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Purpose: Fetal stress and postnatal overfeeding are associated with programming of cardiometabolic diseases, but their relative role is not clearly established. By using specific animal models we aimed to address this issue comparing the perinatal alterations induced in several organs key for cardiometabolic control.Methods: Fetal stress was induced by maternal undernutrition (MUN) during pregnancy. MUN rats were fed ad libitum with standard chow from gestational day 1 to 10; with 50% of their daily intake from day 11 to the end of pregnancy and returned back to ad libitum during lactation (12 pups/litter). Postnatal overfeeding (POF) was induced in the offspring from ad libitum fed dams by reducing litter size during lactation (4 pups/litter). Control animals were offspring from dams ad libitum fed both during pregnancy and lactation (12 pups/litter). Tibial length and body, heart, kidney, liver and fat adipose tissue weights, as well as adipocites size, were assessed in the offspring at weaning (21 days). A group of MUN offspring was followed to measure body weight along perinatal development.Results: MUN offspring showed reduced tibial length and body weight from birth with progressive catch-up growth until month two, when body weight reached similar values to control rats. By weaning MUN offspring rats exhibited: 1) hyperglycemia; 2) heart and liver hypertrophy, 3) reduced kidney weight and 4) increased subcutaneous and periorganic fat deposits and adipocite size. POF offspring also exhibited increased glycemia together with liver, fat and body weight and adipocite size, but heart and kidney weights were not altered. There were no sex-related differences in either MUN or POF models in any of the parameters studied.Conclusions: We conclude that accelerated growth during postnatal life, rather than fetal stress, seems to be critical for metabolic control organs alterations. On the other hand, fetal stress induces changes in heart and kidney at an early age which might have a direct role on later cardiovascular disease development. [ABSTRACT FROM AUTHOR]

Vegetable oils such as palm oil (enriched in saturated fatty acids, SFA) and high-oleic-acid sunflower oil (HOSO, containing mainly monounsaturated fatty acids, MUFA) have emerged as the most common replacements for trans-fats in the food industry. The aim of this study is to analyze the impact of SFA and MUFA-enriched high-fat (HF) diets on endothelial function, vascular remodeling, and arterial stiffness compared to commercial HF diets. Five-week-old male C57BL6J mice were fed a standard (SD), a HF diet enriched with SFA (saturated oil-enriched Food, SOLF), a HF diet enriched with MUFA (unsaturated oil-enriched Food, UOLF), or a commercial HF diet for 8 weeks. Vascular function was analyzed in the thoracic aorta. Structural and mechanical parameters were assessed in mesenteric arteries by pressure myography. SOLF, UOLF, and HF diet reduced contractile responses to phenylephrine and induced endothelial dysfunction in the thoracic aorta. A significant increase in the β-index, and thus in arterial stiffness, was also detected in mesenteric arteries from the three HF groups, due to enhanced deposition of collagen in the vascular wall. SOLF also induced hypotrophic inward remodeling. In conclusion, these data demonstrate a deleterious effect of HF feeding on obesity-related vascular alterations that is exacerbated by SFA.

Fetal undernutrition programs hypertension and cardiovascular diseases, and resistance artery remodeling may be a contributing factor. We aimed to assess if fetal undernutrition induces resistance artery remodeling and the relationship with hypertension. Sprague-Dawley dams were fed ad libitum (Control) or with 50% of control intake between days 11 and 21 of gestation (maternal undernutrition, MUN). In six-month-old male and female offspring we assessed blood pressure (anesthetized and tail-cuff); mesenteric resistance artery (MRA) structure and mechanics (pressure myography), cellular and internal elastic lamina (IEL) organization (confocal microscopy) and plasma MMP-2 and MMP-9 activity (zymography). Systolic blood pressure (SBP, tail-cuff) and plasma MMP activity were assessed in 18-month-old rats. At the age of six months MUN males exhibited significantly higher blood pressure (anesthetized or tail-cuff) and plasma MMP-9 activity, while MUN females did not exhibit significant differences, compared to sex-matched controls. MRA from 6-month-old MUN males and females showed a smaller diameter, reduced adventitial, smooth muscle cell density and IEL fenestra area, and a leftward shift of stress-strain curves. At the age of eighteen months SBP and MMP-9 activity were higher in both MUN males and females, compared to sex-matched controls. These data suggest that fetal undernutrition induces MRA inward eutrophic remodeling and stiffness in both sexes, independent of blood pressure level. Resistance artery structural and mechanical alterations can participate in the development of hypertension in aged females and may contribute to adverse cardiovascular events associated with low birth weight in both sexes.

