Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Transcription factors in fibroblast plasticity and CAF heterogeneity.

In recent years, research focused on the multifaceted landscape and functions of cancer-associated fibroblasts (CAFs) aimed to reveal their heterogeneity and identify commonalities across diverse tumors for more effective therapeutic targeting of pro-tumoral stromal microenvironment. However, a unified functional categorization of CAF subsets remains elusive, posing challenges for the development of targeted CAF therapies in clinical settings.The CAF phenotype arises from a complex interplay of signals within the tumor microenvironment, where transcription factors serve as central mediators of various cellular pathways. Recent advances in single-cell RNA sequencing technology have emphasized the role of transcription factors in the conversion of normal fibroblasts to distinct CAF subtypes across various cancer types.This review provides a comprehensive overview of the specific roles of transcription factor networks in shaping CAF heterogeneity, plasticity, and functionality. Beginning with their influence on fibroblast homeostasis and reprogramming during wound healing and fibrosis, it delves into the emerging insights into transcription factor regulatory networks. Understanding these mechanisms not only enables a more precise characterization of CAF subsets but also sheds light on the early regulatory processes governing CAF heterogeneity and functionality. Ultimately, this knowledge may unveil novel therapeutic targets for cancer treatment, addressing the existing challenges of stromal-targeted therapies.

The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo.

Nucleophosmin (NPM), a nucleolar multifunctional phosphoprotein, acts as a stress sensor in different cell types. NPM can be actively secreted by inflammatory cells, however its biology on endothelium remains unexplored. In this study, we show for the first time that NPM is secreted by human vein endothelial cells (HUVEC) in the early response to serum deprivation and that NPM acts as a pro-inflammatory and angiogenic molecule both in vitro and in vivo. Accordingly, 24 h of serum starvation condition induced NPM relocalization from the nucleus to cytoplasm. Interestingly, NPM was increasingly excreted in HUVEC-derived conditioned media in a time dependent fashion upon stress conditions up to 24 h. The secretion of NPM was unrelated to cell necrosis within 24 h. The treatment with exogenous and recombinant NPM (rNPM) enhanced migration as well as the Intercellular Adhesion Molecule 1 (ICAM-1) but not Vascular cell adhesion protein 1 (VCAM-1) expression and it did not affect cell proliferation. Notably, in vitro tube formation by Matrigel assay was significantly increased in HUVEC treated with rNPM compared to controls. This result was confirmed by the in vivo injection of Matrigel plug assay upon stimulation with rNPM, displaying significant enhanced number of functional capillaries in the plugs. The stimulation with rNPM in HUVEC was also associated to the increased expression of master genes regulating angiogenesis and migration, including Vascular Endothelial Growth Factor-A (VEGF-A), Hepatocyte Growth Factor (HGF), Stromal derived factor-1 (SDF-1), Fibroblast growth factor-2 (FGF-2), Platelet Derived Growth Factor-B (PDGF-B), and Matrix metallopeptidase 9 (MMP9). Our study demonstrates for the first time that NPM is physiologically secreted by somatic cells under stress condition and in the absence of cell necrosis. The analysis of the biological effects induced by NPM mainly related to a pro-angiogenic and inflammatory activity might suggest an important autocrine/paracrine role for NPM in the regulation of both phenomena.

Actin Cytoskeleton and Regulation of TGFß Signaling: Exploring Their Links.

Human tissues, to maintain their architecture and function, respond to injuries by activating intricate biochemical and physical mechanisms that regulates intercellular communication crucial in maintaining tissue homeostasis. Coordination of the communication occurs through the activity of different actin cytoskeletal regulators, physically connected to extracellular matrix through integrins, generating a platform of biochemical and biomechanical signaling that is deregulated in cancer. Among the major pathways, a controller of cellular functions is the cytokine transforming growth factor ß (TGFß), which remains a complex and central signaling network still to be interpreted and explained in cancer progression. Here, we discuss the link between actin dynamics and TGFß signaling with the aim of exploring their aberrant interaction in cancer.

The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation.

