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Background Tyrosine kinase inhibitors (TKI) can be safely discontinued in chronic phase chronic myeloid leukemia (CP‐CML) patients who had achieved a sustained deep molecular response. Based on the results of discontinuation trials, recommendations regarding patient selection for a treatment‐free remission (TFR) attempt had been proposed. The aims of this study were to evaluate the rate of patients eligible for TKI discontinuation and molecular recurrence‐free survival (MRFS) after stop according to recommendations. Methods Over a 10‐year period, newly diagnosed CP‐CML patients and treated with first‐line TKI in the nine French participating centers were included. Eligibility to treatment discontinuation and MRFS were analyzed and compared according to selection criteria defined by recommendations and first‐line treatments. Results From January 2006 to December 2015, 398 patients were considered. Among them, 73% and 27% of patients received imatinib or either second or third generation tyrosine kinase inhibitors as frontline treatment, respectively. Considering the selection criteria defined by recommendations, up to 55% of the patients were selected as optimal candidates for treatment discontinuation. Overall 95/398 (24%) discontinued treatment. MRFS was 51.8% [95% CI 41.41–62.19] at 2 years and 43.8% [31.45–56.15] at 5 years. Patients receiving frontline second‐generation TKI and fulfilling the eligibility criteria suggested by recommendations had the lowest probability of molecular relapse after TKI stop when compare to others. Conclusion One third of CP‐CML patients treated with TKI frontline fulfilled the selection criteria suggested by European LeukemiaNet TFR recommendations. Meeting selection criteria and second‐generation TKI frontline were associated with the highest MRFS., Tyrosine kinase inhibitors (TKI) can be safely discontinued in chronic phase chronic myeloid leukemia patients who had achieved a sustained deep molecular response. Several recommendations regarding the optimal selection of the patients before stop have been proposed. Based on these recommendations, we estimate that one third of the patients treated with frontline TKI will meet these criteria. Moreover, patients treated with frontline second generation TKI and selected as optimal candidate according to these recommendations have the highest probability of molecular recurrence‐free survival when compared to others.

International audience; PurposeTo assess the incidence of BCR‐ABL kinase domain (KD) mutation detection and its prognostic significance in chronic phase chronic myeloid leukemia (CP‐CML) patients treated with tyrosine kinase inhibitors (TKIs).Patients and MethodsWe analyzed characteristics and outcome of 253 CP‐CML patients who had at least one mutation analysis performed using direct sequencing. Of them, 187 patients were early CP (ECP) and 66 were late CP late chronic phase (LCP) and 88% were treated with Imatinib as first‐line TKI.ResultsOverall, 80 (32%) patients harbored BCR‐ABL KD mutations. A BCR‐ABL KD mutation was identified in 57% of patients, who progressed to accelerated or blastic phases (AP‐BP), and 47%, 29%, 35%, 16% and 26% in patients in CP‐CML at the time of mutation analysis who lost a complete hematologic response, failed to achieve or loss of a prior complete cytogenetic and major molecular response, respectively.Overall survival and cumulative incidence of CML‐related death were significantly correlated with the disease phase whatever the absence or presence of a mutation was and for the latter the mutation subgroup (T315I vs P‐loop vs non‐T315I non‐P‐loop) (P

Early molecular response has been associated with clinical outcome in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The BCR-ABL1 transcript rate decline from baseline to 3 months has been demonstrated to be more predictive than a single BCR-ABL1 level at 3 months (M3). However, it cannot be used routinely because ABL1, as an internal gene control, is not reliable for BCR-ABL1 quantification above 10%. This study aimed to compare clinical outcome and molecular response of chronic phase CML patients, depending on the percentage of BCR-ABL1 transcript decrease from month 3 to month 6 using ABL1 as an internal control gene. Two hundred sixteen chronic phase CML patients treated with imatinib 400 mg for whom M3 and month 6 molecular data were available were included in the study. Associations with event-free (EFS), failure-free (FFS), progression-free (PFS), and overall survivals (OS) molecular response 4 log and 4.5 log were assessed. The percentage of BCR-ABL1 decline from month 3 to month 6 was significantly linked to the EFS and the FFS (p