The experimental approach for the study of cardiometabolic disorders requires the use of animal models fed with commercial diets whose composition differs notably, even between diets used for control groups. While chow diets are usually made of agricultural by-products, purified low-fat diets (LF) contain a higher percentage of easy metabolizable carbohydrates, together with a reduced amount of polyunsaturated fatty acids, micronutrients and fiber, all associated with metabolic and vascular dysfunction. We hypothesize that the LF diet, commonly used in control animals, could promote adverse vascular and metabolic outcomes. To address this issue, 5-week-old male C57BL6J mice were fed with a standard (Chow) or a LF diet for 6 weeks. Changes in body weight, adiposity, biochemical parameters, systemic and aortic insulin sensitivity and endothelial function were recorded. LF diet did not modify body weight but significantly impaired systemic glucose tolerance and increased triglycerides and cholesterol levels. Endothelial function and aortic insulin sensitivity were significantly impaired in the LF group, due to a reduction of NO availability. These findings highlight the importance of selecting the proper control diet in metabolic studies. It may also suggest that some cardiometabolic alterations obtained in experimental studies using LF as a control diet may be underestimated.

Bariatric surgery (BS) results in sustained weight loss and may reverse inflammation, metabolic alterations, extracellular matrix remodeling and arterial stiffness. We hypothesize that increased stiffening in omental arteries from obese patients might be associated with an increase in MMP activity and a decrease in p-AMPK, together with systemic oxidative stress and inflammation. Moreover, BS could contribute to reversing these alterations. This study was conducted with 38 patients of Caucasian origin: 31 adult patients with morbid obesity (9 men and 22 women; mean age 46 years and BMI = 42.7 ± 1.0 kg/m 2 ) and 7 non-obese subjects (7 women; mean age 45 years and BMI = 22.7 ± 0.6 kg/m 2 ). Seventeen obese patients were studied before and 12 months after BS. The stiffness index β, an index of intrinsic arterial stiffness, was determined in omental arteries and was significantly higher in obese patients. Levels of phosphorylated AMPK (p-AMPK Thr-172 ) and SIRT-1 were significantly lower in peripheral blood mononuclear cells (PBMCs) from obese patients than those from non-obese patients ( p < 0.05) and were normalized after BS. Total and active MMP-9 activities, LDH, protein carbonyls and uric acid were higher in obese patients and reduced by BS. Moreover, there was a correlation between plasmatic LDH levels and the stiffness index β. BS has a beneficial effect on abnormal MMP-9, LDH and AMPK activities that might be associated with the development of arterial stiffness in obese patients. Since these parameters are easily measured in blood samples, they could constitute potential biomarkers of cardiovascular risk in morbid obesity.

Infusions of murtilla leaves exhibit antioxidant, analgesic, and anti-inflammatory properties. Several compounds that are structurally similar to madecassic acid (MA), a component of murtilla leaf extract (ethyl acetate extract, EAE), have been shown to inhibit protein tyrosine phosphatase 1B (PTP1P). The aim of this study was to evaluate if EAE and two compounds identified in EAE (MA and myricetin [MYR]) could have a beneficial effect on systemic and vascular insulin sensitivity and endothelial function in a model of diet-induced obesity. Experiments were performed in 5-week-old male C57BL6J mice fed with a standard (LF) or a very high-fat diet (HF) for 4 weeks and treated with EAE, MA, MYR, or the vehicle as control (C). EAE significantly inhibited PTP1B. EAE and MA, but not MYR, significantly improved systemic insulin sensitivity in HF mice and vascular relaxation to Ach in aorta segments, due to a significant increase of eNOS phosphorylation and enhanced nitric oxide availability. EAE, MA, and MYR also accounted for increased relaxant responses to insulin in HF mice, thus evidencing that the treatments significantly improved aortic insulin sensitivity. This study shows for the first time that EAE and MA could constitute interesting candidates for treating insulin resistance and endothelial dysfunction associated with obesity.

