NEST3D printed bone-mimicking scaffolds: assessment of the effect of geometrical design on stiffness and angiogenic potential.

Tissue-engineered implants for bone regeneration require consideration regarding their mineralization and vascularization capacity. Different geometries, such as biomimetic designs and lattices, can influence the mechanical properties and the vascularization capacity of bone-mimicking implants. Negative Embodied Sacrificial Template 3D (NEST3D) printing is a versatile technique across a wide range of materials that enables the production of bone-mimicking scaffolds. In this study, different scaffold motifs (logpile, Voronoi, and trabecular bone) were fabricated via NEST3D printing in polycaprolactone to determine the effect of geometrical design on stiffness (10.44 ± 6.71, 12.61 ± 5.71, and 25.93 ± 4.16 MPa, respectively) and vascularization. The same designs, in a polycaprolactone scaffold only, or when combined with gelatin methacryloyl, were then assessed for their ability to allow the infiltration of blood vessels in a chick chorioallantoic membrane (CAM) assay, a cost-effective and time-efficient in ovo assay to assess vascularization. Our findings showed that gelatin methacrylolyl alone did not allow new chorioallantoic membrane tissue or blood vessels to infiltrate within its structure. However, polycaprolactone on its own or when combined with gelatin methacrylolyl allowed tissue and vessel infiltration in all scaffold designs. The trabecular bone design showed the greatest mineralized matrix production over the three designs tested. This reinforces our hypothesis that both biomaterial choice and scaffold motifs are crucial components for a bone-mimicking scaffold.

A novel patient-derived immortalised cell line of myxofibrosarcoma: a tool for preclinical drugs testing and the generation of near-patient models.

BACKGROUND: Myxofibrosarcoma is a rare malignant soft tissue sarcoma characterised by multiple local recurrence and can become of higher grade with each recurrence. Consequently, myxofibrosarcoma represents a burden for patients, a challenge for clinicians, and an interesting disease to study tumour progression. Currently, few myxofibrosarcoma preclinical models are available. METHODS: In this paper, we present a spontaneously immortalised myxofibrosarcoma patient-derived cell line (MF-R 3). We performed phenotypic characterization through multiple biological assays and analyses: proliferation, clonogenic potential, anchorage-independent growth and colony formation, migration, invasion, AgNOR staining, and ultrastructural evaluation. RESULTS: MF-R 3 cells match morphologic and phenotypic characteristics of the original tumour as 2D cultures, 3D aggregates, and on the chorioallantoic membrane of chick embryos. Overall results show a clear neoplastic potential of this cell line. Finally, we tested MF-R 3 sensitivity to anthracyclines in 2D and 3D conditions finding a good response to these drugs. CONCLUSIONS: In conclusion, we established a novel patient-derived myxofibrosarcoma cell line that, together with the few others available, could serve as an important model for studying the molecular pathogenesis of myxofibrosarcoma and for testing new drugs and therapeutic strategies in diverse experimental settings.

Modeling Myxofibrosarcoma: Where Do We Stand and What Is Missing?

Myxofibrosarcoma (MFS) is a malignant soft tissue sarcoma (STS) that originates in the body's connective tissues. It is characterized by the presence of myxoid (gel-like) and fibrous components and typically affects patients after the fifth decade of life. Considering the ongoing trend of increasing lifespans across many nations, MFS is likely to become the most common musculoskeletal sarcoma in the future. Although MFS patients have a lower risk of developing distant metastases compared with other STS cases, MFS is characterized by a high frequency of local recurrence. Notably, in 40-60% of the patients where the tumor recurs, it does so multiple times. Consequently, patients may undergo multiple local surgeries, removing the risk of potential amputation. Furthermore, because the tumor relapses generally have a higher grade, they exhibit a decreased response to radio and chemotherapy and an increased tendency to form metastases. Thus, a better understanding of MFS is required, and improved therapeutic options must be developed. Historically, preclinical models for other types of tumors have been instrumental in obtaining a better understanding of tumor development and in testing new therapeutic approaches. However, few MFS models are currently available. In this review, we will describe the MFS models available and will provide insights into the advantages and constraints of each model.

Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels.