Background: Tyrosine Kinase Inhibitors (TKIs) discontinuation in patients who had achieved a deep molecular response (DMR) offer now the opportunity of prolonged treatment-free remission (TFR). Patients and Methods: Aims of this study were to evaluate the proportion of de novo chronic-phase chronic myeloid leukemia (CP-CML) patients who achieved a sustained DMR and to identify predictive factors of DMR and molecular recurrence-free survival (MRFS) after TKI discontinuation. Results: Over a period of 10 years, 398 CP-CML patients treated with first-line TKIs were included. Median age at diagnosis was 61 years, 291 (73%) and 107 (27%) patients were treated with frontline imatinib (IMA) or second- or third-generation TKIs (2&ndash, 3G TKI), respectively. With a median follow-up of seven years (range, 0.6 to 13.8 years), 182 (46%) patients achieved a sustained DMR at least 24 months. Gender, BCR-ABL1 transcript type, and Sokal and ELTS risk scores were significantly associated with a higher probability of sustained DMR while TKI first-line (IMA vs. 2&ndash, 3G TKI) was not. We estimate that 28% of CML-CP would have been an optimal candidate for TKI discontinuation according to recent recommendations. Finally, 95 (24%) patients have entered in a TFR program. MRFS rates at 12 and 48 months were 55.1% (95% CI, 44.3% to 65.9%) and 46.9% (95% CI, 34.9% to 58.9%), respectively. In multivariate analyses, first-line 2&ndash, 3G TKIs compared to IMA and TKI duration were the most significant factors of MRFS. Conclusions: Our results suggest that frontline TKIs have a significant impact on TFR in patients who fulfill the selection criteria for TKI discontinuation.

Introduction The acquisition of ABL1 Kinase Domain (KD) mutations represent the most frequent resistance mechanism in CP-CML patients (pts) treated with tyrosine kinase inhibitors (TKI). Currently, the standard assay relies on a poorly sensitive technique, Sanger Sequencing (SS). Thus, the detection of these mutations using SS might be too late to trigger a timely treatment change. In a national phase III academic trial (PETALs, EudraCT 2013-004974-82), we evaluated prospectively the value of a more sensitive technique, Next Generation Sequencing (NGS) to detect KD ABL1 mutations in newly diagnosed CP-CML patients randomized to get nilotinib 600 mg/d for 6 years ± Pegylated-IFN-α2a (Peg-IFN) 45 μg/wk for 2 years in combination. Methods Newly diagnosed CP CML pts ≤65 years were randomized 1:1 to get NIL 300 mg BID alone (M0 to M48, arm A) vs Peg-IFN alone for 30 days (M-1→M0) 30 mg/wk as priming, prior to NIL 300 mg BID + Peg-IFN 30 μg/wk 2 weeks, upgraded to 45 μg/wk thereafter, for up to 2 y (M0 to M24, arm B) followed by NIL alone for 4 more years unless pts enter a treatment-free remission phase. In addition to KD mutational analysis performed by SS as per protocol, patients also had KD mutational analysis performed by NGS at M3, M6, M12 and 6-monthly thereafter until achievement of a stable MMR, regardless of response. NGS assay was performed as previously described (Kizilors et al. Lancet Haematol 2019). Results Two hundred pts were randomized (99 in A, 101 in B), of which 96 patients (51/99 in A, 45/101 in B, p=0.399) underwent a KD mutational analysis performed by NGS as part of this study. The remaining 104 patients are currently being screened and the full dataset will be presented. Among the 96 patients tested, there was no difference in the distribution between the 2 arms with respect to gender, age [median 45 years (18-66)] or risk factors distribution (p=0.862 and 0.328 for Sokal and ELTS respectively in patients tested at 3 months). The median follow-up of this cohort is 45.0 (33.2-58.7) months. By 12 months, 11 patients [8/51 (11.8%) in A, 3/45 (6.6%) in B] had developed a KD mutation. After only 3 months of TKI therapy, 3 patients were found mutated (Y253H 2 pts, T315I 1 pt), of whom 2 pts were only detected using NGS. At M6, a KD mutation was found in 8 pts [A: 7 patients, B: 1 pt, (p= 0.055), of which 6/8 were not detected by SS, due to either low level Variant Allele frequency (VAF, n=5) or low level BCR-ABL transcript levels (n=1). Y253H mutations were found in 4 pts, T315I in 2 pts and E255K in 1 pt. Consecutively to KD mutation identification, 6/8 patients lost their response and were withdrawn from study (1 pt with a Y253H detected at M3 progressed to advanced phase), while 1 pt lost MMR at last follow-up and another pt with a mutation sensitive to nilotinib achieved MMR. KD mutations were detected while pts were in optimal response at M6 [BCR-ABL Conclusions This is the first prospective trial to demonstrate that NGS can detect low level KD mutations in CP CML patients treated with first line 2GTKI±Peg-IFN after only 3 to 6 months on therapy before these become detectable by SS and despite achieving an optimal response in BCR-ABL transcript level reduction (according to ELN 2013). The proportion of patients who develop KD mutations by 12 months on upfront 2GTKI should not be underestimated, as their outcome is poor. NGS may trigger early clinical intervention and prevent progression in this group, although a prospective trial is needed in this regard. Finally, the proportion of KD incidence in pts who receive Peg-IFN in addition to nilotinib might be lower compared to those treated with nilotinib alone. Final updated results will be presented. Figure Disclosures de Lavallade: Novartis: Honoraria, Speakers Bureau; Incyte biosciences: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Jackson:Incyte biosciences: Honoraria, Research Funding, Speakers Bureau. Kizilors:Incyte biosciences: Research Funding. Etienne:BMS: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Huguet:Jazz Pharmaceuticals: Honoraria; Servier: Honoraria; Amgen: Honoraria. Guerci-Bresler:Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte biosciences: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Rea:BMS: Honoraria; Incyte Biosciences: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mahon:Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau. Dulucq:BMS: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Nicolini:Incyte Biosciences: Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Sun Pharma Ltd: Consultancy. OffLabel Disclosure: Pegylated Interferon alpha 2 a is not licensed in this indication