Albuminuria is an early marker of renovascular damage associated to an increase in oxidative stress. The Munich Wistar Frömter (MWF) rat is a model of chronic kidney disease (CKD), which exhibits endothelial dysfunction associated to low nitric oxide availability. We hypothesize that the new highly selective, non-steroidal mineralocorticoid receptor (MR) antagonist, finerenone, reverses both endothelial dysfunction and microalbuminuria. Twelve-week-old MWF (MWF-C; MWF-FIN) and aged-matched normoalbuminuric Wistar (W-C; W-FIN) rats were treated with finerenone (FIN, 10 mg/kg/day p.o.) or vehicle (C) for 4-week. Systolic blood pressure (SBP) and albuminuria were determined the last day of treatment. Finerenone lowered albuminuria by >40% and significantly reduced SBP in MWF. Aortic rings of MWF-C showed higher contractions to either noradrenaline (NA) or angiotensin II (Ang II), and lower relaxation to acetylcholine (Ach) than W-C rings. These alterations were reversed by finerenone to W-C control levels due to an upregulation in phosphorylated Akt and eNOS, and an increase in NO availability. Apocynin and 3-amino-1,2,4-triazole significantly reduced contractions to NA or Ang II in MWF-C, but not in MWF-FIN rings. Accordingly, a significant increase of Mn-superoxide dismutase (SOD) and Cu/Zn-SOD protein levels were observed in rings of MWF-FIN, without differences in p22phox, p47phox or catalase levels. Total SOD activity was increased in kidneys from MWF-FIN rats. In conclusion, finerenone improves endothelial dysfunction through an enhancement in NO bioavailability and a decrease in superoxide anion levels due to an upregulation in SOD activity. This is associated with an increase in renal SOD activity and a reduction of albuminuria.

Background and Aims: Fetal undernutrition is a risk factor for heart disease in both genders, despite the protection of women against hypertension development. Using a rat model of maternal undernutrition (MUN) we aimed to assess possible sex differences in the development of cardiac alterations and the implication of hypertension and cardiac oxidative stress., Methods: Male and female offspring from rats fed ad libitum (control) or with 50% of the normal daily intake during the second half of gestation (MUN) were used. Heart weight/body weight ratio (HW/BW), hemodynamic parameters (anaesthetized rats) and plasma brain natriuretic peptide (BNP, ELISA) were assessed in 21-day, 6-month and 22-month old rats. Plasma testosterone (ELISA) and cardiac protein expression of enzymes related to reactive oxygen species synthesis (p22phox, xanthine-oxidase) and degradation (catalase, Cu/Zn-SOD, Mn-SOD, Ec-SOD) were evaluated in 21-day and 6-month old rats (Western Blot). Heart structure and function was studied at the age of 22 months (echocardiography)., Results: At the age of 21 days MUN males exhibited significantly larger HW/BW and cardiac p22phox expression while females had reduced p22phox expression, compared to their respective sex-matched controls. At the age of 6-months, MUN males showed significantly larger blood pressure and cardiac xanthine-oxidase expression; MUN females were normotensive and had a lower cardiac expression of antioxidant enzymes, compared to their respective sex-matched controls. At the age of 22 months, both MUN males and females showed larger HW/BW and left ventricular mass and lower ejection fraction compared to sex-matched controls; only MUN males exhibited hypertension and a larger plasma BNP compared to aged male controls., Conclusions: 1) During perinatal life females exposed to fetal undernutrition are protected from cardiac alterations, but in ageing they exhibit ventricular hypertrophy and functional loss, like MUN males; 2) cardiac oxidative stress might be implicated in the observed heart alterations in both sexes and 3) the severity of cardiac damage might be greater in males due to hypertension.

Caloric restriction (CR) ameliorates cardiac dysfunction associated with obesity. However, most of the studies have been performed under severe CR (30-65% caloric intake decrease) for several months or even years in aged animals. Here, we investigated whether mild (20% food intake reduction) and short-term (2-weeks) CR prevented the obese cardiomyopathy phenotype and improved the metabolic profile of young (14 weeks of age) genetically obese Zucker fa/fa rats. Heart weight (HW) and HW/tibia length ratio was significantly lower in fa/fa rats after 2 weeks of CR than in counterparts fed ad libitum . Invasive pressure measurements showed that systolic blood pressure, maximal rate of positive left ventricle (LV) pressure, LV systolic pressure and LV end-diastolic pressure were all significantly higher in obese fa/fa rats than in lean counterparts, which were prevented by CR. Magnetic resonance imaging revealed that the increase in LV end-systolic volume, stroke volume and LV wall thickness observed in fa/fa rats was significantly lower in animals on CR diet. Histological analysis also revealed that CR blocked the significant increase in cardiomyocyte diameter in obese fa/fa rats. High resolution magic angle spinning magnetic resonance spectroscopy analysis of the LV revealed a global decrease in metabolites such as taurine, creatine and phosphocreatine, glutamate, glutamine and glutathione, in obese fa/fa rats, whereas lactate concentration was increased. By contrast, fatty acid concentrations in LV tissue were significantly elevated in obese fa/fa rats. CR failed to restore the LV metabolomic profile of obese fa/fa rats. In conclusion, mild and short-term CR prevented an obesity-induced cardiomyopathy phenotype in young obese fa/fa rats independently of the cardiac metabolic profile.

