Noemí Cabré

Immunohistochemical Analysis of Paraoxonases and Chemokines in Arteries of Patients with Peripheral Artery Disease

Oxidative damage to lipids and lipoproteins is implicated in the development of atherosclerotic vascular diseases, including peripheral artery disease (PAD). The paraoxonases (PON) are a group of antioxidant enzymes, termed PON1, PON2, and PON3 that protect lipoproteins and cells from peroxidation and, as such, may be involved in protection against the atherosclerosis process. PON1 inhibits the production of chemokine (C–C motif) ligand 2 (CCL2) in endothelial cells incubated with oxidized lipoproteins. PON1 and CCL2 are ubiquitously distributed in tissues, and this suggests a joint localization and combined systemic effect. The aim of the present study has been to analyze the quantitative immunohistochemical localization of PON1, PON3, CCL2 and CCL2 receptors in a series of patients with severe PAD. Portions of femoral and/or popliteal arteries from 66 patients with PAD were obtained during surgical procedures for infra-inguinal limb revascularization. We used eight normal arteries from donors as controls. PON1 and PON3, CCL2 and the chemokine-binding protein 2, and Duffy antigen/chemokine receptor, were increased in PAD patients. There were no significant changes in C–C chemokine receptor type 2. Our findings suggest that paraoxonases and chemokines play an important role in the development and progression of atherosclerosis in peripheral artery disease.

Nutrients in Energy and One-Carbon Metabolism: Learning from Metformin Users

Metabolic vulnerability is associated with age-related diseases and concomitant co-morbidities, which include obesity, diabetes, atherosclerosis and cancer. Most of the health problems we face today come from excessive intake of nutrients and drugs mimicking dietary effects and dietary restriction are the most successful manipulations targeting age-related pathways. Phenotypic heterogeneity and individual response to metabolic stressors are closely related food intake. Understanding the complexity of the relationship between dietary provision and metabolic consequences in the long term might provide clinical strategies to improve healthspan. New aspects of metformin activity provide a link to many of the overlapping factors, especially the way in which organismal bioenergetics remodel one-carbon metabolism. Metformin not only inhibits mitochondrial complex 1, modulating the metabolic response to nutrient intake, but also alters one-carbon metabolic pathways. Here, we discuss findings on the mechanism(s) of action of metformin with the potential for therapeutic interpretations.

Galectin-3 in Peripheral Artery Disease. Relationships with Markers of Oxidative Stress and Inflammation.

Galectin-3 is a modulator of oxidative stress, inflammation, and fibrogenesis involved in the pathogenesis of vascular diseases. The present study sought to characterize, in patients with peripheral artery disease (PAD), the localization of galectin-3 in arterial tissue, and to analyze the relationships between the circulating levels of galectin-3 and oxidative stress and inflammation. It also sought to compare the diagnostic accuracy of galectin-3 with that of other biochemical markers of this disease. We analyzed femoral or popliteal arteries from 50 PAD patients, and four control arteries. Plasma from 86 patients was compared with that from 72 control subjects. We observed differences in the expression of galectin-3 in normal arteries, and arteries from patients with PAD, with a displacement of the expression from the adventitia to the media, and the intima. In addition, plasma galectin-3 concentration was increased in PAD patients, and correlated with serologic markers of oxidative stress (F2-isoprostanes), and inflammation [chemokine (C−C motif) ligand 2, C-reactive protein, β-2-microglobulin]. We conclude that the determination of galectin-3 has good diagnostic accuracy in the assessment of PAD and compares well with other analytical parameters currently in use.

Metformin administration induces hepatotoxic effects in paraoxonase-1-deficient mice.

Metformin is the first-line pharmacological treatment of diabetes. In these patients, metformin reduces body weight and decreases the risk of diabetes-related complications such as cardiovascular disease. However, whether metformin elicits beneficial effects on liver histology is a controversial issue and, as yet, there is no consensus. Paraoxonase-1 (PON1), an enzyme synthesized mainly by the liver, degrades lipid peroxides and reduces oxidative stress. PON1 activities are decreased in chronic liver diseases. We evaluated the effects of metformin in the liver of PON1-deficient mice which, untreated, present a mild degree of liver steatosis. Metformin administration aggravated inflammation in animals given a standard mouse chow and in those fed a high-fat diet. Also, it was associated with a higher degree of steatosis in animals fed a standard chow diet. This report is a cautionary note regarding the prescription of metformin for the treatment of diabetes in patients with concomitant liver impairment.

