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Purpose: The oncogene TCL1 plays a key role in the development of B chronic lymphocytic leukemia (B-CLL), but it is not known whether TCL1 could be modulated by therapeutic approaches.Experimental Design: B-CLL patient samples (n = 35) and B leukemic cell lines (EHEB, JVM2, JVM3, MEC1, MEC2, and BJAB) with different p53 status were exposed to Nutlin-3, a small-molecule inhibitor of the p53–MDM2 interaction. Modulations of the steady-state mRNA levels of TCL1 were analyzed by quantitative real-time PCR and Western blotting in both primary B-CLL samples and leukemic cell lines. In addition, transfection experiments with either p53 siRNA or with a TCL1 expression plasmid were carried out in the EHEB B-CLL cell line.Results: Upon ex vivo treatment with Nutlin-3, TCL1 was significantly (P < 0.05) decreased in 23 of 28 B-CLL p53wild-type. The functionality of the p53 pathway in the same leukemic cell samples was underscored by the concomitant ability of Nutlin-3 to significantly (P < 0.05) upregulate the p53 target gene MDM2 in the p53wild-type leukemic cells. The dependence of TCL1 downregulation by a functional p53 pathway was confirmed in a panel of B lymphoblastoid cell lines and by p53 knockdown experiments with p53 siRNA. The importance of TCL1 in promoting leukemic cell survival was underscored in transfection experiments, in which TCL1 overexpression significantly counteracted the Nutlin-3–mediated induction of apoptosis in EHEB.Conclusions: Our data indicate that the Nutlin-3 downregulates TCL1 mRNA and protein, which likely represents an important molecular determinant in the proapoptotic activity of Nutlin-3. Clin Cancer Res; 17(17); 5649–55. ©2011 AACR.

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Purpose: Because the nongenotoxic inhibitor of the p53/MDM2 interactions Nutlin-3 has shown promising in vitro therapeutic activity against a variety of p53wild-type cancer cells, in this study we evaluated an innovative strategy able to specifically target Nutlin-3 toward CD20+ malignant cells.Experimental Design: The cytotoxic effects of Nutlin-3 encapsulated into poly(lactide-co-glycolide) nanoparticles (NP-Nut) and into rituximab (anti-CD20 antibody)-engineered NP (NP-Rt-Nut) as well as of NPs engineered with rituximab alone (NP-Rt) were initially analyzed in vitro in JVM-2 B-leukemic cells, by assessing both the functional activation of the p53 pathway (by Nutlin-3) and/or the activation of the complement cascade (by rituximab). Moreover, the potential therapeutic efficacy of NP-Nut, NP-Rt, and NP-Rt-Nut were comparatively assessed in vivo in CD20+ JVM-2 leukemic xenograft SCID mice.Results: Functional in vitro assays showed that NP-Nut and NP-Rt-Nut exhibited a comparable ability to activate the p53 pathway in the p53wild-type JVM-2 leukemic cells. On the other hand, NP-Rt and NP-Rt-Nut, but not NP nor NP-Nut, were able to promote activation of the complement cascade. Of note, the in vivo intratumoral injection in JVM-2 B-leukemic/xenograft mice showed that NP-Rt-Nut displayed the maximal therapeutic activity promoting a survival rate significantly higher not only with respect to control animals, treated either with vehicle or with empty NP, but also with respect to animals treated with NP-Nut or NP-Rt.Conclusions: Our data show for the first time the potential antileukemic activity of rituximab-engineered Nutlin-3–loaded NPs in xenograft SCID mice. Clin Cancer Res; 19(14); 3871–80. ©2013 AACR.