Background: The hypothesis of this study is that long-term high-fat diets (HFD) induce perivascular adipose tissue (PVAT) dysfunction characterized by a redox imbalance, which might contribute to aggravate endothelial dysfunction in obesity., Methods and Results: C57BL/6J mice were fed either control or HFD (45% kcal from fat) for 32 weeks. Body weight, lumbar and mesenteric adipose tissue weights were significantly higher in HFD animals compared to controls. The anticontractile effect of PVAT in mesenteric arteries (MA) was lost after 32 week HFD and mesenteric endothelial-dependent relaxation was significantly impaired in presence of PVAT in HFD mice (Emax = 71.0±5.1 vs Emax = 58.5±4.2, p<0.001). The inhibitory effect of L-NAME on Ach-induced relaxation was less intense in the HFD group compared with controls suggesting a reduction of endothelial NO availability. Expression of eNOS and NO bioavailability were reduced in MA and almost undetectable in mesenteric PVAT of the HFD group. Superoxide levels and NOX activity were higher in PVAT of HFD mice. Apocynin only reduced contractile responses to NA in HFD animals. Expression of ec-SOD and total SOD activity were significantly reduced in PVAT of HFD mice. No changes were observed in Mn-SOD, Cu/Zn-SOD or catalase. The ratio [GSSG]/([GSH]+[GSSG]) was 2-fold higher in the mesenteric PVAT from HFD animals compared to controls., Conclusions: We suggest that the imbalance between pro-oxidant (NOX, superoxide anions, hydrogen peroxide) and anti-oxidant (eNOS, NO, ecSOD, GSSG) mechanisms in PVAT after long-term HFD might contribute to the aggravation of endothelial dysfunction.

Dietary treatment with high-fat diets (HFD) triggers diabetes and hyperleptinemia, concomitantly with a partial state of leptin resistance that affects hepatic and adipose tissue but not the heart. In this context, characterized by widespread steatosis, cardiac lipid content remains unchanged. As previously reported, HFD-evoked hyperleptinemia could be a pivotal element contributing to increase fatty-acid (FA) metabolism in the heart and to prevent cardiac steatosis. This metabolic adaptation might theoretically reduce energy efficiency in cardiomyocytes and lead to cardiac electrophysiological remodeling. Therefore the aim of the current study has been to investigate the impact of long-term HFD on cardiac metabolism and electrophysiological properties of the principal ionic currents responsible of the action potential duration in mouse cardiomyocytes. Male C57BL/6J mice were fed a control (10 kcal% from fat) or HFD (45 kcal% from fat) during 32 weeks. Quantification of enzymatic activities regulating mitochondrial uptake of pyruvate and FA showed an increase of both carnitine-palmitoyltransferase and citrate synthase activities together with a decrease of lactate dehydrogenase and pyruvate dehydrogenase activities. Increased expression of uncoupling protein-3, Mn-, and Cu/Zn-superoxide dismutases and catalase were also detected. Total glutathione/oxidized glutathione ratios were unaffected by HFD. These data suggest that HFD triggers adaptive mechanisms aimed at (i) facilitating FA catabolism, and (ii) preventing oxidative stress. All these changes did not affect the duration of action potentials in cardiomyocytes and only slightly modified electrocardiographic parameters.

Most blood vessels are surrounded by adipose tissue. Similarly to the adventitia, perivascular adipose tissue (PVAT) was considered only as a passive structural support for the vasculature, and it was routinely removed for isolated blood vessel studies. In 1991, Soltis and Cassis demonstrated for the first time that PVAT reduced contractions to noradrenaline in rat aorta. Since then, an important number of adipocyte-derived factors with physiological and pathophysiological paracrine vasoactive effects have been identified. PVAT undergoes structural and functional changes in obesity. During early diet-induced obesity, an adaptative overproduction of vasodilator factors occurs in PVAT, probably aimed at protecting vascular function. However, in established obesity, PVAT loses its anticontractile properties by an increase of contractile, oxidative, and inflammatory factors, leading to endothelial dysfunction and vascular disease. The aim of this review is to focus on PVAT dysfunction mechanisms in obesity.