The dynamic interplay between cancer cells and cancer-associated fibroblasts (CAFs) is regulated by multiple signaling pathways, which can lead to cancer progression and therapy resistance. We have previously demonstrated that hMENA, a member of the actin regulatory protein of Ena/VASP family, and its tissue-specific isoforms influence a number of intracellular signaling pathways related to cancer progression. Here, we report a novel function of hMENA/hMENAΔv6 isoforms in tumor-promoting CAFs and in the modulation of pro-tumoral cancer cell/CAF crosstalk via GAS6/AXL axis regulation. LC-MS/MS proteomic analysis reveals that CAFs that overexpress hMENAΔv6 secrete the AXL ligand GAS6, favoring the invasiveness of AXL-expressing pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) cells. Reciprocally, hMENA/hMENAΔv6 regulates AXL expression in tumor cells, thus sustaining GAS6-AXL axis, reported as crucial in EMT, immune evasion, and drug resistance. Clinically, we found that a high hMENA/GAS6/AXL gene expression signature is associated with a poor prognosis in PDAC and NSCLC. We propose that hMENA contributes to cancer progression through paracrine tumor-stroma crosstalk, with far-reaching prognostic and therapeutic implications for NSCLC and PDAC.

The pattern of hMENA isoforms is regulated by TGF-ß1 in pancreatic cancer and may predict patient outcome.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease in need of prognostic markers to address therapeutic choices. We have previously shown that alternative splicing of the actin regulator, hMENA, generates hMENA11a, and hMENAΔv6 isoforms with opposite roles in cell invasion. We examined the expression pattern of hMENA isoforms by immunohistochemistry, using anti-pan hMENA and specific anti-hMENA11a antibodies, in 285 PDACs, 15 PanINs, 10 pancreatitis, and normal pancreas. Pan hMENA immunostaining, absent in normal pancreas and low-grade PanINs, was weak in PanIN-3 and had higher levels in virtually all PDACs with 64% of cases showing strong staining. Conversely, the anti-invasive hMENA11a isoform only showed strong staining in 26% of PDAC. The absence of hMENA11a in a subset (34%) of pan-hMENA-positive tumors significantly correlated with poor outcome. The functional effects of hMENA isoforms were analyzed by loss and gain of function experiments in TGF-ß1-treated PDAC cell lines. hMENA11a knock-down in PDAC cell lines affected cell-cell adhesion but not invasion. TGF-ß1 cooperated with ß-catenin signaling to upregulate hMENA and hMENAΔv6 expression but not hMENA11a In the absence of hMENA11a, the hMENA/hMENAΔv6 up-regulation is crucial for SMAD2-mediated TGF-ß1 signaling and TGF-ß1-induced EMT. Since the hMENA isoform expression pattern correlates with patient outcome, the data suggest that hMENA splicing and related pathways are novel key players in pancreatic tumor microenvironment and may represent promising targets for the development of new prognostic and therapeutic tools in PDAC.

Transcriptional control of skin reepithelialization.

The wound healing process is characterized by a series of overlapping phases, such as coagulation, inflammation, reepithelialization/granulation tissue generation and remodeling. It is important to obtain a deeper insight into the cutaneous wound repair mechanisms, in order to develop novel pharmacological tools for the treatment of chronic non-healing ulcers which are a frequent and high morbidity complication of diabetes, ischaemia, venous insufficiency, and other local or systemic factors. Several transcription factors, many of which belong to gene families, are known to play a role in cutaneous wound repair through the orchestration of cellular responses which promote the reconstitution of skin integrity. The aim of this review is to provide an updated analysis of the transcription factor role in the reepithelialization process, in the context of skin wound repair.

Ex vivo acidic preconditioning enhances bone marrow ckit+ cell therapeutic potential via increased CXCR4 expression.