Purpose: To elucidate new molecular mechanisms able to downregulate the mRNA levels of key oncogenes, such as B-Myb and E2F1, in a therapeutic perspective.Experimental Design:B-Myb and E2F1 mRNA levels were evaluated in primary B chronic lymphocytic leukemia (B-CLL, n = 10) and acute myeloid leukemia (AML, n = 5) patient cells, in a variety of p53wild-type and p53mutated/deleted leukemic cell lines, as well as in primary endothelial cells and fibroblasts. Knockdown experiments with siRNA for p53 and E2F1 and overexpression experiments with miR34a were conducted to elucidate the role of these pathways in promoting B-Myb downregulation.Results:In vitro exposure to Nutlin-3, a nongenotoxic activator of p53, variably downregulated the expression of B-Myb in primary leukemic cells and in p53wild-type myeloid (OCI, MOLM) and lymphoblastoid (SKW6.4, EHEB) but not in p53mutated (NB4, BJAB, MAVER) or p53deleted (HL-60) leukemic cell lines. The transcriptional repression of B-Myb was also observed in primary normal endothelial cells and fibroblasts. B-Myb downregulation played a critical role in the cell-cycle block in G1 phase induced by Nutlin-3, as shown by transfection experiments with specific siRNA. Moreover, we have provided experimental evidence suggesting that miR-34a is a central mediator in the repression of B-Myb both directly and through E2F1.Conclusions: Owing to the role of B-Myb and E2F1 transcription factors in controlling cell-cycle progression of leukemic cells, the downregulation of these oncogenes by miR-34a suggests the usefulness of therapeutic approaches aimed to modulate the levels of miR-34a. Clin Cancer Res; 17(9); 2712–24. ©2011 AACR.

Purpose: To analyze the effect of the combination of Dasatinib, a multikinase inhibitor, plus Nutlin-3, a nongenotoxic activator of the p53 pathway, in primary B chronic lymphocytic leukemia (B-CLL) patient samples and B leukemic cell line models.Experimental Design: The induction of cytotoxicity was evaluated in both primary B-CLL cell samples (n = 20) and in p53wild-type (EHEB, JVM-2) and p53deleted/mutated (MEC-2, BJAB) B leukemic cell lines. The role of Akt in modulating leukemic cell survival/apoptosis in response to Dasatinib or Dasatinib + Nutlin-3 was documented by functional experiments carried out using specific pharmacological inhibitors and by overexpression of membrane-targeted constitutively active form of Akt.Results: The combination of Dasatinib + Nutlin-3 exhibited a synergistic cytotoxicity in the majority (19 out of 20) of B-CLL samples, including patients carrying 17p- (n = 4), and in both p53wild-type and p53deleted/mutated B leukemic cell lines. At the molecular level, Dasatinib significantly counteracted the Nutlin-3-mediated induction of the p53 transcriptional targets MDM2 and p21 observed in p53wild-type leukemic cells. Conversely, Nutlin-3 did not interfere with the ability of Dasatinib to decrease the phosphorylation levels of ERK1/2, p38/MAPK, and Akt in both p53wild-type and p53deleted/mutated B leukemic cell lines. A critical role of Akt downregulation in mediating the antileukemic activity of Dasatinib and Dasatinib + Nutlin-3 was demonstrated in experiments carried out by specifically modulating the Akt pathway.Conclusions: These findings suggest that Dasatinib + Nutlin-3 might represent an innovative therapeutic combination for both p53wild-type and p53deleted/mutated B-CLL. Clin Cancer Res; 17(4); 1–9. ©2010 AACR.

PDF file - 121K, Supplementary Figure 2. Comparative in vitro assessment of p53 activation.

PDF file - 83K, Supplementary Table 1. Physicochemical properties and technological characterization of the NP-Nut, NP-Rt and NP-Rt-Nut preparations.

PDF file - 307K, Supplementary Figure 1. Evidence of NP entry into cells.

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PDF file - 70K, Supplementary Figure 3. Analysis of survival of JVM-2 xenograft mice.

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Supplementary Table S1.