Leptin causes vasodilatation both by endothelium-dependent and -independent mechanisms. Leptin is synthesized by perivascular adipose tissue (PVAT). The hypothesis of this study is that a decrease of leptin production in PVAT of spontaneously hypertensive rats (SHR) might contribute to a diminished paracrine anticontractile effect of the hormone. We have determined in aorta from Wistar-Kyoto (WKY) and SHR (i) leptin mRNA and protein levels in PVAT, (ii) the effect of leptin and PVAT on contractile responses, and (iii) leptin-induced relaxation and nitric oxide (NO) production. Leptin mRNA and protein expression were significantly lower in PVAT from SHR. Concentration-response curves to angiotensin II were significantly blunted in presence of PVAT as well as by exogenous leptin (10(-9) M) only in WKY. This anticontractile effect was endothelium-dependent. Vasodilatation induced by leptin was smaller in SHR than in WKY, and was also endothelium-dependent. Moreover, release of endothelial NO in response to acute leptin was higher in WKY compared to SHR, but completely abolished in the absence of endothelium. In conclusion, the reduced anticontractile effect of PVAT in SHR might be attributed to a reduced PVAT-derived leptin and to an abrogated effect of leptin on endothelial NO release probably due to an impaired activation of endothelial NO synthase.

High-fat (HF) diets trigger an increase in adipose tissue and body weight (BW) and disordered eating behavior. Our study deals with the hypothesis that circadian distribution of energy intake is more relevant for BW dynamics than diet composition. Four-week-old mice were exposed for 8 wk to a HF diet and compared with animals receiving control chow. HF mice progressively increased BW, decreased the amount of nocturnal (1800-0900 h) calories (energy or food intake) (30%) and increased diurnal (0900-1800 h) caloric intake (energy or food intake), although total daily intake was identical between groups. Animals were killed at 3-h intervals and plasma insulin, leptin, corticosterone, glucose, and fatty acid levels quantified. Adipose tissue was weighed, and enzymatic activities integral to the pentose phosphate pathway (PPP) assayed in lumbar adipose tissue. Phosphorylated AMP-dependent protein kinase and fatty acid synthase were quantified by Western blotting. In HF mice, there was a shift in the circadian oscillations of plasma parameters together with an inhibition of PPP activity and a decrease in phosphorylated AMP-dependent protein kinase and fatty acid synthase. In a second experiment, HF mice were forced to adhere to a circadian pattern of food intake similar to that in control animals. In this case, BW, adipose tissue, morning plasma parameters and PPP activity appeared to be normal. These data indicate that disordered feeding behavior can trigger BW gain independently of food composition and daily energy intake. Because PPP is the main source of reduced nicotinamide adenine dinucleotide phosphate, we suggest that PPP inhibition might be an early marker of adipose dysfunction in diet-induced obesity.

Perivascular adipose tissue (PVAT) plays a paracrine role in regulating vascular tone. We hypothesize that PVAT undergoes adaptative mechanisms during initial steps of diet-induced obesity (DIO) which contribute to preserve vascular function. Four-week-old male C57BL/6J mice were assigned either to a control [low-fat (LF); 10% kcal from fat] or to a high-fat diet (HF; 45% kcal from fat). After 8 wk of dietary treatment vascular function was analyzed in the whole perfused mesenteric bed (MB) and in isolated mesenteric arteries cleaned of PVAT. Relaxant responses to acetylcholine (10(-9)-10(-4) m) and sodium nitroprusside (10(-12)-10(-5) m) were significantly ameliorated in the whole MB from HF animals. However, there was no difference between HF and LF groups in isolated mesenteric arteries devoid of PVAT. The enhancement of relaxant responses detected in HF mice was not attributable to an increased release of nitric oxide (NO) from the endothelium nor to an increased sensitivity and/or activity of muscular guanilylcyclase. Mesenteric PVAT of HF animals showed an increased bioavailability of NO, detected by 4,5-diaminofluorescein diacetate (DAF2-DA) staining, which positively correlated with plasma leptin levels. DAF-2DA staining was absent in PVAT from ob/ob mice but was detected in these animals after 4-wk leptin replacement. The main finding in this study is that adaptative NO overproduction occurs in PVAT during early DIO which might be aimed at preserving vascular function.

