Fabio Pellegatta

Effector Memory T cells Are Associated With Atherosclerosis in Humans and Animal Models

Background— Adaptive T-cell response is promoted during atherogenesis and results in the differentiation of naïve CD4+T cells to effector and/or memory cells of specialized T-cell subsets. Aim of this work was to investigate the relationship between circulating CD4+T-cell subsets and atherosclerosis. Methods and Results— We analyzed 57 subsets of circulating CD4+T cells by 10-parameter/8-color polychromatic flow cytometry (markers: CD3/CD4/CD45RO/CD45RA/CCR7/CCR5/CXCR3/HLA-DR) in peripheral blood from 313 subjects derived from 2 independent cohorts. In the first cohort of subjects from a free-living population (n=183), effector memory T cells (TEM: CD3+CD4+CD45RA−CD45RO+CCR7− cells) were strongly related with intima-media thickness of the common carotid artery, even after adjustment for age (r=0.27; P<0.001). Of note, a significant correlation between TEM and low-density lipoproteins was observed. In the second cohort (n=130), TEM levels were significantly increased in patients with chronic stable angina or acute myocardial infarction compared with controls. HLA-DR+TEM were the TEM subpopulation with the strongest association with the atherosclerotic process (r=0.37; P<0.01). Finally, in animal models of atherosclerosis, TEM (identified as CD4+CD44+CD62L−) were significantly increased in low-density lipoprotein receptor and apolipoprotein E deficient mice compared with controls and were correlated with the extent of atherosclerotic lesions in the aortic root (r=0.56; P<0.01). Conclusions— Circulating TEM cells are associated with increased atherosclerosis and coronary artery disease in humans and in animal models and could represent a key CD4+T-cell subset related to the atherosclerotic process. (J Am Heart Assoc. 2012;1:27-41.)

Human Endothelial Cells Exposed to Oxidized LDL Express hsp70 Only When Proliferating

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, is cytotoxic and triggers the expression of various heat shock proteins (hsps), among which is hsp70, in cultured animal and human cells. hsps constitutively act as molecular chaperones and in situations of stress protect other cellular proteins from potential denaturation caused by cytotoxic stimuli. The sensitivity of endothelial cells to OxLDL toxicity and accordingly the level of hsp70 expression depend on cell density. While confluent cells were relatively resistant to OxLDL toxicity and were not induced to express hsp70 when challenged with the lipoprotein (up to 800 micrograms/mL), sparse cells exhibited a concentration- and time-dependent expression of inducible hsp70, which increased up to fivefold to sixfold in unchallenged cells. Neither the activity of receptors recognizing OxLDL nor potentially protective cell products affected the stress response. Rather, we demonstrated that cell proliferation, which is high for sparse cultures and wound-healing monolayers, is responsible for these observations. We also demonstrated that the lipid moiety of OxLDL essentially accounts for the hsp-inducing effect of the lipoprotein. OxLDL has been detected in atherosclerotic lesions, which also show an increase of immunoreactive hsp72/73. We speculate that, in vivo, rapidly growing cells, such as those of lesion-prone areas, are more sensitive to the toxicity of OxLDL than are quiescent cells and that an increased expression of hsp70 may allow proliferating cells an increased chance of survival.

Oxidized LDL induce hsp70 expression in human smooth muscle cells

Heat shock protein 70 (hsp70) has been detected in atherosclerotic lesions, in which endothelial cells and smooth muscle cells are involved. In a previous report we showed that Ox‐LDL, a causal factor in atherosclerosis, could induce hsp70 expression in cultured human endothelial cells [Zhu et al. B.B.R.C 1994, 200: 389]. Here, with immunofluorescence and immunoblotting techniques, we show that Ox‐LDL are capable of inducing hsp70 expression also in human smooth muscle cells, and that this induction is dependent on cell density and on the concentration of Ox‐LDL. The induced expression of hsp70 was higher in human umbilical vein smooth muscle cells than in a human smooth muscle cell line. Conversely, Ox‐LDL was cytotoxic to both types of cells, more so to the human smooth muscle cell line. These observations indicate that Ox‐LDL may be a stress responsible for hsp70 expression in atherosclerotic plaques and the presence of hsp70 in plaques may be a useful marker for continuous oxidative damage in the arterial wall.