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients, however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

Cultivation of asparagus (Asparagus officinalis L., Asp) for food and medicinal use has taken place since the early Roman Empire. Today, Asp represents a worldwide diffuse perennial crop. Lower portions of the spears represent a food industry waste product that can be used to extract bioactive molecules. In this study, aqueous extracts derived from the non-edible portion of the plant (hard stem) were prepared and characterized for chemical content. Furthermore, the biocompatibility and bioactivity of Asp aqueous extracts were assessed in vitro on normal fibroblasts and on breast cancer cell lines. Results showed no interference with fibroblast viability, while a remarkable cytostatic concentration-dependent activity, with significant G1/S cell cycle arrest, was specifically observed in breast cancer cells without apoptosis induction. Asp extracts were also shown to significantly inhibit cell migration. Further analyses showed that Asp extracts were characterized by specific pro-oxidant activity against tumoral cells, and, importantly, that their combination with menadione resulted in a significant enhancement of oxidants production with respect to menadione alone in breast cancer cells but not in normal cells. This selectivity of action on tumoral cells, together with the easiness of their preparation, makes the aqueous Asp extracts very attractive for further investigation in breast cancer research, particularly to investigate their role as possible co-adjuvant agents of clinical drug therapies.

Ligands and receptors of the tumor necrosis factor (TNF) superfamily regulate immune responses and homeostatic functions with potential diagnostic and therapeutic implications. Kidney disease represents a global public health problem, whose prevalence is rising worldwide, due to the aging of the population and the increasing prevalence of diabetes, hypertension, obesity, and immune disorders. In addition, chronic kidney disease is an independent risk factor for the development of cardiovascular disease, which further increases kidney-related morbidity and mortality. Recently, it has been shown that some TNF superfamily members are actively implicated in renal pathophysiology. These members include TNF-related apoptosis-inducing ligand (TRAIL), its decoy receptor osteoprotegerin (OPG), and TNF-like weaker inducer of apoptosis (TWEAK). All of them have shown the ability to activate crucial pathways involved in kidney disease development and progression (e.g. canonical and non-canonical pathways of the transcription factor nuclear factor-kappa B), as well as the ability to regulate cell proliferation, differentiation, apoptosis, necrosis, inflammation, angiogenesis, and fibrosis with double-edged effects depending on the type and stage of kidney injury. Here we will review the actions of TRAIL, OPG, and TWEAK on diabetic and non-diabetic kidney disease, in order to provide insights into their full clinical potential as biomarkers and/or therapeutic options against kidney disease.

// Rebecca Voltan 1, * , Erika Rimondi 2, * , Elisabetta Melloni 1 , Gian Matteo Rigolin 3 , Fabio Casciano 1 , Maria Vittoria Arcidiacono 1 , Claudio Celeghini 2 , Antonio Cuneo 3 , Giorgio Zauli 1 , Paola Secchiero 1 1 Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy 2 Department of Life Sciences, University of Trieste, Trieste, Italy 3 Department of Medical Sciences, Section of Hematology, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Paola Secchiero, email: paola.secchiero@unife.it Keywords: B leukemic cells, Ibrutinib, MDM-2 inhibitors, apoptosis, combination therapy Received: July 20, 2016 Accepted: September 09, 2016 Published: September 20, 2016 ABSTRACT Objective: The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. Methods: The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Results: Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Conclusions: Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.

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.

Progressive breakthroughs in nanomedicine have been instrumental for the clinical translation of actively targeted drug-delivery approaches. Besides storing large payloads of drugs within the nanoparticle core, the conjugation of targeting moieties confers specific targeting ability to the nanoplatforms. In this respect, clinical results suggest that actively targeted nanocarriers can exhibit an overall improved antitumor efficacy, minimizing off-target toxicity.This review article summarizes the advances in active targeting of nanocarriers to cancer cells. Specifically, we discuss the various types of nanocarriers, describe the receptors that are frequently overexpressed in solid tumors, and discuss how this approach can be used to improve clinical outcomes. We particularly focus on ongoing clinical trials of actively targeted nanoparticles that are yet to be clinically approved.Further investment in active targeting will likely pose clinical benefits. We envisage a future requiring the use of longitudinal measures in the clinical setting to profile the patients that are likely to benefit from actively targeted nanocarriers. At the preclinical stage, a complete picture of intratumoral barriers combined with a quantitative approach of the intratumoral fate of nanomaterials will be instrumental in defining more effective strategies to improve their clinical translation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Snail mucus is a mixture of active substances commonly thought to have healthy properties for the treatment of skin disorders. Although snail mucus is an ingredient of several cosmetic and para-pharmaceutic products, a comprehensive characterization of chemical composition and biological effects is still missing. Crude purified extracts from Helix aspersa muller mucus (HelixComplex) were prepared and, after chemical characterization, tested on in vitro experimental models. Differently from what expected, HelixComplex was characterized by the presence of small amounts of glycolic acid and allantoin. By using different in vitro assays on fibroblast cultures, we found that HelixComplex lacked of cytotoxicity, protected cells from apoptosis (p