Atherosclerosis is a slowly evolutive age-linked disease of large arteries, characterized by a local lipid deposition associated with a chronic inflammatory response, leading potentially to acute plaque rupture, thrombosis and ischemic heart disease. Atherogenesis is a complex sequence of events associating first expression of adhesion molecules, recruitment of mononuclear cells to the endothelium, local activation of leukocytes and inflammation, lipid accumulation and foam cell formation. Low density lipoproteins (LDLs) become atherogenic after undergoing oxidation by vascular cells, that transform them into highly bioreactive oxidized LDL (oxidized LDLs). Oxidized LDLs are involved in foam cell formation, and trigger proatherogenic events such as overexpression of adhesion molecules, chemoattractant agents growth factors and cytokines involved in the inflammatory process, cell proliferation and apoptosis. Moreover, this toxic effect of oxidized LDLs plays probably a role in plaque erosion/rupture and subsequent atherothrombosis. Several biological effects of oxidized LDLs are mediated through changes in the activity of transcription factors and subsequently in gene expression. Oxidized LDLs exert a biphasic effect on the redox-sensitive transcription factor NF-kappaB, which can be activated thereby up-regulating proinflammatory gene expression, such as adhesion molecules, tissue factor, scavenger receptor LOX-1. On the other hand, higher concentrations of oxidized LDLs may inhibit NF-kappaB activation triggered by inflammatory agents such as LPS, and may thereby exert an immunosuppressive effect. This review is an attempt to clarify the mechanism by which oxidized LDLs may up- or down-regulate NF-kappaB, the role of NF-kappaB activation (or inhibition), and the consequences of the oxidized LDLs-mediated NF-kappaB dysregulation and their potential involvement in atherosclerosis.

Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL act in part through alteration of intracellular signalling pathways in cells of the vascular wall. We recently reported that the EGF receptor (EGFR) signalling pathway is activated by lipid peroxidation products (among them 4-hydroxynonenal, 4-HNE) contained in oxLDL. The use of phenolic antioxidants, such as trolox, alpha-tocopherol, caffeic acid and tyrphostins A-25, A-46 or A-1478, showed that the oxLDL-induced EGFR activation is constituted by two separate components, the first (early) one being antioxidant-insensitive, the second (late) being antioxidant-sensitive. 4-HNE derivatization of EGFR and EGFR activation induced by exogenous 4-HNE, suggest that the early (0.5 - 3 h) component of oxLDL-induced EGFR activation is mediated (at least in part) by 4-HNE (and possibly by other oxidized lipids). This early component is antioxidant-insensitive. The second component (4 - 5 h) of the oxLDL-induced EGFR activation is antioxidant-sensitive, since it is blocked by antioxidants such as trolox, caffeic acid or PDTC, which act by blocking the cellular oxidative stress (H(2)O(2) generation) evoked by oxLDL. Conversely, exogenous H(2)O(2) induced EGFR autophosphorylation (thus mimicking the second component) and was also inhibited by antioxidants. This effect is mediated in part through inhibition by oxidative stress of protein tyrosine phosphatases involved in EGFR dephosphorylation.

