cross-linking modes with protamine due to the higher steric hindrance of the polymer chains. Moreover, it is possible to further slow down protein and EV release by changing the scaffold shape from parallelepiped to cylindrical. In conclusion, it is possible to control the release kinetics of proteins and EVs by changing the composition of the alginate hydrogel, the scaffold's shape, and hydrogel cross-linking. Such scaffold prototypes for bone regenerative medicine are now available for further testing of safety and efficacy.

Anomalous aortic origin of the coronary artery (AAOCA) is a rare disease associated with sudden cardiac death, usually related to physical effort in young people. Clinical routine tests fail to assess the ischemic risk, calling for novel diagnostic approaches. To this aim, some recent studies propose to assess the coronary blood flow (CBF) in AAOCA by computational simulations but they are limited by the use of data from literature retrieved from normal subjects. To overcome this limitation and obtain a reliable assessment of CBF, we developed a fully patient-specific lumped parameter model based on clinical imaging and in-vivo data retrieved during invasive coronary functional assessment of subjects with AAOCA. In such a way, we can estimate the CBF replicating the two hemodynamic conditions in-vivo analyzed. The model can mimic the effective coronary behavior with high accuracy and could be a valuable tool to quantify CBF in AAOCA. It represents the first step required to move toward a future clinical application with the aim of improving patient care. The study was registered at Clinicaltrial.gov with (ID: NCT05159791, date 2021-12-16).

Repair of peripheral nerve injuries is an intensive area of challenge and research in modern reconstructive microsurgery. Intensive research is being carried out to develop effective alternatives to the standard nerve autografting, avoiding its drawbacks. The aim of the study was to evaluate the effectiveness of a newly designed mechanical device for the reconstruction of the sciatic nerve in rats in comparison to nerve autografting and to assess the pain during the period of distraction neurogenesis.

Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).

Prostate cancer (PC) is one of the most frequently diagnosed cancers and a leading cause of cancer-related deaths in Western society. Current PC therapies prevalently target the functions of androgen receptor (AR) and may only be effective within short time periods, beyond which the majority of PC patients progress to castration-resistant PC (CRPC) and metastatic disease. The role of estradiol/estradiol receptor (ER) axis in prostate transformation and PC progression is well established. Further, considerable efforts have been made to investigate the mechanism by which somatostatin (SST) and somatostatin receptors (SSTRs) influence PC growth and progression. A number of therapeutic strategies, such as the combination of SST analogs with other drugs, show, indeed, strong promise. However, the effect of the combined treatment of SST analogs and estradiol on proliferation, epithelial mesenchyme transition (EMT) and migration of normal- and cancer-derived prostate cells has not been investigated so far. We now report that estradiol plays anti-proliferative and pro-apoptotic effect in non-transformed EPN prostate cells, which express both ERα and ERβ. A weak apoptotic effect is observed in transformed CPEC cells that only express low levels of ERβ. Estradiol increases, mainly through ERα activation, the expression of SSTRs in EPN, but not CPEC cells. As such, the hormone enhances the anti-proliferative effect of the SST analog, pasireotide in EPN, but not CPEC cells. Estradiol does not induce EMT and the motility of EPN cells, while it promotes EMT and migration of CPEC cells. Addition of pasireotide does not significantly modify these responses. Altogether, our results suggest that pasireotide may be used, alone or in combination with other drugs, to limit the growth of prostate proliferative diseases, provided that both ER isoforms (α and β) are present. Further investigations are needed to better define the cross talk between estrogens and SSTRs as well as its role in PC.