The TNF-related apoptosis inducing ligand TRAIL is a member of the TNF superfamily that has been firstly studied and evaluated for its anti-cancer activity, and the insights into its biology have already led to the identification of several TRAIL-based anticancer strategies with strong clinical therapeutic potentials. Nonetheless, the TRAIL system is far more complex and it can lead to a wider range of biological effects other than the ability of inducing apoptosis in cancer cells. By virtue of the different receptors and the different signalling pathways involved, TRAIL plays indeed a role in the regulation of different processes of the innate and adaptive immune system and this feature makes it an intriguing molecule under consideration in the development/progression/treatment of several immunological disorders. In this context, central nervous system represents a peculiar anatomic site where, despite its "status" of immune-privileged site, both innate and adaptive inflammatory responses occur and are involved in several pathological conditions. A number of studies have evaluated the role of TRAIL and of TRAIL-related pathways as pro-inflammatory or protective stimuli, depending on the specific pathological condition, confirming a twofold nature of this molecule. In this light, the aim of this review is to summarize the main preclinical evidences of the potential/involvement of TRAIL molecule and TRAIL pathways for the treatment of central nervous system disorders and the key suggestions coming from their assessment in preclinical models as proof of concept for future clinical studies.

Background Multiple sclerosis (MS) is an inflammatory, demyelinating and degenerative disorder of the central nervous system (CNS). Several observations support interactions between vascular and neurodegenerative mechanisms in multiple sclerosis (MS). To investigate the contribution of the extracranial venous compartment, we analysed expression profiles of internal jugular vein (IJV), which drains blood from CNS, and related plasma protein levels. Methods We studied a group of MS patients (n = 19), screened by echo-color Doppler and magnetic resonance venography, who underwent surgical reconstruction of IJV for chronic cerebrospinal venous insufficiency (CCSVI). Microarray-based transcriptome analysis was conducted on specimens of IJV wall from MS patients and from subjects undergoing carotid endarterectomy, as controls. Protein levels were determined by multiplex assay in: i) jugular and peripheral plasma from 17 MS/CCSVI patients; ii) peripheral plasma from 60 progressive MS patients, after repeated sampling and iii) healthy individuals. Results Of the differentially expressed genes (≥ 2 fold-change, multiple testing correction, P

Background: Chronic kidney disease (CKD) patients experience a high risk of cardiovascular disease (CV); however, the factors involved in CV-related morbidity and mortality in these patients have not been fully defined. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a cytokine, which exhibits pleiotropic activities on endothelial, vascular smooth muscle and inflammatory cells, with relevant effects on atheromatous plaque formation. On this basis, the present study aims to investigate the role of TRAIL in atheromatosis progression in CKD patients. Methods: Circulating TRAIL levels were measured in 378 CKD patients belonging to the Spanish National Observatory of Atherosclerosis in Nephrology (NEFRONA) study. All patients were free of previous CV events. Carotid and femoral B-mode ultrasound was performed to detect the presence of plaque at baseline and after 24 months of follow-up. Results: The lowest levels of TRAIL at baseline were significantly (p

The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53 wild-type JVM-2 cells, but not in p53 mutated BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.