Objective— The purpose of this study is to further document alteration of signal transduction pathways, more particularly of hedgehog (Hh) signaling, causing impaired ischemic muscle repair in old mice. Approach and Results— We used 12-week-old (young mice) and 20- to 24-month-old C57BL/6 mice (old mice) to investigate the activity of Hh signaling in the setting of hindlimb ischemia–induced angiogenesis and skeletal muscle repair. In this model, delayed ischemic muscle repair observed in old mice was associated with an impaired upregulation of Gli1. Sonic Hh expression was not different in old mice compared with young mice, whereas desert Hh (Dhh) expression was downregulated in the skeletal muscle of old mice both in healthy and ischemic conditions. The rescue of Dhh expression by gene therapy in old mice promoted ischemia-induced angiogenesis and increased nerve density; nevertheless, it failed to promote myogenesis or to increase Gli1 mRNA expression. After further investigation, we found that, in addition to Dhh, smoothened expression was significantly downregulated in old mice. We used smoothened haploinsufficient mice to demonstrate that smoothened knockdown by 50% is sufficient to impair activation of Hh signaling and ischemia-induced muscle repair. Conclusions— The present study demonstrates that Hh signaling is impaired in aged mice because of Dhh and smoothened downregulation. Moreover, it shows that hegdehog-dependent regulation of angiogenesis and myogenesis involves distinct mechanisms.

Oxidized low-density lipoproteins (oxLDL) exhibit proinflammatory properties and play a role in atherosclerosis plaque formation, rupture, and subsequent thrombosis. OxLDL alter the activity of the transcription factor NF-kappaB that is involved in the expression of immune and inflammatory genes. In contrast, high-density lipoproteins (HDL) are anti-atherogenic and exhibit anti-inflammatory properties. This work aimed to investigate how oxLDL activate NF-kappaB and whether and how HDL may prevent NF-kappaB activation. In cultured rabbit smooth muscle cells, mitogenic concentrations of mildly oxLDL trigger a rapid and transient NF-kappaB activation, which is strongly inhibited by HDL. Growth factors, phosphatidylinositol 3-kinase/Akt, and sphingosine kinase pathways are not implicated in the oxLDL-induced NF-kappaB activation and are not targets of HDL. OxLDL induce reactive oxygen species (ROS) generation and proteasome activation, which are implicated in NF-kappaB activation, as suggested by the inhibitory effect of the antioxidants N-acetyl-L-cysteine and pyrrolidinedithiocarbamate and the proteasome inhibitor PSI. HDL were able to prevent the intracellular ROS rise triggered by oxLDL or H2O2, thereby inhibiting the subsequent proteasome activation, IkappaB degradation, and NF-kappaB activation. In conclusion, the oxLDL-induced NF-kappaB activation involves ROS generation and proteasome activation, both events being inhibited by HDL. This 'antioxidant' and potentially anti-inflammatory effect of HDL may participate in their general anti-atherogenic properties.

Background— Inflammatory processes play a critical role in myocarditis, dilated cardiomyopathy, and heart failure. The expression of the inflammatory chemokine osteopontin (OPN) is dramatically increased in cardiomyocytes and inflammatory cells during myocarditis and heart failure in human and animals. However, its role in the development of heart diseases is not known. Methods and Results— To understand whether OPN is involved in cardiomyopathies, we generated a transgenic mouse (MHC-OPN) that specifically overexpresses OPN in cardiomyocytes with cardiac-specific promoter-directed OPN expression. Young MHC-OPN mice were phenotypically indistinguishable from their control littermates, but most of them died prematurely with a half-life of 12 weeks of age. Electrocardiography revealed conduction defects. Echocardiography showed left ventricular dilation and systolic dysfunction. Histological analysis revealed cardiomyocyte loss, severe fibrosis, and inflammatory cell infiltration. Most of these inflammatory cells were activated T cells with Th1 polarization and cytotoxic activity. Autoantibodies against OPN, cardiac myosin, or troponin I, were not found in the serum of MHC-OPN mice. Conclusions— These data show that OPN expression in the heart induces in vivo T-cell recruitment and activation leading to chronic myocarditis, the consequence of which is myocyte destruction and hence, dilated cardiomyopathy. Thus, OPN might therefore constitute a potential therapeutic target to limit heart failure.