Leukocyte-associated Ig-like receptor-1 prevents granulocyte-monocyte colony stimulating factor-dependent proliferation and Akt1/PKB alpha activation in primary acute myeloid leukemia cells

The leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1), a surface leukocyte receptor containing two immune receptor tyrosine‐based inhibitory motif (ITIM) is expressed on acute myeloid leukemia (AML) blasts isolated from peripheral blood or bone marrow of 17 patients (2u2004M0, 3u2004M1, 5u2004M2, 2u2004M4 and 5u2004M5 acording to French, American and British classification). Further, we provide evidence thatLAIR‐1 engagement inhibits granulocyte‐monocyte colony‐stimulating factor (GM‐CSF)‐induced proliferation of AML blasts. Indeed, leukemia cells stimulated with GM‐CSF were blocked in the G0/G1 phaseof the cell cycle and underwent apoptosis within 4 days after the engagement of LAIR‐1. Remarkably, LAIR‐1 was functional also in AML blasts which do not express CD33, mainly M4 and M5. Importantly, the LAIR‐1 ligation led to a strong inhibition of both GM‐CSF receptor‐mediated intracellular calcium increases, phosphorylation and activation of Akt1/protein kinase B alpha, a substrate of the phosphatidylinositol‐3 kinase. This last inhibitory effect was prevented by a synthetic peptide spanning the ITIM portion of LAIR‐1, suggesting the involvement of SHP‐1 phosphatase in LAIR‐1‐mediated inhibitory signal. Altogether, these findings indicate that the engagement of LAIR‐1 can down‐regulate GM‐CSF‐mediated survival and proliferation of AML blasts, suggesting an additional therapeutic approach to the treatment of AML patients.

Telomere shortening over 6 years is associated with increased subclinical carotid vascular damage and worse cardiovascular prognosis in the general population

Leucocyte telomere length (LTL) is an important determinant of telomere function and cellular replicative capacity. The aim of the present study was to examine prospectively the associations between telomere shortening (TS) and both the progression of atherosclerosis and the incidence of cardiovascular events (CVEs).

Protective effect of EDTA preadministration on renal ischemia

BackgroundChelation therapy with sodium edetate (EDTA) improved renal function and slowed the progression of renal insufficiency in patients subjected to lead intoxication. This study was performed to identify the underlying mechanism of the ability of EDTA treatment to protect kidneys from damage.MethodsThe effects of EDTA administration were studied in a rat model of acute renal failure induced by 60 minutes ischemia followed or not by 60 minutes reperfusion. Renal ischemic damage was evaluated by histological studies and by functional studies, namely serum creatinine and blood urea nitrogen levels. Treatment with EDTA was performed 30 minutes before the induction of ischemia. Polymorphonuclear cell (PMN) adhesion capability, plasmatic nitric oxide (NO) levels and endothelial NO synthase (eNOS) renal expression were studied as well as the EDTA protection from the TNFα-induced vascular leakage in the kidneys. Data was compared by two-way analysis of variance followed by a post hoc test.ResultsEDTA administration resulted in the preservation of both functional and histological parameters of rat kidneys. PMN obtained from peripheral blood of EDTA-treated ischemized rats, displayed a significant reduction in the expression of the adhesion molecule Mac-1 with respect to controls. NO was significantly increased by EDTA administration and eNOS expression was higher and more diffuse in kidneys of rats treated with EDTA than in the controls. Finally, EDTA administration was able to prevent in vivo the TNFα-induced vascular leakage in the kidneys.ConclusionThis data provides evidence that EDTA treatment is able to protect rat kidneys from ischemic damage possibly through the stimulation of NO production.

Oxysterols from oxidized LDL are cytotoxic but fail to induce hsp70 expression in endothelial cells

Oxidized low density lipoprotein (OxLDL) possesses several proatherogenic characteristics, among which a marked cytotoxicity. In vitro, cytotoxicity of OxLDL to endothelial cells is associated with an increase in the expression of the inducible form of heat shock protein 70 (hsp70), generally regarded as a cytoprotective protein. Oxidized derivatives of cholesterol which form upon LDL oxidation are cytotoxic. Moreover, most of the OxLDL cytotoxicity is due to its lipid moiety, in particular to oxysterols. In this report we demonstrate that although oxysterols identified in OxLDL are cytotoxic, they cannot trigger the increase in hsp70 expression observed with intact oxidized lipoproteins. We speculate therefore that oxysterols may represent the most toxic form of oxidized lipids in LDL because they cannot activate a rescue mechanism (i.e. the hsp response) and may contribute significantly to cell death within atherosclerotic plaques.

In human endothelial cells amino acids inhibit insulin-induced Akt and ERK1/2 phosphorylation by an mTOR-dependent mechanism.