Resistance to hormone therapy and disease progression is the major challenge in clinical management of prostate cancer (PC). Drugs currently used in PC therapy initially show a potent antitumor effects, but PC gradually develops resistance, relapses and spreads. Most patients who fail primary therapy and have recurrences eventually develop castration-resistant prostate cancer (CRPC), which is almost incurable. The nerve growth factor (NGF) acts on a variety of non-neuronal cells by activating the NGF tyrosine-kinase receptor, tropomyosin receptor kinase A (TrkA). NGF signaling is deregulated in PC. In androgen-dependent PC cells, TrkA mediates the proliferative action of NGF through its crosstalk with the androgen receptor (AR). Epithelial PC cells, however, acquire the ability to express NGF and TrkA, as the disease progresses, indicating a role for NGF/TrkA axis in PC progression and androgen-resistance. We here report that once activated by NGF, TrkA mediates proliferation, invasiveness and epithelial-mesenchymal transition (EMT) in various CRPC cells. NGF promotes organoid growth in 3D models of CRPC cells, and specific inhibition of TrkA impairs all these responses. Thus TrkA represents a new biomarker to target in CRPC.

Transient receptor potential melastatin-8 (TRPM8) represents an emerging target in prostate cancer, although its mechanism of action remains unclear. Here, we have characterized and investigated the effects of TRPM8 modulators in prostate cancer aggressiveness disclosing the molecular mechanism underlying their biological activity. Patch-clamp and calcium fluorometric assays were used to characterize the synthesized compounds. Androgen-stimulated prostate cancer-derived cells were challenged with the compounds and the DNA synthesis was investigated in a preliminary screening. The most effective compounds were then employed to inhibit the pro-metastatic behavior of in various PC-derived cells, at different degree of malignancy. The effect of the compounds was then assayed in prostate cancer cell-derived 3D model and the molecular targets of selected compounds were lastly identified using transcriptional and non-transcriptional reporter assays. TRPM8 antagonists inhibit the androgen-dependent prostate cancer cell proliferation, migration and invasiveness. They are highly effective in reverting the androgen-induced increase in prostate cancer cell spheroid size. The compounds also revert the proliferation of castrate-resistant prostate cancer cells, provided they express the androgen receptor. In contrast, no effects were recorded in prostate cancer cells devoid of the receptor. Selected antagonists interfere in non-genomic androgen action and abolish the androgen-induced androgen receptor/TRPM8 complex assembly as well as the increase in intracellular calcium levels in prostate cancer cells. Our results shed light in the processes controlling prostate cancer progression and make the transient receptor potential melastatin-8 as a 'druggable' target in the androgen receptor-expressing prostate cancers.

Steroid hormones and growth factors control neuritogenesis through their cognate receptors under physiological and pathological conditions. We have already shown that nerve growth factor and androgens induce neurite outgrowth of PC12 cells through a reciprocal crosstalk between the NGF receptor, TrkA and the androgen receptor. Here, we report that androgens or NGF induce neuritogenesis in PC12 cells through inactivation of RhoA. Ectopic expression of the dominant negative RhoA N19 promotes, indeed, the neurite-elongation of unchallenged and androgen- or NGF-challenged PC12 cells and the increase in the expression levels of βIII tubulin, a specific neuronal marker. Pharmacological inhibition of the Ser/Thr kinase ROCK, an RhoA effector, induces neuritogenesis in unchallenged PC12 cells, and potentiates the effect of androgens and NGF, confirming the role of RhoA/ROCK axis in the neuritogenesis induced by androgen and NGF, through the phosphorylation of Akt. These findings suggest that therapies based on new selective androgen receptor modulators and/or RhoA/ROCK inhibitors might exert beneficial effects in the treatment of neuro-disorders, neurological diseases and ageing-related processes.

to investigate the factors implied in the development of postoperative complications in both self-expandable and balloon-expandable transcatheter heart valves by means of finite element analysis (FEA).