Acute leptin increase as well as endogenous hyperleptinemia evoked by high-fat diets (HF) activate fatty acid metabolism in nonadipose tissues. This supports the notion that hyperleptinemia is pivotal to prevent/delay steatosis during periods of positive energy balance. We have previously shown that long-term HF spares ectopic accumulation of lipids specifically in the miocardium. Because carnitine palmitoyltransferase I (CPT-I) allows mitochondrial uptake/oxidation of fatty acids, we have hypothesized that leptin drives cardiac CPT-I activity. In the current study, hyperleptinemia was induced in C57BL/6J mice either by exogenous leptin administration or by means of HF, and the ability of malonyl-coenzyme A (malonyl-CoA) (the main endogenous inhibitor of CPT-I) to inhibit cardiac CPT was analyzed. IC(50) values of malonyl-CoA were 8.1 +/- 1.5 micromol/liter in controls vs. 69.3 +/- 5.2 micromol/liter (P < 0.01) in leptin-treated mice. This effect was also observed in cardiac explants incubated with leptin and was blocked by triciribine, a compound shown to inhibit protein kinase B (Akt) phosphorylation (pAkt). In accordance, acute leptin evoked an increase of cardiac pAkt levels, which correlated with CPT sensitivity to malonyl-CoA. Otherwise, the inhibitory effect of malonyl-CoA was hindered in HF hyperleptinemic mice, and in this case, pAkt levels also correlated with CPT sensitivity to malonyl-CoA. Our data show that leptin reduces the sensitivity of cardiac CPT-I to malonyl-CoA and suggest the involvement of an Akt-related signaling pathway in this effect. This mechanism appears to be sensitive to both acute and chronic hyperleptinemia. We conclude that this action of leptin is pivotal to drive cardiac metabolism under situations associated to hyperleptinemia.

Background: Persistent high risk HPV infection can lead to cervical cancer, the second most common malignant tumor in women worldwide. NK cells play a crucial role against tumors and virus-infected cells through a fine balance between activating and inhibitory receptors. Expression of triggering receptors NKp30, NKp44, NKp46 and NKG2D on NK cells correlates with cytolytic activity against tumor cells, but these receptors have not been studied in cervical cancer and precursor lesions. The aim of the present work was to study NKp30, NKp46, NKG2D, NKp80 and 2B4 expression in NK cells from patients with cervical cancer and precursor lesions, in the context of HPV infection., Methods: NKp30, NKp46, NKG2D, NKp80 and 2B4 expression was analyzed by flow cytometry on NK cells from 59 patients with cervical cancer and squamous intraepithelial lesions. NK cell cytotoxicity was evaluated in a 4 hour CFSE/7-AAD flow cytometry assay. HPV types were identified by PCR assays., Results: We report here for the first time that NK cell-activating receptors NKp30 and NKp46 are significantly down-regulated in cervical cancer and high grade squamous intraepithelial lesion (HGSIL) patients. NCRs down-regulation correlated with low cytolytic activity, HPV-16 infection and clinical stage. NKG2D was also down-regulated in cervical cancer patients., Conclusion: Our results suggest that NKp30, NKp46 and NKG2D down-regulation represent an evasion mechanism associated to low NK cell activity, HPV-16 infection and cervical cancer progression.

Regulation of body weight (BW) results from the interplay between different hormonal systems acting at central and peripheral level. This study aims at characterizing the involvement of cholecystokinin (CCK) in BW and energy balance regulation. We have characterized, in free-feeding rats, the effect of CCK-8 on 1) food intake, BW, and adiposity; 2) skeletal muscle metabolism; 3) leptin signaling pathway within the arcuate nucleus of the hypothalamus; and 4) the permeability of brain barriers to leptin. We demonstrate here that CCK-8 acutely decreases BW by a mechanism partially dependent on central leptin pathways, based on the following results: 1) the effect of CCK was less intense in rats lacking functional leptin receptors (Zucker fa/fa), 2) CCK-8 facilitated the uptake of leptin from peripheral circulation to cerebrospinal fluid (CSF), 3) the concentration of leptin in CSF of rats receiving CCK was more elevated in those animals showing higher loss of BW, and 4) CCK activated leptin signaling pathways within the hypothalamus as well as phosphorylation of AMP-activated protein kinase in skeletal muscle. We also suggest that gain of BW may be linked to individual susceptibility to the effect of CCK, because we observed that in animals treated with this hormone, the increase of BW negatively correlated with leptin concentration within the CSF. Our data show that CCK has a negative impact on energy balance and suggest that CCK facilitates the access of leptin to hypothalamic areas, thus allowing leptin to act on hypothalamic targets involved in BW control.

