Fetal programming and lactation: modulating gene expression in response to undernutrition during intrauterine life.

BACKGROUND: Adverse environmental conditions during intrauterine life, known as fetal programming, significantly contribute to the development of diseases in adulthood. Fetal programming induced by factors like maternal undernutrition leads to low birth weight and increases the risk of cardiometabolic diseases. METHODS: We studied a rat model of maternal undernutrition during gestation (MUN) to investigate gene expression changes in cardiac tissue using RNA-sequencing of day 0-1 litters. Moreover, we analyzed the impact of lactation at day 21, in MUN model and cross-fostering experiments, on cardiac structure and function assessed by transthoracic echocardiography, and gene expression changes though qPCR. RESULTS: Our analysis identified specific genes with altered expression in MUN rats at birth. Two of them, Agt and Pparg, stand out for being associated with cardiac hypertrophy and fibrosis. At the end of the lactation period, MUN males showed increased expression of Agt and decreased expression of Pparg, correlating with cardiac hypertrophy. Cross-fostering experiments revealed that lactation with control breastmilk mitigated these expression changes reducing cardiac hypertrophy in MUN males. CONCLUSIONS: Our findings highlight the interplay between fetal programming, gene expression, and cardiac hypertrophy suggesting that lactation period is a potential intervention window to mitigate the effects of fetal programming. IMPACT: Heart remodeling involves the alteration of several groups of genes and lactation period plays a key role in establishing gene expression modification caused by fetal programming. We could identify expression changes of relevant genes in cardiac tissue induced by undernutrition during fetal life. We expose the contribution of the lactation period in modulating the expression of Agt and Pparg, relevant genes associated with cardiac hypertrophy. This evidence reveal lactation as a crucial intervention window for preventing or countering fetal programming.

Evaluation of Parameters Which Influence Voluntary Ingestion of Supplements in Rats.

Drug safety and efficacy studies frequently use oral gavage, but repetitive usage may cause problems. Administration through voluntary ingestion represents an opportunity for refinement. We aimed to develop a protocol for voluntary ingestion of gelatin-based supplements in rats, assessing the influence of age, sex, fasting (4 h), and additives (vanilla, VF; sucralose, S), and to test it in lactating dams. Three-week-old and 5-month-old Sprague-Dawley rats were placed individually in an empty cage containing a gelatin cube and trained daily (5 days/week), recording the day the whole cube was consumed (latency). Rats trained prior to gestation were offered a gelatin containing 250 mg/kg cocoa shell extract (CSE) during lactation. Rats that did not eat the cube after 8 training days were considered non-habituated, with a proportion similar in young males (7.1%), young females (11.1%), and adult females (10.3%), but significantly higher in adult males (39.3%). Excluding non-habituated rats, latency was 2-3 days, without differences between young and adult rats (p = 0.657) or between males and females (p = 0.189). VF or VF + S in the gelatin did not modify latency, while fasting significantly reduced it in females (p = 0.007) but not in males (p = 0.501). During lactation, trained females ate the CSE-gelatin within 1-5 min without litter problems. Conclusions: Acceptance of a gelatin-based supplement is negatively influenced by male sex, facilitated by fasting, and not modified by additives. Training is remembered after 2 months and does not interfere with lactation. Gelatin-based voluntary ingestion is suitable to administer drugs that need to pass through the digestive system, ensuring adequate dosage, and is important to detect non-habituated rats prior to the study. The current protocol may be implemented by training the rats in their own cage.

Cocoa Shell Extract Reduces Blood Pressure in Aged Hypertensive Rats via the Cardiovascular Upregulation of Endothelial Nitric Oxide Synthase and Nuclear Factor (Erythroid-Derived 2)-like 2 Protein Expression.