Colorectal cancer (CRC) is the third most deadly and fourth most diagnosed cancer worldwide. Despite the progress in early diagnosis and advanced therapeutic options, CRC shows a poor prognosis with a 5 year survival rate of ~ 45%. PRDM2/RIZ, a member of PR/SET domain family (PRDM), expresses two main molecular variants, the PR-plus isoform (RIZ1) and the PR-minus (RIZ2). The imbalance in their expression levels in favor of RIZ2 is observed in many cancer types. The full length RIZ1 has been extensively investigated in several cancers where it acts as a tumor suppressor, whereas few studies have explored the RIZ2 oncogenic properties. PRDM2 is often target of frameshift mutations and aberrant DNA methylation in CRC. However, little is known about its role in CRC.

Aging induces a slow and progressive decrease in muscle mass and function, causing sarcopenia. Androgens control muscle trophism and exert important anabolic functions through the binding to the androgen receptor. Therefore, analysis of the androgen receptor-mediated actions in skeletal muscle might provide new hints for a better understanding of sarcopenia pathogenesis. In this study, we report that expression of the androgen receptor in skeletal muscle biopsies from 20 subjects is higher in young, as compared with old subjects. Co-immunoprecipitation experiments reveal that the androgen receptor is complexed with filamin A mainly in young, that in old subjects. Therefore, we have in depth analyzed the role of such complex using C2C12 myoblasts that express a significant amount of the androgen receptor. In these cells, hormone stimulation rapidly triggers the assembly of the androgen receptor/filamin A complex. Such complex prevents the senescence induced by oxidative stress in C2C12 cells, as disruption of the androgen receptor/filamin A complex by Rh-2025u stapled peptide re-establishes the senescent phenotype in C2C12 cells. Simultaneously, androgen stimulation of C2C12 cells rapidly triggers the activation of various signaling effectors, including Rac1, focal adhesion kinase, and mitogen-activated kinases. Androgen receptor blockade by bicalutamide or perturbation of androgen receptor/filamin A complex by Rh-2025u stapled peptide both reverse the hormone activation of signaling effectors. These findings further reinforce the role of the androgen receptor and its extranuclear partners in the rapid hormone signaling that controls the functions of C2C12 cells. Further investigations are needed to promote clinical interventions that might ameliorate muscle cell function as well the clinical outcome of age-related frailty.

The relationship among increased aortic arch angulation, aortic flow dynamics, and vessel wall stiffness remains unclear. This experimental ex vivo study investigated how increased aortic arch angulation affects aortic stiffness and stent-graft induced aortic stiffening, assessed by pulse wave velocity (PWV).

Triple-negative breast cancer is a heterogeneous disease that still lacks specific therapeutic approaches. The identification of new biomarkers, predictive of the disease's aggressiveness and pharmacological response, is a challenge for a more tailored approach in the clinical management of patients. Nerve growth factor, initially identified as a key factor for neuronal survival and differentiation, turned out to be a multifaceted molecule with pleiotropic effects in quite divergent cell types, including cancer cells. Many solid tumors exhibit derangements of the nerve growth factor and its receptors, including the tropomyosin receptor kinase A. This receptor is expressed in triple-negative breast cancer, although its role in the pathogenesis and aggressiveness of this disease is still under investigation. We now report that triple-negative breast cancer-derived MDA-MB-231 and MDA-MB-453 cells express appreciable levels of tropomyosin receptor kinase A and release a biologically active nerve growth factor. Activation of tropomyosin receptor kinase by nerve growth factor treatment positively affects the migration, invasion, and proliferation of triple-negative breast cancer cells. An increase in the size of triple-negative breast cancer cell spheroids is also detected. This latter effect might occur through the nerve growth factor-induced release of matrix metalloproteinase 9, which contributes to the reorganization of the extracellular matrix and cell invasiveness. The tropomyosin receptor kinase A inhibitor GW441756 reverses all these responses. Co-immunoprecipitation experiments in both cell lines show that nerve growth factor triggers the assembly of the TrkA/β1-integrin/FAK/Src complex, thereby activating several downstream effectors. GW441756 prevents the complex assembly induced by nerve growth factor as well as the activation of its dependent signaling. Pharmacological inhibition of the tyrosine kinases Src and FAK (focal adhesion kinase), together with the silencing of β1-integrin, shows that the tyrosine kinases impinge on both proliferation and motility, while β1-integrin is needed for motility induced by nerve growth factor in triple-negative breast cancer cells. The present data support the key role of the nerve growth factor/tropomyosin receptor kinase A pathway in triple-negative breast cancer and offer new hints in the diagnostic and therapeutic management of patients.