In several cellular systems, amino acids synergize with insulin in promoting protein synthesis through the activation of the protein kinases p70/S6-K and PHAS-1. Such activations are mediated by the upstream kinase: mammalian target of rapamycin (mTor). In this work we have investigated the intracellular pathways involved in insulin-induced and amino acid-induced p70/S6-K activations in human endothelial cells. In human umbilical vein endothelial cells, insulin induces the phosphorylation of p70/S6-K at 5 minutes decreasing thereafter, whereas amino acids alone or associated with insulin phosphorylate p70/S6-K at all the time points analyzed (60 minutes). Insulin and amino acids phosphorylate p70/S6-K by mTor-dependent and phosphotidylinositol 3-kinase-dependent mechanisms, whereas the mitogen-activated protein kinase pathway is involved only when p70/S6-K is activated by insulin. Insulin induces the phosphorylation of Akt and extracellular signal-regulated protein kinase (ERK) 1/2, whereas amino acids did not. Moreover, amino acids suppress the phosphorylations induced by insulin. The inhibitory effects of amino acids are reverted by the mTor inhibitor rapamycin. Insulin-induced phosphorylation of Akt (at 15 and 30 minutes) is not accompanied by the phosphorylation of the downstream kinase p70/S6-K, indicating the existence of a negative feedback at this level. Our data demonstrate that at the level of human endothelial cells, amino acids synergize with insulin in the phosphorylation of the kinase that lies downstream mTor, as p70/S6-K, whereas they inhibit the upstream kinases Akt and extracellular signal-regulated protein kinase 1/2 when activated by insulin, by an mTor-dependent mechanism.

PCSK9 deficiency results in increased ectopic fat accumulation in experimental models and in humans

Background Proprotein convertase subtilisin kexin type 9 (PCSK9) regulates low-density lipoprotein and very low-density lipoprotein receptor expression in several tissues. Here we evaluated whether PCSK9 may modulate the handling of triglycerides in the liver and peripheral tissues. Methods Subjects from the PLIC cohort were genotyped for the loss-of-function PCSK9 R46L variant and characterized for clinical and biochemical parameters, total and android fat mass, hepatic steatosis and epicardial fat thickness. Visceral adipose tissue and subcutaneous adipose tissue in PCSK9 KO and wild type mice were quantified by nuclear magnetic resonance imaging. Results Carriers of the R46L variant (nu2009=u200913) had lower low-density lipoprotein cholesterol levels, higher body mass index and increased percentage of total and android fat masses compared with non-carriers (nu2009=u2009521). R46L variant associated with a two-fold increase prevalence of hepatic steatosis and higher epicardial fat thickness. These observations were replicated in PCSK9 KO mice, which showed increased visceral adipose tissue (but not subcutaneous adipose tissue) when fed chow or high-fat diet for 20 weeks, compared with wild type mice. Conclusions These data suggest that genetically determined PCSK9 deficiency might be associated with ectopic fat accumulation.

Statins decrease thrombin generation in patients with hypercholesterolemia

OBJECTIVEnStatins are cholesterol-lowering agents with antithrombotic effect possibly unrelated to their lipid-lowering properties. Traditional global coagulation tests failed, however, to reveal clinically relevant change after treatment. We therefore sought to investigate whether statins were able to modify thrombin generation in hypercholesterolemia.nnnMETHODSnFifty-one patients who needed treatment with statins were enrolled in this study. Thrombin generation, assessed as endogenous thrombin potential (the amount of thrombin generated after triggering coagulation with small amount of tissue factor) was measured at pre- and two months post-treatment with statins.nnnRESULTSnThe median (inter-quartile range) level of total cholesterol that was 325 mg/dL (278-405) decreased significantly [211 mg/dL (197-247)] at post-treatment (p<0.001); the median level of HDL cholesterol that was 49 mg/dL (43-56) increased significantly [55 mg/dL (47-66)] at post-treatment (p<0.001). The median endogenous thrombin potential (inter-quartile range) before treatment was 2372 nM·min (2008-2617) and decreased to 2,048 nM·min (1764-2375) (p<0.001) after treatment.nnnCONCLUSIONnThe results support the hypothesis of a direct link between statins and coagulation through their capacity to lower thrombin generation in patients with hypercholesterolemia.nnnPRACTICE IMPLICATIONSnThe antithrombotic properties of statins could be mediated (at least in part) by their endogenous thrombin potential lowering effect. This interesting hypothesis warrants evaluation by clinical trials.