Cocoa shell is a by-product of cocoa manufacturing. We obtained an aqueous extract (CSE) rich in polyphenols and methylxanthines with antioxidant and vasodilatory properties. We aimed to evaluate the effects of CSE supplementation in aged hypertensive rats on blood pressure and the mechanism implicated. Eighteen-month-old male and female rats exposed to undernutrition during the fetal period who developed hypertension, with a milder form in females, were used (MUN rats). Systolic blood pressure (SBP; tail-cuff plethysmography) and a blood sample were obtained before (basal) and after CSE supplementation (250 mg/kg; 2 weeks, 5 days/week). Plasma SOD, catalase activity, GSH, carbonyls, and lipid peroxidation were assessed (spectrophotometry). In hearts and aortas from supplemented and non-supplemented age-matched rats, we evaluated the protein expression of SOD-2, catalase, HO-1, UCP-2, total and phosphorylated Nrf2 and e-NOS (Western blot), and aorta media thickness (confocal microscopy). MUN males had higher SBP compared with females, which was reduced via CSE supplementation with a significant difference for group, sex, and interaction effect. After supplementation with plasma, GSH, but not catalase or SOD, was elevated in males and females. Compared with non-supplemented rats, CSE-supplemented males and females exhibited increased aorta e-NOS and Nrf2 protein expression and cardiac phosphorylated-Nrf2, without changes in SOD-2, catalase, HO-1, or UCP-2 in cardiovascular tissues or aorta remodeling. In conclusion, CSE supplementation induces antihypertensive actions related to the upregulation of e-NOS and Nrf2 expression and GSH elevation and a possible direct antioxidant effect of CSE bioactive components. Two weeks of supplementation may be insufficient to increase antioxidant enzyme expression.

Effect of Supplementation with Coffee and Cocoa By-Products to Ameliorate Metabolic Syndrome Alterations Induced by High-Fat Diet in Female Mice.

Coffee and cocoa manufacturing produces large amounts of waste. Generated by-products contain bioactive compounds with antioxidant and anti-inflammatory properties, suitable for treating metabolic syndrome (MetS). We aimed to compare the efficacy of aqueous extracts and flours from coffee pulp (CfPulp-E, CfPulp-F) and cocoa shell (CcShell-E, CcShell-F) to ameliorate MetS alterations induced by a high-fat diet (HFD). Bioactive component content was assessed by HPLC/MS. C57BL/6 female mice were fed for 6 weeks with HFD followed by 6 weeks with HFD plus supplementation with one of the ingredients (500 mg/kg/day, 5 days/week), and compared to non-supplemented HFD and Control group fed with regular chow. Body weight, adipocyte size and browning (Mitotracker, confocal microscopy), plasma glycemia (basal, glucose tolerance test-area under the curve, GTT-AUC), lipid profile, and leptin were compared between groups. Cocoa shell ingredients had mainly caffeine, theobromine, protocatechuic acid, and flavan-3-ols. Coffee pulp showed a high content in caffeine, protocatechuic, and chlorogenic acids. Compared to Control mice, HFD group showed alterations in all parameters. Compared to HFD, CcShell-F significantly reduced adipocyte size, increased browning and high-density lipoprotein cholesterol (HDL), and normalized basal glycemia, while CcShell-E only increased HDL. Both coffee pulp ingredients normalized adipocyte size, basal glycemia, and GTT-AUC. Additionally, CfPulp-E improved hyperleptinemia, reduced triglycerides, and slowed weight gain, and CfPulp-F increased HDL. In conclusion, coffee pulp ingredients showed a better efficacy against MetS, likely due to the synergic effect of caffeine, protocatechuic, and chlorogenic acids. Since coffee pulp is already approved as a food ingredient, this by-product could be used in humans to treat obesity-related MetS alterations.

Maternal and Neonatal Factors Modulating Breast Milk Cytokines in the First Month of Lactation.