In myelinating Schwann cells, connection between myelin layers is mediated by gap junction channels (GJCs) formed by docked connexin 32 (Cx32) hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy without a cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high-resolution cryo-electron cryo-myography (cryo-EM) structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: In the W3S and R22G mutant HCs, the amino-terminal gating helix partially occludes the pore, consistent with a diminished HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.

Experimental evidence of a limited impact of new-generation perfluoroalkyl substance C6O4 on differentiating human dopaminergic neurons from induced pluripotent stem cells.

Perfluoroalkyl substances (PFASs) are persistent pollutants, raising concerns for human health. Legacy PFAS perfluoro-octanoic acid (PFOA) accumulate in brains of people at high environmental exposure, especially in areas enriched with dopaminergic neurons (DN). In vitro exposure to 10 ng/mL PFOA for 24 h was also associated with an altered molecular and functional phenotype of DN differentiated from human induced pluripotent stem cells (hiPSCs). Acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)- 1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1), known as C6O4, is a new generation PFAS proposed to have a safer profile. Here we investigated the effect of C6O4 exposure on the molecular phenotype of hiPSC-derived DN. Cells were exposed to C6O4 for 24 h, at the concentration of 10 ng/mL, at neuronal commitment (DP1), neuronal precursor (DP2) and the mature dopaminergic (DP3) phases of differentiation. Liquid-chromatography/mass-spectrometry showed negligible cell accumulation of C6O4 at each differentiation stage and by staining with Merocyanine-540 we observed unaltered cell membrane fluidity. Immunofluorescence showed that the expression of tyrosine hydroxylase (TH) and ßIII-Tubulin was unaffected by the exposure to C6O4 at each differentiation phase (respectively: DP1, p = 0.332; DP2, p = 0.623; DP3, p = 0.816, with respect to control unexposed conditions). Exposure to C6O4 is presumed to have minor effects on cell molecular/functional phenotype of developing human DN cells, requiring confirm on in vivo models.

Molecular mechanisms of skin wound healing in non-diabetic and diabetic mice in excision and pressure experimental wounds.

Experimental models for chronic skin lesions are excision and pressure ulcer, defined as "open" and "closed" lesions, respectively, only the latter characterized by tissue hypoxia. Moreover, systemic diseases, such as diabetes mellitus, affect wound repair. Thus, models for testing new therapies should be carefully selected according to the expected targets. In this study, we present an extensive and comparative histological, immunohistochemical, and molecular characterization of these two lesions in diabetic (db/db) and non-diabetic (C57BL/6 J) mice. In db/db mice, we found significant reduction in PGP9.5-IR innervation, reduction of capillary network, and reduced expression of NGF receptors. We found an increase in VEGF receptor Kdr expression, and the PI3K-Akt signaling pathway at the core of the altered molecular network. Db/db mice with pressure ulcers showed an impairment in the molecular regulation of hypoxia-related genes (Hif1a, Flt1, and Kdr), while extracellular matrix encoding genes (Itgb3, Timp1, Fn1, Col4a1) were upregulated by hyperglycemia and lesions. Overall, the molecular analysis suggests that db/db mice have a longer inflammatory phase of the wound repair process, delaying the progression toward the proliferation and remodeling phases.

Impairment of human dopaminergic neurons at different developmental stages by perfluoro-octanoic acid (PFOA) and differential human brain areas accumulation of perfluoroalkyl chemicals.

Perfluoroalkyl substances (PFASs) are synthetic chemicals widely used in industrial and consumer products. The environmental spreading of PFASs raises concerns for their impact on human health. In particular, the bioaccumulation in humans due to environmental exposure has been reported also in total brain samples and PFAS exposure has been associated with neurodevelopmental disorders. In this study we aimed to investigate the specific PFAS bioaccumulation in different brain areas. Our data reported major accumulation in the brainstem region, which is richly populated by dopaminergic neurons (DNs), in brain autopsy samples from people resident in a PFAS-polluted area of Italy. Since DNs are the main source of dopamine (DA) in the mammalian central nervous system (CNS), we evaluated the possible functional consequences of perfluoro-octanoic acid (PFOA) exposure in a human model of DNs obtained by differentiation of human induced pluripotent stem cells (hiPSCs). Particularly, we analyzed the specific effect of the exposure to PFOA for 24 h, at the concentration of 10 ng/ml, at 3 different steps of dopaminergic differentiation: the neuronal commitment phase (DP1), the neuronal precursor phase (DP2) and the mature dopaminergic differentiation phase (DP3). Interestingly, compared to untreated cells, exposure to PFOA was associated with a reduced expression of Tyrosine Hydroxylase (TH) and Neurofilament Heavy (NFH), both markers of dopaminergic maturation at DP2 phase. In addition, cells at DP3 phase exposed to PFOA showed a severe reduction in the expression of the Dopamine Transporter (DAT), functionally involved in pre-synaptic dopamine reuptake. In this proof-of-concept study we show a significant impact of PFOA exposure, mainly on the most sensitive stage of neural dopaminergic differentiation, prompting the way for further investigations more directly relevant to risk assessment of these chemicals.