AIMS: The chemokine receptor CXCR4 modulates endothelial progenitor cell migration, homing, and differentiation, and plays a key role in cardiovascular regeneration. Here we examined the effect of ex vivo acidic preconditioning (AP) on CXCR4 expression and on the regenerative potential of mouse bone marrow (BM) ckit(+) cells. METHODS AND RESULTS: Acidic preconditioning was achieved by exposing BM ckit(+) cells to hypercarbic acidosis (pH 7.0) for 24 h; control cells were kept at pH 7.4. Acidic preconditioning enhanced CXCR4 and stromal cell-derived factor 1 (SDF-1) mRNA levels, as well as CXCR4 phosphorylation. Acidic preconditioning ability to modulate CXCR4 expression depended on cytosolic calcium [Ca(2+)]i mobilization and on nitric oxide (NO), as determined by [Ca(2+)]i buffering with BAPTA, and by treatment with the NO donor (DETA/NO) and the NO synthase inhibitor (L-NAME). Further, AP increased SDF-1-driven chemotaxis, transendothelial migration, and differentiation toward the endothelial lineage in vitro. In a mouse model of hindlimb ischaemia, control and AP ckit(+) cells were transplanted into the ischaemic muscle; AP cells accelerated blood flow recovery, increased capillary, and arteriole number as well as the number of regenerating muscle fibres vs. control. These effects were abolished by treating AP cells with L-NAME. CONCLUSION: Acidic preconditioning represents a novel strategy to enhance BM ckit(+) cell therapeutic potential via NO-dependent increase in CXCR4 expression.

C/EBPγ regulates wound repair and EGF receptor signaling.

We aimed at identifying novel regulators of skin wound healing (WH), in an epidermal scratch WH assay, by a small interfering RNA (siRNA) silencing approach. Several transcription factors have been previously reported to affect wound repair. We here show that gene silencing of the transcription factor CAAT enhancer-binding protein γ (C/EBPγ), STAT3, REL, RELA, RELB, SP1, and NFkB impaired WH in vitro, in keratinocytes, whereas E2F and CREBBP silencing accelerated the WH process. We further characterized C/EBPγ, as its silencing yielded the maximal impairment (52.2 ± 12.5%) of scratch wounding (SW). We found that C/EBPγ silencing inhibited both EGF- and serum-induced keratinocyte migration, whereas C/EBPγ overexpression enhanced cell migration to EGF and to serum via the EGFR. Further, C/EBPγ silencing impaired scratch-induced Y1068 and Y1173 EGFR phosphorylation, as well as Y118 paxillin phosphorylation, key molecules regulating cell migration and epidermal WH. Moreover, C/EBPγ levels were induced in keratinocytes, following both SW and EGF stimulation. C/EBPγ siRNA silencing in vivo impaired WH at 3, 5, 7, and 14 days following excisional wounding in mice inhibited both re-epithelialization and granulation tissue formation, and induced a decrease of arteriole number. In conclusion, we here report that C/EBPγ positively regulates wound repair both in vitro and in vivo, at least in part, by affecting EGFR signaling.

Increase of plasma IL-9 and decrease of plasma IL-5, IL-7, and IFN-γ in patients with chronic heart failure.

BACKGROUND: Several cytokines are associated with the development and/or progression of chronic heart failure (CHF). Our aim was to look more closely at the cytokine networks involved in CHF, and to assess whether disease etiology affects cytokine expression. The study population was comprised of a) 69 patients with stable CHF, New York Heart Association (NYHA) II/IV classes, secondary to ischaemic (ICM) and non ischaemic dilated (NIDCM) cardiomyopathy and b) 16 control subjects. We analyzed and compared the plasma levels of 27 pro- and anti-inflammatory mediators, in the study population and assessed for any possible correlation with echocardiographic parameters and disease duration. METHODS: 27 cytokines and growth factors were analyzed in the plasma of ICM- (n = 42) and NIDCM (n = 27) NYHA class II-IV patients vs age- and gender-matched controls (n = 16) by a beadbased multiplex immunoassay. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test for multiple comparison. RESULTS: Macrophage inflammatory protein (MIP)-1ß, Vascular endothelial growth factor (VEGF), interleukin (IL)-9, Monocyte chemotactic protein (MCP)-1, and IL-8 plasma levels were increased in both ICM and NIDCM groups vs controls. In contrast, IL-7, IL-5, and Interferon (IFN)-γ were decreased in both ICM and NIDCM groups as compared to controls. Plasma IL-6 and IL-1 ß were increased in ICM and decreased in NIDCM, vs controls, respectively.IL-9 levels inversely correlated, in ICM patients, with left ventricular ejection fraction (LVEF) while IL-5 plasma levels inversely correlated with disease duration, in NYHA III/IV ICM patients.This is the first time that both an increase of plasma IL-9, and a decrease of plasma IL-5, IL-7 and IFN-γ have been reported in ICM as well as in NIDCM groups, vs controls. Interestingly, such cytokines are part of a network of genes whose expression levels change during chronic heart failure. The altered expression levels of MIP-1 ß, VEGF, MCP-1, IL-1 ß, IL-6, and IL-8, found in this study, are in keeping with previous reports. CONCLUSIONS: The increase of plasma IL-9, and the decrease of plasma IL-5, IL-7 and IFN-γ in ICM as well as in NIDCM groups vs controls may contribute to get further insights into the inflammatory pathways involved in CHF.