Breast milk (BM) cytokines support and modulate infant immunity, being particularly relevant in premature neonates with adverse outcomes (NAO). This study aimed to examine, in a cohort of Spanish breastfeeding women, changes in BM cytokines in the first month of lactation, their modulation by neonatal factors (sex, gestational age, and NAO), maternal factors (obstetric complications, C-section, and diet), and their relationship with oxidative status. Sixty-three mother-neonate dyads were studied at days 7 and 28 of lactation. Dietary habits were assessed by a 72-h dietary recall, and the maternal dietary inflammatory index (mDII) was calculated. BM cytokines (IL-10, IL-13, IL-8, MCP-1, and TNFα) were assessed by ultra-sensitive chemiluminescence. Total antioxidant capacity was assessed by the ABTS method and lipid peroxidation by the MDA+HNE kit. From days 7 to 28 of lactation, the levels of IL-10 and TNFα remained stable, while IL-13 increased (ß = 0.85 ± 0.12, p < 0.001) and IL-8 and MCP-1 levels decreased (ß = -0.64 ± 0.27, p = 0.019; ß = -0.98 ± 0.22, p < 0.001; respectively). Antioxidant capacity and lipid peroxidation also decrease during lactation. Neonatal sex did not influence any of the cytokines, but BM from mothers with male infants had a higher antioxidant capacity. Gestational age was associated with male sex and NAO, being inversely correlated with the BM proinflammatory cytokines IL-8, MCP-1, and TNFα. From days 7 to 28 of lactation, BM from women with NAO infants increased MCP-1 levels and had a larger drop in antioxidant capacity, with the opposite trend in lipid peroxidation. MCP-1 was also significantly higher in women undergoing C-section; this cytokine declined in women who decreased mDII during lactation, while IL-10 increased. Linear mixed regression models evidenced that the most important factors modulating BM cytokines were lactation period and gestational age. In conclusion, during the first month of lactation, BM cytokines shift towards an anti-inflammatory profile, influenced mainly by prematurity. BM MCP-1 is associated with maternal and neonatal inflammatory processes.

Radical Scavenging and Cellular Antioxidant Activity of the Cocoa Shell Phenolic Compounds after Simulated Digestion.

The cocoa industry generates a considerable quantity of cocoa shell, a by-product with high levels of methylxanthines and phenolic compounds. Nevertheless, the digestion process can extensively modify these compounds' bioaccessibility, bioavailability, and bioactivity as a consequence of their transformation. Hence, this work's objective was to assess the influence of simulated gastrointestinal digestion on the concentration of phenolic compounds found in the cocoa shell flour (CSF) and the cocoa shell extract (CSE), as well as to investigate their radical scavenging capacity and antioxidant activity in both intestinal epithelial (IEC-6) and hepatic (HepG2) cells. The CSF and the CSE exhibited a high amount of methylxanthines (theobromine and caffeine) and phenolic compounds, mainly gallic acid and (+)-catechin, which persisted through the course of the simulated digestion. Gastrointestinal digestion increased the antioxidant capacity of the CSF and the CSE, which also displayed free radical scavenging capacity during the simulated digestion. Neither the CSF nor the CSE exhibited cytotoxicity in intestinal epithelial (IEC-6) or hepatic (HepG2) cells. Moreover, they effectively counteracted oxidative stress triggered by tert-butyl hydroperoxide (t-BHP) while preventing the decline of glutathione, thiol groups, superoxide dismutase, and catalase activities in both cell lines. Our study suggests that the cocoa shell may serve as a functional food ingredient for promoting health, owing to its rich concentration of antioxidant compounds that could support combating the cellular oxidative stress associated with chronic disease development.

Vascular nitrosative stress in hypertension induced by fetal undernutrition in rats.

Fetal undernutrition predisposes to hypertension development. Since nitric oxide (NO) is a key factor in blood pressure control, we aimed to investigate the role of NO alterations in hypertension induced by fetal undernutrition in rats. Male and female offspring from dams exposed to undernutrition during the second half of gestation (MUN) were studied at 21 days (normotensive) and 6 months of age (hypertension developed only in males). In aorta, we analyzed total and phosphorylated endothelial NO synthase (eNOS, p-eNOS), 3-nitrotyrosine (3-NT), and Nrf2 (Western blot). In plasma we assessed L-arginine, asymmetric and symmetric dimethylarginine (ADMA, SDMA; LC-MS/MS), nitrates (NOx, Griess reaction), carbonyl groups, and lipid peroxidation (spectrophotometry). In iliac arteries, we studied superoxide anion production (DHE staining, confocal microscopy) and vasodilatation to acetylcholine (isometric tension). Twenty-one-day-old MUN offspring did not show alterations in vascular e-NOS or 3NT expression, plasma L-Arg/ADMA ratio, or NOx. Compared to control group, 6-month-old MUN rats showed increased aortic expression of p-eNOS/eNOS and 3-NT, being Nrf2 expression lower, elevated plasma L-arginine/ADMA, NOx and carbonyl levels, increased iliac artery DHE staining and reduced acetylcholine-mediated relaxations. These alterations in MUN rats were sex-dependent, affecting males. However, females showed some signs of endothelial dysfunction. We conclude that increased NO production in the context of a pro-oxidative environment, leads to vascular nitrosative damage and dysfunction, which can participate in hypertension development in MUN males. Females show a better adaptation, but signs of endothelial dysfunction, which can explain hypertension in ageing.