Exposure to Perfluoro-Octanoic Acid Associated With Upstream Uncoupling of the Insulin Signaling in Human Hepatocyte Cell Line.

Perfluoro-alkyl substances (PFAS) are chemical pollutants with prevalent stability and environmental persistence. Exposure to PFAS, particularly perfluoro-octanoic acid (PFOA), has been associated with increased diabetes-related cardiovascular mortality in subjects residing areas of high environmental contamination, however the exact pathogenic mechanism remains elusive. Here we used HepG2 cells, an in vitro model of human hepatocyte, to investigate the possible role of PFOA exposure in the alteration of hepatic glucose metabolism. HepG2 cells were exposed for 24 hours to PFOA at increasing concentration from 0 to 1000 ng/mL and then stimulated with 100 nm Insulin (Ins). The consequent effect on glycogen synthesis, glucose uptake and Glut-4 glucose transporter translocation was then evaluated by, respectively, Periodic Acid Schiff (PAS) staining, 2-deoxyglucose (2-DG) uptake assay and immunofluorescence. Exposure to PFOA was associated with reduced glycogen synthesis and glucose uptake, at concentration equal or greater than, respectively, 0,1 ng/mL and 10 ng/mL, with parallel impaired membrane translocation of Glut-4 upon Ins stimulation. Western blot analysis showed early uncoupling of Insulin Receptor (InsR) activation from the downstream Akt and GSK3 phosphorylation. Computational docking analysis disclosed the possible stabilizing effect of PFOA on the complex between InsR and GM3 ganglioside, previously shown to be associated with the low grade chronic inflammation-related insulin resistance. Consistently, long term treatment with glucosyl-ceramide synthase inhibitor PDMP was able to largely restore glycogen synthesis, glucose uptake and Glut-4 translocation upon Ins stimulation in HepG2 exposed to PFOA. Our data support a novel pathogenic mechanism linking exposure to PFOA to derangement of hepatocyte cell metabolism.

NGF and Endogenous Regeneration: From Embryology Toward Therapies.

The self-repair ability of tissues and organs in case of injury and disease is a fundamental biological mechanism and an important therapeutic target. The tissue plasticity and the presence of adult stem cell niches open a new path in the development of pharmacological and non-pharmacological treatments finalized to improve the intrinsic regeneration.In this context, nerve growth factor (NGF) is widely studied for its capability of driving endogenous regeneration of ectoderm-derived tissues, directly acting on the cell targets and through the regulation of the stem cell niches. In fact, this growth factor is very promising for its key role in the development and multiplicity of the cellular targets.In this chapter, we have traveled across the recent history of NGF pleiotropic role in ectodermal tissue generation and repair, from embryonic development to skin wound healing, axonal regrowth, and remyelination.The better understanding of both the biological mechanisms underlying regeneration and the physiological role of NGF in development and injury response will open new therapeutic strategies, driven by the potential applications of this growth factor as an agent for improving endogenous regeneration processes.

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway.

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRß), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.

Improved Functional Recovery in Rat Spinal Cord Injury Induced by a Drug Combination Administered with an Implantable Polymeric Delivery System.