The objective of this work was to characterize the adaptation of cardiac metabolism to a lipid overload in a model of diet-induced obesity (DIO) in mice. After 8 wk dietary treatment, mice receiving a high-fat diet exhibited an increase in the amount of adipose tissue, accompanied by a surge in plasma leptin concentration (from 5.4-16.0 ng/ml). This was associated with: 1) an induction of uncoupling protein-2 (120%), 2) an increase in the phosphorylated form of AMP-activated protein kinase (120%), and 3) a reduction in lactate concentration and lactate dehydrogenase activity in myocardial tissue (40%). Because DIO induces leptin resistance, we analyzed leptin receptor functionality by measuring phospho-signal transducer and activator of transcription 3 in response to acute leptin (1 mg/kg). We observed that leptin receptor signaling remained unaltered within the heart but was fully impaired within the hypothalamus. Taken together, these data show that during DIO development, there is a metabolic shift in the heart aimed at increasing fatty acid oxidation to the detriment of carbohydrates. This effect seems to be leptin-dependent, suggesting that the increased adiposity observed during the onset of obesity might contribute to impairing ectopic lipidic deposition in the heart.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an adhesion molecule expressed in a wide variety of tissues including epithelial cells, leukocytes, and tumors that may establish both homotypic and heterotypic interactions. The aim of this work was to study the protein expression pattern of CEACAM1 in cervical cancer and precursor lesions in the context of human papillomavirus (HPV) infection. We used immunohistochemistry to analyze CEACAM1 expression in formalin-fixed, paraffin-embedded cervical tissues from 15 healthy women, 15 patients with low-grade squamous intraepithelial lesions (SIL), 15 patients with high-grade SIL, and 15 patients with squamous carcinomas. HPV types were identified by PCR. CEACAM1 was either undetectable (13/15) or low (2/15) in normal cervical tissues. By contrast, CEACAM1 expression was increased in high-grade SIL (10 samples staining intermediate/high and 4 samples staining low) as compared with low-grade SIL with undetectable (n=3) or low (n=12) expression. CEACAM1 expression was undetectable or low in cervical carcinoma. Our results suggest that CEACAM1 may be an interesting progression marker in SIL and cervical cancer, in particular due to reported immunoregulatory properties.

Resistance artery narrowing and stiffening are key elements in the pathogenesis of essential hypertension, but their origin is not completely understood. In mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR), we have shown that inward remodeling is associated with abnormal elastic fiber organization, leading to smaller fenestrae in the internal elastic lamina. Our current aim is to determine whether this alteration is an early event that precedes vessel narrowing, or if elastic fiber reorganization in SHR arteries occurs because of the remodeling process itself. Using MRA from 10-day-old, 30-day-old, and 6-mo-old SHR and normotensive Wistar Kyoto rats, we investigated the time course of the development of structural and mechanical alterations (pressure myography), elastic fiber organization (confocal microscopy), and amount of elastin (radioimmunoassay for desmosine) and collagen (picrosirius red). SHR MRA had an impairment of fenestrae enlargement during the first month of life. In 30-day-old SHR, smaller fenestrae and more packed elastic fibers in the internal elastic lamina were paralleled by increased wall stiffness. Collagen and elastin levels were unaltered at this age. MRA from 6-mo-old SHR also had smaller fenestrae and a denser network of adventitial elastic fibers, accompanied by increased collagen content and vessel narrowing. At this age, elastase digestion was less effective in SHR MRA, suggesting a lower susceptibility of elastic fibers to enzymatic degradation. These data suggest that abnormal elastic fiber deposition in SHR increases resistance artery stiffness at an early age, which might participate in vessel narrowing later in life.

We have previously developed a method for estimating elastin content and organization in resistance arteries, where it is a minor component. The aim of the present study was to validate the method against a quantitative assay and to determine the relative importance of elastin content and organization for intrinsic elasticity of small arteries. Mesenteric third order branches (from 10-day-old, 1- and 6-month-old rats) and middle cerebral arteries (from 6-month-old rats) were pressurized. beta-Values were calculated from stress-strain relationships and used as indicators of intrinsic stiffness. The same pressure-fixed arteries were used to estimate elastin content and organization in the internal elastic lamina with confocal microscopy. Collagen and elastin contents were determined by Picrosirius Red staining and radioimmunoassay for desmosine, respectively. Confocal and desmosine assays gave similar results: no difference in elastin content of mesenteric vessels from 1- and 6-month-old rats, and a significant reduction in cerebral compared to mesenteric arteries. For all parameters (elastin and collagen content, fenestrae area and internal elastic lamina thickness) the best correlation was found between beta-values and fenestrae size. These data suggest that in small arteries: (1) confocal microscopy can be used as a method for the simultaneous study of changes in elastin content and organization; and (2) elastin organization might be a key determinant of intrinsic elastic properties.