Trace Elements and Paraoxonase-1 Activity in Lower Extremity Artery Disease

Oxidative stress and inflammation are candidate mechanisms to explain the potential role of exposure to metals and reduced activity of paraoxonase-1 (PON1) in age-related diseases. Both may be risk factors contributing to atherosclerosis. In the present study, inductively coupled mass spectrometry was used to explore multiple trace elements, while in-house methods were employed to measure PON1-related variables in patients with lower extremity artery disease (LEAD). Healthy controls were matched for sex, age, body weight, and relevant genotype variants. Serum concentrations of As, Ba, Cu, and Sr were higher in patients than those in controls, with a strong predictive ability to discriminate between groups. Differences in serum Pb, Cd, and Zn were negligible. Serum Cu increased when the disease was more severe, but a negative trend was noted for serum As, B, Ba, and Zn. The only variable associated with ankle-brachial index was serum Zn. Serum PON1 activity was significantly lower in LEAD patients. When the ability of serum trace elements to modulate PON1 activity was explored, the analysis revealed a unique association with serum Zn. The current results strongly suggest that Zn may have a protective effect in non-coronary atherosclerosis and indicate that this element may exert its anti-inflammatory and antioxidant functions through interactions with PON1 activity. These findings deserve confirmation and further research. In particular, the periodic evaluation of serum trace elements and the prescription of Zn supplements are easy measures to implement and that can improve the treatment of patients with LEAD.

Metformin directly targets the H3K27me3 demethylase KDM6A/UTX

Metformin, the first drug chosen to be tested in a clinical trial aimed to target the biology of aging per se, has been clinically exploited for decades in the absence of a complete understanding of its therapeutic targets or chemical determinants. We here outline a systematic chemoinformatics approach to computationally predict biomolecular targets of metformin. Using several structure‐ and ligand‐based software tools and reference databases containing 1,300,000 chemical compounds and more than 9,000 binding sites protein cavities, we identified 41 putative metformin targets including several epigenetic modifiers such as the member of the H3K27me3‐specific demethylase subfamily, KDM6A/UTX. AlphaScreen and AlphaLISA assays confirmed the ability of metformin to inhibit the demethylation activity of purified KDM6A/UTX enzyme. Structural studies revealed that metformin might occupy the same set of residues involved in H3K27me3 binding and demethylation within the catalytic pocket of KDM6A/UTX. Millimolar metformin augmented global levels of H3K27me3 in cultured cells, including reversion of global loss of H3K27me3 occurring in premature aging syndromes, irrespective of mitochondrial complex I or AMPK. Pharmacological doses of metformin in drinking water or intraperitoneal injection significantly elevated the global levels of H3K27me3 in the hepatic tissue of low‐density lipoprotein receptor‐deficient mice and in the tumor tissues of highly aggressive breast cancer xenograft‐bearing mice. Moreover, nondiabetic breast cancer patients receiving oral metformin in addition to standard therapy presented an elevated level of circulating H3K27me3. Our biocomputational approach coupled to experimental validation reveals that metformin might directly regulate the biological machinery of aging by targeting core chromatin modifiers of the epigenome.

Effect of radiotherapy on activity and concentration of serum paraoxonase-1 in breast cancer patients

Paraoxonase-1 (PON1) is an intra-cellular antioxidant enzyme found also in the circulation associated with high-density lipoproteins. The activity of this enzyme has been shown to be decreased in breast cancer (BC) patients. The aims of our study were to investigate the changes produced by radiotherapy (RT) on activity and concentration of serum PON1 in BC patients, and to evaluate the observed variations in relation to clinical and pathological characteristics of patients and tumors, and the response to treatment. We studied 200 women with BC who were scheduled to receive RT following excision of the tumor. Blood for analyses was obtained before and after the irradiation procedure. The control group was composed of 200 healthy women. Relative to control, BC patients had significantly lower serum PON1 activities pre-RT, while PON1 concentrations were at similar levels. RT was associated with a significant increase in serum PON1 activities and concentrations. We observed significant differences in serum PON1 concentrations post-RT between patients with luminal A or luminal B tumors. Serum PON1 concentration post-RT was markedly lower in BC patients with metastases. We conclude that benefit from RT accrues to the BC patients not only through its direct effect on cancer cells but also indirectly by improving the organism’s anti-oxidant defense mechanisms. In addition, our preliminary evidence suggests that the measurement of serum PON1 concentration post-RT could be an efficient prognostic biomarker, and may be used as an index of the efficacy of the RT.