// Paola Secchiero 1, * , Rebecca Voltan 1, * , Erika Rimondi 1 , Elisabetta Melloni 1 , Emmanouil Athanasakis 2 , Veronica Tisato 1 , Stefania Gallo 1 , Gian Matteo Rigolin 3 and Giorgio Zauli 1 1 Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy 2 Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy 3 Department of Medical Sciences, Section of Hematology, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Giorgio Zauli, email: giorgio.zauli@unife.it Paola Secchiero, email: paola.secchiero@unife.it Keywords: B-leukemic cells, Ibrutinib, γ-secretase inhibitors, NOTCH1, combination therapy Received: May 05, 2017 Accepted: June 20, 2017 Published: July 22, 2017 ABSTRACT Ibrutinib blocks B-cell receptor signaling and interferes with leukemic cell-to-microenvironment interactions. Ibrutinib plays a key role in the management of B-CLL and is recommended for first line treatment of high-risk CLL patients with 17p deletion. Therefore, elucidating the factors governing sensitivity/resistance to Ibrutinib represents a relevant issue. For this purpose, in 3 B-CLL patient samples harboring functional TP53 mutations, the frequency of the mutated clones was monitored during in vivo Ibrutinib therapy, revealing a progressive decline of the frequency of TP53 mut clones during 12 months of treatment. In parallel, the anti-leukemic activity of Ibrutinib was assessed in vitro on B-CLL patient cell cultures in combination with γ-secretase inhibitors (GSI). In the in vitro assays, the combination of Ibrutinib+GSI exhibited enhanced cytotoxicity on B-CLL cells also in the presence of stroma and it was coupled to the down-regulation of the stroma-activated NOTCH1 and c-MYC pathways. Moreover, the combined treatment was effective in reducing CXCR4 expression and functions. Therefore, the ability of GSI to enhance the Ibrutinib anti-leukemic activity in B-CLL cells, by down-regulating the NOTCH1 and c-MYC pathways, warrants further experimentation for its potential therapeutic applications.

Background: Despite in vitro studies demonstrated expression of cytokines when endothelial cells are stressed with oscillatory flow, in vivo evaluations are still lacking.The aim of this blinded case-control study was to find correlations between altered venous hemodynamics and induced endothelial signaling, in an in vivo setting. Methods: Fifty-four chronic venous disease (CVD) patients (C24EpAsPr) who underwent varicose veins surgery were included into the study. Into the venous segments that subsequently were going to be surgically ablated, we assessed the followings by echo-color-Doppler (ECD): peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index (RI), and reflux time (RT). The vein samples were sent to the lab, where both an electronic microscopic evaluation and a vascular endothelial cells (VEC) cultivation, with released cytokines quantification and endothelial migration and proliferation characterization, were performed. Five saphenous vein samples were used as controls. Results: The hemodynamic assessment provided the following: PSV (29 + 13 cm/s), EDV (-8 + 6 cm/s), RI (1.31 + 0.19), RT (2.66 + 0.46 s). The microscopic evaluation demonstrated in CVD a derangement on the endothelium layer directly correlating with the CEAP class severity. An erythrocyte progressive sticking was observed as a possible consequence of increased adhesion molecules expression. Twenty-one VEC samples came out from the purification process. In CVD, we assessed a significant increased level of NF-kB, a nuclear transcriptional factor linked with expression of adhesion molecules and several pro-inflammatory cytokines. OPG and VEGF were assessed to be significantly higher both in the supernatants and in the serum assays. We investigated possible correlation between hemodynamic parameters and cytokines assessed in the VEC. The spontaneous and TNFa-induced cytokines release quantification highlighted statistical significant correlations with several hemodynamic parameters. The most significant one was between PDGF and RT, both in basal (r 1⁄4 0.5446, P 1⁄4 .0150) and TNFa-induced (r 1⁄4 0.5448, P 1⁄4 .0150) conditions. While the VEC migration capacity was lower in CVD than in controls, its proliferation trend was higher (P < .05). Conclusions: To our knowledge this is the first study correlating hemodynamic physic parameters with biochemical endothelial signaling, in vivo. As in vitro demonstrated, PDGF is one of the main actors in smooth muscle cell remodeling, reactive oxygen species production, and venous wall derangement. Our data confirm this action in vivo, adding the correlation with the physic forces expressed by the refluxing flow. For the first time, several cytokines involved in biochemical signaling demonstrates to be modulated by hemodynamic parameters. These preliminary results could pave the way for a deeper comprehension of the translation path, which, from the oscillatory flow, leads to the morphological and biochemical alterations detected in the CVD endothelial cells.