Induction of myogenic differentiation by SDF-1 via CXCR4 and CXCR7 receptors.

The stromal cell-derived factor (SDF)-1/CXC receptor 4 (CXCR4) axis has been shown to play a role in skeletal muscle development, but its contribution to postnatal myogenesis and the role of the alternate SDF-1 receptor, CXC receptor 7 (CXCR7), are poorly characterized. Western blot analysis and real-time polymerase chain reaction (PCR) were performed to evaluate in vitro the effect of SDF-1 and CXCR4 and CXCR7 inhibition on myogenic differentiation. Proliferating myoblasts express CXCR4, CXCR7, and SDF-1; during myogenic differentiation, CXCR4 and CXCR7 levels are downregulated, and SDF-1 release is decreased. SDF-1 anticipates myosin heavy chain accumulation and myotube formation in both C2C12 myoblasts and satellite cells. Interestingly, inhibition of CXCR4 and CXCR7 signaling, either by drugs or RNA interfererence, blocks myogenic differentiation. Further, the CXCR4 antagonist, 4F-benzoyl-TN14003, inhibits myoblast cell cycle withdrawal and decreases the retinoblastoma gene (pRb) product accumulation in its hypophosphorylated form. Our experiments demonstrate that SDF-1 regulates myogenic differentiation via both CXCR4 and CXCR7 chemokine receptors.

Role of HIF-1alpha in proton-mediated CXCR4 down-regulation in endothelial cells.

AIMS: Acidification is associated with a variety of pathological and physiological conditions. In the present study, we aimed at investigating whether acidic pH may regulate endothelial cell (EC) functions via the chemokine receptor CXCR4, a key modulator of EC biological activities. METHODS AND RESULTS: Exposure of ECs to acidic pH reversibly inhibited mRNA and protein CXCR4 expression, CXCL12/stromal cell-derived factor (SDF)-1-driven EC chemotaxis in vitro, and CXCR4 expression and activation in vivo in a mouse model. Further, CXCR4 signalling impaired acidosis-induced rescue from apoptosis in ECs. The inhibition of CXCR4 expression occurred transcriptionally and was hypoxia-inducible factor (HIF)-1alpha-dependent as demonstrated by both HIF-1alpha and HIF-1alpha dominant negative overexpression, by HIF-1alpha silencing, and by targeted mutation of the -29 to -25 hypoxia response element (HRE) in the -357/-59 CXCR4 promoter fragment. Moreover, chromatin immunoprecipitation (ChIP) analysis showed endogenous HIF-1alpha binding to the CXCR4 promoter that was enhanced by acidification. CONCLUSION: The results of the present study identify CXCR4 as a key player in the EC response to acidic pH and show, for the first time, that HRE may function not only as an effector of hypoxia, but also as an acidosis response element, and raise the possibility that this may constitute a more general mechanism of transcriptional regulation at acidic pH.

Gene expression profiles in peripheral blood mononuclear cells of chronic heart failure patients.

The present study was aimed at identifying chronic heart failure (CHF) biomarkers from peripheral blood mononuclear cells (PBMCs) in patients with ischemic (ICM) and nonischemic dilated (NIDCM) cardiomyopathy. PBMC gene expression profiling was performed by Affymetrix in two patient groups, 1) ICM (n = 12) and 2) NIDCM (n = 12) New York Heart Association (NYHA) III/IV CHF patients, vs. 3) age- and sex-matched control subjects (n = 12). Extracted RNAs were then pooled and hybridized to a total of 11 microarrays. Gene ontology (GO) analysis separated gene profiling into functional classes. Prediction analysis of microarrays (PAM) and significance analysis of microarrays (SAM) were utilized in order to identify a molecular signature. Candidate markers were validated by quantitative real-time polymerase chain reaction. We identified a gene expression profiling that distinguished between CHF patients and control subjects. Interestingly, among the set of genes constituting the signature, chemokine receptor (CCR2, CX(3)CR1) and early growth response (EGR1, 2, 3) family members were found to be upregulated in CHF patients vs. control subjects and to be part of a gene network. Such findings were strengthened by the analysis of an additional 26 CHF patients (n = 14 ICM and n = 12 NIDCM), which yielded similar results. The present study represents the first large-scale gene expression analysis of CHF patient PBMCs that identified a molecular signature of CHF and putative biomarkers of CHF, i.e., chemokine receptor and EGR family members. Furthermore, EGR1 expression levels can discriminate between ICM and NIDCM CHF patients.

MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3.

MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation.

Role of rat alpha adducin in angiogenesis: null effect of the F316Y polymorphism.

OBJECTIVE: Rat alpha adducin point mutation (F316Y) has been associated with primary systemic arterial hypertension. As microcirculatory abnormalities are present in most forms of hypertension, the aim of the present study was to investigate whether rat alpha adducin may regulate endothelial cell (EC) functions in vitro and in vivo. METHODS AND RESULTS: The overexpression of rat wild type alpha adducin (WT-Add1) in ECs induced capillary-like structure development in Matrigel in vitro and enhanced capillary formation in Matrigel implants in vivo in CD1 mice. In contrast, the overexpression of the mutated form (MUT-Add1) of rat alpha adducin had a Null effect in vitro and lacked any significant activity in vivo. Further, adenovirus-mediated rat WT-Add1 but not MUT-Add1 gene transfer to murine ischemic hindlimb enhanced capillary formation in skeletal muscles. Gene profiling of human umbilical vein endothelial cells overexpressing alpha adducin was performed in order to identify putative effector molecules of alpha adducin-mediated activities on ECs. Interestingly, among a number of genes involved in angiogenesis regulation, retinoic acid-induced protein (RAI17) was found to be upregulated in WT-Add1 vs MUT-Add1 overexpressing cells, possibly representing a key molecule/axis for the functional Add1-induced effect. CONCLUSIONS: Rat WT alpha adducin enhanced EC functions both in vitro and in vivo. The expression of the F316Y variant, associated with the hypertensive phenotype, had a Null effect and might contribute to endothelial rarefaction/dysfunction in hypertension. RAI17 was found to be a putative effector molecule differentially regulated by the overexpression of the two forms of Add1 in endothelial cells.

Laminar shear stress inhibits CXCR4 expression on endothelial cells: functional consequences for atherogenesis.

Laminar shear stress (LSS) represents a major athero-protective stimulus. However, the mechanisms for this effect are poorly characterized. As chemokine receptors modulate endothelial cell functions, we hypothesized that at least some LSS effects on endothelial cells (ECs) may be due to LSS-dependent changes in chemokine receptor expression and function. Exposure of Human umbilical vein endothelial cells (HUVECs) to 15 dynes/cm2/sec(-1) LSS strongly inhibited CXC chemokine receptor 4 (CXCR4) expression at the transcriptional level and impaired stromal-derived factor (SDF)-1/CXCL12-driven chemotaxis. On the contrary, low shear stress (SS; 4 dynes/cm2/sec(-1)) only marginally affected CXCR4 expression when compared with static control cells. Differently from CXCR4, the expression of SDF-1 mRNA was not affected by LSS treatment. CXCR4 overexpression induced a dose-dependent endothelial cell apoptosis that was enhanced by SDF-1 treatment and was caspase-dependent. CXCR4 overexpression inhibited the LSS-mediated antiapoptotic effect on ECs and was associated to impairment of LSS-induced ERK1/2 phosphorylation. These findings suggest that LSS-induced CXCR4 down-regulation may contribute to endothelial cell survival. Interestingly, the expression of the proatherogenic chemokines MCP-1 and IL-8 was induced by SDF-1 treatment and by CXCR4 overexpression in HUVECs. Further, the known LSS-induced inhibition of MCP-1 expression was impaired in CXCR4 overexpressing ECs. Finally, CXCR4 was abundantly expressed by human atherosclerotic plaque endothelium that is exposed to low/absent shear stress, while it was poorly expressed by minimally diseased carotid artery endothelium. In conclusion, LSS-dependent CXCR4 down-regulation may contribute to atheroprotection by favoring the integrity of the endothelial barrier and by inhibiting MCP-1 and IL-8 expression.