Objective: We have studied the modulatory role of the adventitia on vascular tone and nitric oxide (NO) availability in response to noradrenaline (NA) and angiotensin II (Ang II)., Methods: Changes in isometric tension were determined in carotid arteries from 3-month-old Sprague-Dawley rats denuded from adventitia (-A) and compared to intact rings (+A). NO availability was assessed by the fluorescent NO indicator, 4,5-diaminofluorescein diacetate (DAF-2)., Results: Responses to NA (10(-10) to 10(-6) M) were: (i) significantly lower in -A compared to +A rings; (ii) equally enhanced in +A and -A rings without endothelium; and (iii) reduced in +A and -A rings incubated with superoxide dismutase (SOD; 15 U/ml). Responses to Ang II (10(-10) to 10(-7) M) were: (i) similar between +A and -A segments; (ii) equally reduced in both groups by SOD; and (iii) increased by endothelial denudation in both +A and -A arteries. Blockade of AT2 receptors with PD 123,319 (10(-7) M) significantly increased Ang II-induced contractions in +A rings. In segments preincubated with losartan (10(-5) M) and precontracted with NA (10(-7) M), Ang II elicited a relaxation that was abolished by l-NAME (10(-4) M), PD 123,319 (10(-7) M), and endothelium or adventitial removal. NO availability was increased in carotid rings stimulated with Ang II, but not with NA. This NO release was blocked by PD 123,319 (10(-7) M) and endothelium denudation., Conclusions: These results suggest that the adventitia differently modulates responses to vasoconstrictors and that it is a key layer in Ang II-induced contractions, mediating NO release from the endothelium via AT2 receptors. This increase in NO counterbalances basal superoxide release.

Chronic hypertension is associated with resistance artery remodelling and mechanical alterations. However, the contribution of elastin has not been thoroughly studied. Our objective was to evaluate the role of elastin in vascular remodelling of mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR). MRA segments from Wistar Kyoto rats (WKY) and SHR were pressurised under passive conditions at a range of physiological pressures with pressure myography. Confocal microscopy was used to determine differences in the quantity and organisation of elastin in intact pressure-fixed arteries. To assess the contribution of elastin to MRA structure and mechanics, myograph-mounted vessels were studied before and after elastase incubation. When compared with WKY, MRA from SHR showed: (1) a smaller lumen, (2) decreased distensibility at low pressures, (3) a leftward shift of the stress-strain relationship, (4) redistribution of elastin within the internal elastic lamina (IEL) leading to smaller fenestrae but no change in fenestrae number or elastin amount. Elastase incubation (1) fragmented the structure of IEL in a concentration-dependent fashion, (2) abolished all the structural and mechanical differences between strains, and (3) decreased distensibility at low pressures. The study shows the overriding role of elastin in determining vascular dimensions and mechanical properties in a resistance artery. In addition, it informs hypertensive remodelling. MRA remodelling and increased stiffness are accompanied by elastin restructuring within the IEL and elastin degradation reverses structural and mechanical alterations of SHR MRA. Differences in elastin organisation are, therefore, a central element in small artery remodelling in hypertension.

The aim of this study was to investigate the effect of chronic losartan or captopril on vascular smooth muscle cell (VSMC) phenotype and vascular function in spontaneously hypertensive rats. Male 12-week-old rats were treated for 16 weeks with losartan (15 mg/kg per day) or captopril (60 mg/kg per day) in their drinking water. Systolic blood pressure, measured by the tail-cuff method, was reduced approximately 40 mm Hg in both treatment groups compared with a nontreated control group. Cell structure and proliferation studies were performed in VSMCs obtained from rat carotid arteries. Cells from the losartan-treated group showed a significant reduction in size, total protein content, and nucleus number, as well as proliferation after stimulation with 10% fetal calf serum and an increased percentage of cells in the G(1) phase compared with the control and captopril-treated groups. Functional studies were performed in isolated carotid arteries from these groups. Contractions elicited by 75 mmol/L KCl or 10(-)(7) mol/L norepinephrine and relaxations elicited by acetylcholine were similar in all groups. Concentration-response curves to angiotensin I or angiotensin II (10(-)(10) to 3x10(-)(7) mol/L) were almost abolished in the losartan-treated group and were not modified by preincubation with the angiotensin type 2 receptor antagonist PD 123,319. These results suggest that long-term losartan treatment significantly changes VSMC phenotype and proliferative status, apparently unrelated to blood pressure lowering or to endothelial function improvements.