Spinal cord injury (SCI) is an incurable condition, in which a cascade of cellular and molecular events triggered by inflammation and excitotoxicity impairs endogenous regeneration, namely remyelination and axonal outgrowth. We designed a treatment solution based on an implantable biomaterial (electrospun poly (l-lactic acid) [PLLA]) loaded with ibuprofen and triiodothyronine (T3) to counteract inflammation, thus improving endogenous regeneration. In vivo efficacy was tested by implanting the drug-loaded PLLA in the rat model of T8 contusion SCI. We observed the expected recovery of locomotion beginning on day 7. In PLLA-implanted rats (i.e., controls), the recovery stabilized at 21 days post-lesion (DPL), after which no further improvement was observed. On the contrary, in PLLA + ibuprofen (Ibu) + T3 (PLLA-Ibu-T3) rats a further recovery and a significant treatment effect were observed, also confirmed by the gait analysis on 49 DPL. Glutamate release at 24 h and 8 DPL was reduced in PLLA-Ibu-T3- compared to PLLA-implanted rats, such as the estimated lesion volume at 60 DPL. The myelin- and 200-neurofilament-positive area fraction was higher in PLLA-Ibu-T3-implanted rats, where the percentage of astrocytes was significantly reduced. The implant of a PLLA electrospun scaffold loaded with Ibu and T3 significantly improves the endogenous regeneration, leading to an improvement of functional locomotion outcome in the SCI.

A dynamic culture platform enhances the efficiency of the 3D HUVEC-based tube formation assay.

Cell-based in vitro biological models traditionally use monolayer cell cultures grown over plastic surfaces bathing in static media. Higher fidelity to a natural biological tissue is expected to result from growing the cells in a three-dimensional (3D) matrix. However, due to the decreased rate of diffusion inherent to increased distances within a tridimensional space, proper fluidic conditions are needed in this setting to better approximate a physiological environment. To this aim, we here propose a prototypal dynamic cell culture platform for the automatic medium replacement, via periodic perfusion flow, in a human umbilical vein endothelial cell (HUVECs) culture seeded in a Geltrex™ matrix. A state-of-the-art angiogenesis assay performed in these dynamic conditions showed sizable effects with respect to conventional static control cultures, with significantly enhanced pro-(dual antiplatelet therapy [DAPT]) and anti-(EDTA) angiogenic compound activity. In particular, dynamic culture conditions (a) enhance the 3D-organization of HUVECs into microtubule structure; (b) accelerate and improve endothelial tube formation by HUVECs in the presence of DAPT; (c) are able to completely revert the blocking effects of EDTA. These evidence emphasize the need of setting proper fluidic conditions for a better approximation of a physiological environment as an appropriate evolution of current cell culture paradigms.

Growth and Neurotrophic Factors in Embryonic Stem Cells.

In this chapter we illustrate protocols to investigate growth and neurotrophic factors in human and rodent (rat and mouse)-derived embryonic stem cells. The conventional two-dimensional cell monolayer system to grow embryonic stem cells is presented, focusing on the coating strategies also using extracellular matrix components. Then, different approaches for three-dimensional stem cell culture are presented, using hydrogels and scaffolds. Quantitative polymerase chain reaction, immunocytochemistry, immunoenzymatic ELISA assay, and multiparametric assays to quantify growth and neurotrophic factor production are presented.

Estrogen receptor ß-dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis.

Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17ß-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor ß (ERß), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERß. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERß to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.

Distinct gene expression profiles associated with Notch ligands Delta-like 4 and Jagged1 in plaque material from peripheral artery disease patients: a pilot study.

BACKGROUND: The lack of early diagnosis, progression markers and effective pharmacological treatment has dramatic unfavourable effects on clinical outcomes in patients with peripheral artery disease (PAD). Addressing these issues will require dissecting the molecular mechanisms underlying this disease. We sought to characterize the Notch signaling and atherosclerosis relevant markers in lesions from femoral arteries of symptomatic PAD patients. METHODS: Plaque material from the common femoral, superficial femoral or popliteal arteries of 20 patients was removed by directional atherectomy. RNA was obtained from 9 out of 20 samples and analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: We detected expression of Notch ligands Delta-like 4 (Dll4) and Jagged1 (Jag1), of Notch target genes Hes1, Hey1, Hey2, HeyL and of markers of plaque inflammation and stability such as vascular cell adhesion molecule 1 (VCAM1), smooth muscle 22 (SM22), cyclooxygenase 2 (COX2), Bcl2, CD68 and miRNAs 21-5p, 125a-5p, 126-5p,146-5p, 155-5p, 424-5p. We found an "inflamed plaque" gene expression profile characterized by high Dll4 associated to medium/high CD68, COX2, VCAM1, Hes1, miR126-5p, miR146a-5p, miR155-5p, miR424-5p and low Jag1, SM22, Bcl2, Hey2, HeyL, miR125a-5p (2/9 patients) and a "stable plaque" profile characterized by high Jag1 associated to medium/high Hey2, HeyL, SM22, Bcl2, miR125a and low Dll4, CD68, COX2, VCAM1, miR126-5p, miR146a-5p, miR155-5p, miR424-5p (3/9 patients). The remaining patients (4/9) showed a plaque profile with intermediate characteristics. CONCLUSIONS: This study reveals the existence of a gene signature associated to Notch activation by specific ligands that could be predictive of PAD progression.