Anomalous aortic origin of the coronary artery can be associated with sudden cardiac death and ischemic events. Anatomic static characteristics mainly dictated surgical indications, although adverse events are usually related to dynamic physical effort. We developed a computational model able to simulate anomalous coronary behavior, and we aimed to assess its clinical applicability and to investigate coronary characteristics at increasing loading stress conditions.

Breast cancer (BC) is still characterized by high morbidity and mortality. A specific BC subtype named triple negative BC (TNBC) lacks estrogen and progesterone receptors (ER and PR, respectively) and is characterized by the absence of overexpression/amplification of human epidermal growth factor receptor 2 (HER2). The androgen receptor (AR) is expressed in TNBC, although its function in these cancers is still debated. Moreover, few therapeutic options are currently available for the treatment of TNBC. In this study, we have used TNBC-derived MDA-MB231 and MDA-MB453 cells that, albeit at different extent, both express AR. Androgen challenging induces migration and invasiveness of these cells. Use of the anti-androgen bicalutamide or AR knockdown experiments show that these effects depend on AR. Furthermore, the small peptide, S1, which mimics the AR proline-rich motif responsible for the interaction of AR with SH3-Src, reverses the effects in both cell lines, suggesting that the assembly of a complex made up of AR and Src drives the androgen-induced motility and invasiveness. Co-immunoprecipitation experiments in androgen-treated MDA-MB231 and MDA-MB453 cells show that the AR/Src complex recruits p85α, the regulatory subunit of PI3-K. In such a way, the basic machinery leading to migration and invasiveness is turned-on. The S1 peptide inhibits motility and invasiveness of TNBC cells and disrupts the AR/Src/p85α complex assembly in MDA-MB231 cells. This study shows that the rapid androgen activation of Src/PI3-K signaling drives migration and invasiveness of TNBC cells and suggests that the S1 peptide is a promising therapeutic option for these cancers.

 The benefits of thoracic endovascular aortic repair (TEVAR) have encouraged stent graft deployment more proximally in the aortic arch. This study quantifies the hemodynamic impact of TEVAR in proximal landing zone 2 on the thoracic aorta and the proximal supra-aortic branches.

Positive Regulatory Domain (PRDM) gene family members commonly express two main molecular variants, the PR-plus isoform usually acting as tumor suppressor and the PR-minus one functioning as oncogene. Accordingly, PRDM2/RIZ encodes for RIZ1 (PR-plus) and RIZ2 (PR-minus). In human cancers, genetic or epigenetic modifications induce RIZ1 silencing with an expression level imbalance in favor of RIZ2 that could be relevant for tumorigenesis. Additionally, in estrogen target cells and tissues, estradiol increases RIZ2 expression level with concurrent increase of cell proliferation and survival. Several attempts to study RIZ2 function in HeLa or MCF-7 cells by its over-expression were unsuccessful. Thus, we over-expressed RIZ2 in HEK-293 cells, which are both RIZ1 and RIZ2 positive but unresponsive to estrogens. The forced RIZ2 expression increased cell viability and growth, prompted the G2-to-M phase transition and organoids formation. Accordingly, microarray analysis revealed that RIZ2 regulates several genes involved in mitosis. Consistently, RIZ silencing in both estrogen-responsive MCF-7 and -unresponsive MDA-MB-231 cells induced a reduction of cell proliferation and an increase of apoptosis rate. Our findings add novel insights on the putative RIZ2 tumor-promoting functions, although additional attempts are warranted to depict the underlying molecular mechanism.