Metformin Potentiates the Benefits of Dietary Restraint: A Metabolomic Study

Prevention of the metabolic consequences of a chronic energy-dense/high-fat diet (HFD) represents a public health priority. Metformin is a strong candidate to be incorporated in alternative therapeutic approaches. We used a targeted metabolomic approach to assess changes related to the multi-faceted metabolic disturbances provoked by HFD. We evaluated the protective effects of metformin and explored how pro-inflammatory and metabolic changes respond when mice rendered obese, glucose-intolerant and hyperlipidemic were switched to diet reversal with or without metformin. Mice treated with metformin and diet-reversal showed a dramatically improved protection against HFD-induced hepatic steatosis, a beneficial effect that was accompanied by a lowering of liver-infiltrating pro-inflammatory macrophages and lower release of pro-inflammatory cytokines. Metformin combined with diet reversal promoted effective weight loss along with better glucose control, lowered levels of circulating cholesterol and triglycerides, and reduced adipose tissue content. Our findings underscored the ability of metformin to target the contribution of branched chain amino acids to adipose tissue metabolism while suppressing mitochondrial-dependent biosynthesis in hepatic tissue. The relationship between adipose tissue and liver might provide clinical potential for combining metformin and dietary modifications to protect against the metabolic damage occurring upon excessive dietary fat intake.

Relationships Between Metformin, Paraoxonase-1 and the Chemokine (C-C Motif) Ligand 2

Metformin is a biguanide used in the treatment of type 2 diabetes mellitus and obesity. The main mechanism of action is to decrease the intestinal glucose absorption and the hepatic glucose production, however, it does not influence insulin secretion. Metformin also increases the affinity of the insulin receptor, reduces high insulin levels and improves insulin resistance. Additionally, it promotes weight loss. Metformin is a pleiotropic compound but acts, largely, by activating 5 adenosine monophosphate (AMP)-activated protein kinase (AMPK). Data suggest that the therapeutic effects of this compound are mediated, at least in part, through an upregulation of paraoxonase-1 (PON1) synthesis. PON1 is a thiolactonase that degrades lipid peroxides, and downregulates the chemokine (C-C motif) ligand 2 (CCL2) which is a pro-inflammatory chemokine that stimulates the migration of monocytes to areas of inflammation where they differentiate into macrophages. However, the prescription of metformin in patients with liver disease is controversial since, in some cases, this drug causes worsening of liver function. Patients with chronic liver disease have decreased hepatic PON1 activity. A study in mice deficient in PON1 showed that in this experimental model, metformin administration increased the severity of steatosis, increased CCL2 expression, did not activate AMPK, and increased the expression of the apoptosis marker caspase-9. These results suggest that PON1 is essential for the successful activation of AMPK in the liver, and for metformin to demonstrate its therapeutic function.

Trace element concentrations in breast cancer patients

Alterations in the circulating levels of trace elements have been observed in breast cancer (BC) patients. However, the relationships between these alterations and the metabolic and clinical consequences of BC are unknown. The treatment-of-choice of BC is surgery followed by radiation therapy (RT). The present study was aimed at investigating: 1) the concentrations of several trace elements in BC patients, and their relationships with the intrinsic molecular subtypes of tumors; 2) the toxicological effect of RT. We studied 49 women with BC who were scheduled to receive RT following excision of the tumor. Plasma samples were obtained before and after the irradiation procedure. The control group was composed of 49 healthy women. Patients had significantly lower pre-RT concentrations of B, Cu, and Zn, and significantly higher concentrations of Sr than the control group. Irradiation was associated with a striking increase in plasma B concentrations, while Cu, Fe, Sr and Zn concentrations were not significantly different from pre-RT levels, albeit Sr and Zn showed non-significant trends towards increases. The plasma concentrations of B, Cu, Fe, Sr, and Zn were associated with the tumor expression of hormone receptors, epidermal growth factor receptor 2, Ki67 antigen, as well as dermatitis and asthenia, all of which represent the main toxicological responses to RT.