Purpose Sonic Hedgehog (Shh), a morphogen involved in embryonic development, stimulates repair of ischemic myocardium and skeletal muscle (Pola et al 2003 and Kusano et al 2005). Shh was shown to induce both the formation of new vessels and their muscularization. However the mechanisms involved in these processes are still poorly understood. We investigated the hypothesis that the angiogenic and chemotactic protein osteopontin (OPN) was involved in the angiogenic effect of Shh. Methods and Results We used a plasmid transfection strategy to increase Shh expression in the ischemic region of the limb and we compared the effect of Shh overexpression in wild type (OPN+/+) and OPN deficient (OPN-/-) 12-weeks-old mice transfected either with Shh plasmid (pShh) or control plasmid (β-gal). Shh overexpression induced a significant increase in the number of new vessels (+ 22 %± 4.2) in OPN+/+ mice in comparaison with β-gal transfected wild type mice, moreover the Shh effect was lost in OPN-/- mice. Analysis of vessel muscularization showed that Shh mainly increased the number of small non-muscularized vessels. In parallel, we observed an increased number of OPN expressing cells in the ischemic lesion mainly localized close to or inside debasing/regenerating muscle fibres. These cells expressed Mac3 and F4/80 markers suggesting a macrophage phenotype. We suggested that shh could increase the migration of macrophages in an OPN-dependent manner in order to accelerate the repair of the ischemic tissue. The experiments related to the function of OPN in ischemic limb stimulated or not by Shh is currently under investigation. Conclusion Our results demonstrated that OPN is required for the Shh angiogenic effect in the ischemic tissue and suggest that osteopontin expression may be associated with a better migration of macrophages in the ischemic lesion under Shh stimulation. Kusano KF et al. Nat Med. 2005 ; 11:1197-1204. Pola R et al. Circulation. 2003 ; 108:479-485.

Objective— Estradiol (E 2 ) is known to accelerate reendothelialization and thus prevent intimal thickening and in-stent restenosis after angioplasty. Transplantation experiments with ERα −/− mice have previously shown that E 2 acts through local and bone marrow cell compartments to enhance endothelial healing. However, the downstream mechanisms induced by E 2 to mediate endothelial repair are still poorly understood. Methods and Results— We show here that after endovascular carotid artery injury, E 2 -enhanced endothelial repair is lost in osteopontin-deficient mice (OPN −/− ). Transplantation of OPN −/− bone marrow into wild-type lethally irradiated mice, and vice versa, suggested that osteopontin plays a crucial role in both the local and the bone marrow actions of E 2 . In the vascular compartment, using transgenic mice expressing doxycyclin regulatable-osteopontin, we show that endothelial cell specific osteopontin overexpression mimics E 2 -enhanced endothelial cell migration and proliferation in the regenerating endothelium. In the bone marrow cell compartment, we demonstrate that E 2 enhances bone marrow–derived mononuclear cell adhesion to regenerating endothelium in vivo, and that this effect is dependent on osteopontin. Conclusions— We demonstrate here that E 2 acceleration of the endothelial repair requires osteopontin, both for bone marrow–derived cell recruitment and for endothelial cell migration and proliferation.

Within the broad variety of compounds generated via oxidative reactions in low density lipoproteins (LDL) and subsequently found in the atherosclerotic plaque, are aldehydes still esterified to the parent lipid and termed core-aldehydes. The most represented cholesterol core-aldehyde in LDL is 9-oxononanoyl cholesterol (9-ONC), an oxidation product of cholesteryl linoleate. Here we report that 9-ONC, at concentration actually detectable in biological material, significantly up-regulates the expression and the synthesis of the pro-fibrogenic cytokine transforming growth factor beta1 (TGFbeta1) by cultured macrophages. As previously demonstrated for other lipid oxidation products present in LDL, namely a biologically representative mixture of oxysterols and the unesterified aldehyde 4-hydroxynonenal, these effects on TGFbeta1 by 9-ONC further points to LDL lipid oxidation as a powerful source of pro-fibrogenic stimuli.

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor κB (NF-κB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.