Serum From Advanced Heart Failure Patients Promotes Angiogenic Sprouting and Affects the Notch Pathway in Human Endothelial Cells.

It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.

In vitro endothelial cell proliferation assay reveals distinct levels of proangiogenic cytokines characterizing sera of healthy subjects and of patients with heart failure.

Although myocardial angiogenesis is thought to play an important role in heart failure (HF), the involvement of circulating proinflammatory and proangiogenic cytokines in the pathogenesis and/or prognosis of HF has not been deeply investigated. By using a highly standardized proliferation assay with human endothelial cells, we first demonstrated that sera from older (mean age 52 ± 7.6 years; n = 46) healthy donors promoted endothelial cell proliferation to a significantly higher extent compared to sera obtained from younger healthy donors (mean age 29 ± 8.6 years; n = 20). The promotion of endothelial cell proliferation was accompanied by high serum levels of several proangiogenic cytokines. When we assessed endothelial cell proliferation in response to HF patients' sera, we observed that a subset of sera (n = 11) promoted cell proliferation to a significantly lesser extent compared to the majority of sera (n = 18). Also, in this case, the difference between the patient groups in the ability to induce endothelial cell proliferation correlated to significant (P < 0.05) differences in serum proangiogenic cytokine levels. Unexpectedly, HF patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis.

The role of notch in the cardiovascular system: potential adverse effects of investigational notch inhibitors.

Targeting the Notch pathway is a new promising therapeutic approach for cancer patients. Inhibition of Notch is effective in the oncology setting because it causes a reduction of highly proliferative tumor cells and it inhibits survival of cancer stem cells, which are considered responsible for tumor recurrence and metastasis. Additionally, since Delta-like ligand 4 (Dll4)-activated Notch signaling is a major modulator of angiogenesis, anti-Dll4 agents are being investigated to reduce vascularization of the tumor. Notch plays a major role in the heart during the development and, after birth, in response to cardiac damage. Therefore, agents used to inhibit Notch in the tumors (gamma secretase inhibitors and anti-Dll4 agents) could potentially affect myocardial repair. The past experience with trastuzumab and other tyrosine kinase inhibitors used for cancer therapy demonstrates that the possible cardiotoxicity of agents targeting shared pathways between cancer and heart and the vasculature should be considered. To date, Notch inhibition in cancer patients has resulted only in mild gastrointestinal toxicity. Little is known about the potential long-term cardiotoxicity associated to Notch inhibition in cancer patients. In this review, we will focus on mechanisms through which inhibition of Notch signaling could lead to cardiomyocytes and endothelial dysfunctions. These adverse effects could contrast with the benefits of therapeutic responses in cancer cells during times of increased cardiac stress and/or in the presence of cardiovascular risk factor.

17ß-estradiol enhances signalling mediated by VEGF-A-delta-like ligand 4-notch1 axis in human endothelial cells.

Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17ß-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.

The role of Notch pathway in cardiovascular diseases.

The recent increase in human lifespan, coupled with unhealthy diets and lifestyles have led to an unprecedented increase in cardiovascular diseases. Even in the presence of a wide range of therapeutic options with variable efficacy, mortality due to heart failure is still high and there is a need to identify new therapeutic targets. Genetic and in vitro studies have implicated the Notch signalling in the development and maintenance of the cardiovascular system through a direct effect on biological functions of vascular cells (endothelial and vascular smooth muscle cells) and cardiomyocytes. Notch signalling is also involved in the modulation of inflammation, which plays a major role in causing and exacerbating cardiovascular diseases. The Notch pathway could represent a new therapeutic target for the treatment of cardiovascular diseases.