Objectives— The atherogenic oxidized low-density lipoprotein (oxLDL) induces the formation of carbonyl-protein adducts and activates the endothelial growth factor receptor (EGFR) signaling pathway, which is now regarded as a central element for signal transduction. We aimed to investigate whether and by which mechanism the anti-atherogenic high-density lipoprotein (HDL) prevents these effects of oxLDL. Methods and Results— In vascular cultured cells, HDL and apolipoprotein A-I inhibit oxLDL-induced EGFR activation and subsequent signaling by acting through 2 separate mechanisms. First, HDL, like the aldehyde scavenger dinitrophenyl hydrazine, prevented the formation of oxLDL-induced carbonyl–protein adducts and 4-hydroxynonenal (HNE)–EGFR adducts. Secondly, HDL enhanced the cellular antioxidant defenses by preventing (through a scavenger receptor class B-1 (SR-BI)–dependent mechanism) the increase of intracellular reactive oxygen species (ROS) and subsequent EGFR activation triggered by oxLDL or H 2 O 2 . A pharmacological approach suggests that this protective effect of HDL is independent of cellular glutathione level and glutathione peroxidase activity, but it requires catalase activity. Finally, we report that oxLDL upregulates both membrane type 1 (MT1)-matrix metalloproteinase-1 (MT1-MMP) and MMP-2 through an EGFR-dependent mechanism and that HDL inhibits these events. Conclusions— HDLs block in vitro oxLDL-induced EGFR signaling and subsequent MMP-2 activation by inhibiting carbonyl adducts formation and cellular oxidative stress. These effects of HDL may participate to reduce cell activation, excessive remodeling, and alteration of the vascular wall.

The interaction of 4-hydroxynonenal (HNE) with a variety of kinases variously involved in cell signaling is now a matter of active investigation. In particular, findings with regard to the effect of HNE on different components of the protein kinase C family and the mitogen-activated protein kinase complex already provide reliable indications of a potential role of this aldehyde as a cell signal messenger. Such a role appears further supported by the clear-cut evidence of up-regulation of receptor tyrosine kinases and down-regulation of the nuclear factor kappa B system, produced by HNE concentrations actually detectable in pathophysiology.

Publisher Summary The chapter presents simple techniques utilizing fluorescent probes suitable for intracellular reactive oxygen species (ROS) detection. These probes do not discriminate directly among the various ROS produced by living cells nor indicate the cellular source of ROS. This can be accomplished by exploiting the differential reactivities of the probes and with the use of inhibitors and scavengers. Regarding the increasing importance of ROS in cellular biology and pathophysiology, several fluorescent probes and new methods have been developed to monitor ROS generation in intact living cells. Fluorescent probes used for determining intracellular ROS in intact living cells must fulfill specific requirements: (1) effective cell loading with the cell permeant fluorogenic probe; (2) oxidation of the fluorogenic probe by cellular ROS (but not by spontaneous autooxidation); (3) stability and retention (low leakage) of the fluorescent probe inside the cell; (4) easy detection of the fluorescent probe with commonly used fluorescent microscopes; and (5) lack of self-toxicity. The commonly used fluorescent probes, derived from fluorescein, rhodamine, ethidium, or other fluorescent dyes, are dihydrogenated into colorless and nonfluorescent dihydro derivatives. Examples of ROS production measured by spectrofluorometry in adherent vascular cells and by flow cytometry in blood phytohemagglutinin-stimulated lymphocytes are presented in the chapter.

Osteopontin (OPN) is not expressed in healthy heart while, its expression is dramatically increased in cardiomyocytes and inflammatory cells during cardiomyopathies and heart failure. However its role in the development of heart diseases is not known.To understand whether OPN is involved in cardiomyopathy, we created a transgenic mouse (MHC-OPN) expressing recombinant OPN (rOPN) specifically in cardiomyocytes using aMHC promoter-directed OPN expression and tTA technology. In these mice, rOPN expression could be regulated by doxycyclin oral administration.After birth, MHC-OPN young mice were phenotypically indistinguishable from their littermate controls, but most of them died early between the 8 th and 15 th weeks after birth with a half life of 12 weeks. However, less than 10 % MHC-OPN mice survived and were still alive 30 weeks after birth. Inhibition of recombinant OPN expression by doxycyclin at the beginning of T cell infiltration (5 weeks after birth) or when DCM was initiated (11 weeks after birth) reduced myocarditis and thus avoided the early death.Electrocardiography demonstrated atrio-ventricular and intra ventricular defects. Moreover, echocardiography showed left ventricular dilation without hypertrophy and a systolic dysfunction, as indicated by reduced left ventricular fractional shortening (control mice: 29,8±2,0 %, n=13 ; and MHC-OPN mice: 13,7±4,0 %, n=5 ; T test p