Slower Growth during Lactation Rescues Early Cardiovascular and Adipose Tissue Hypertrophy Induced by Fetal Undernutrition in Rats.

Low birth weight (LBW) and accelerated growth during lactation are associated with cardiometabolic disease development. LBW offspring from rats exposed to undernutrition during gestation (MUN) develops hypertension. In this rat model, we tested if slower postnatal growth improves early cardiometabolic alterations. MUN dams were fed ad libitum during gestation days 1-10, with 50% of the daily intake during days 11-21 and ad libitum during lactation. Control dams were always fed ad libitum. Pups were maintained with their own mother or cross-fostered. Body weight and length were recorded weekly, and breastmilk was obtained. At weaning, the heart was evaluated by echocardiography, and aorta structure and adipocytes in white perivascular fat were studied by confocal microscopy (size, % beige-adipocytes by Mitotracker staining). Breastmilk protein and fat content were not significantly different between groups. Compared to controls, MUN males significantly accelerated body weight gain during the exclusive lactation period (days 1-14) while females accelerated during the last week; length growth was slower in MUN rats from both sexes. By weaning, MUN males, but not females, showed reduced diastolic function and hypertrophy in the heart, aorta, and adipocytes; the percentage of beige-type adipocytes was smaller in MUN males and females. Fostering MUN offspring on control dams significantly reduced weight gain rate, cardiovascular, and fat hypertrophy, increasing beige-adipocyte proportion. Control offspring nursed by MUN mothers reduced body growth gain, without cardiovascular modifications. In conclusion, slower growth during lactation can rescue early cardiovascular alterations induced by fetal undernutrition. Exclusive lactation was a key period, despite no modifications in breastmilk macronutrients, suggesting the role of bioactive components. Our data support that lactation is a key period to counteract cardiometabolic disease programming in LBW and a potential intervention window for the mother.

Maintenance over Time of the Effect Produced by Esmolol on the Structure and Function of Coronary Arteries in Hypertensive Heart Diseases.

We previously observed that esmolol treatment for 48 h reduced vascular lesions in spontaneously hypertensive rats (SHRs). Therefore, we investigated whether this beneficial effect is persistent after withdrawal. Fourteen-month-old SHRs (SHR-Es) were treated with esmolol (300 µg/kg/min) or a vehicle for 48 h. Two separate groups were also given identical treatment, but they were then monitored for a further 1 week and 1 month after drug withdrawal. We analyzed the geometry and composition of the coronary artery, vascular reactivity and plasma redox status. Esmolol significantly decreased wall thickness (medial layer thickness and cell count), external diameter and cross-sectional area of the artery, and this effect persisted 1 month after drug withdrawal. Esmolol significantly improved endothelium-dependent relaxation by ACh (10-9-10-4 mol/L); this effect persisted 1 week (10-9-10-4 mol/L) and 1 month (10-6-10-4 mol/L) after withdrawal. Esmolol reduced the contraction induced by 5-HT (3 × 10-8-3 × 10-5 mol/L), and this effect persisted 1 week after withdrawal (10-6-3 × 10-5 mol/L). Esmolol increased nitrates and reduced glutathione, and it decreased malondialdehyde and carbonyls; this enhancement was maintained 1 month after withdrawal. This study shows that the effect of esmolol on coronary remodeling is persistent after treatment withdrawal in SHRs, and the improvement in plasma oxidative status can be implicated in this effect.

Women during Lactation Reduce Their Physical Activity and Sleep Duration Compared to Pregnancy.

Sleep, mental health and physical activity are fundamental for wellbeing, and some of these factors are interrelated. However, these aspects are not usually considered during pregnancy and lactation, which are particularly vulnerable periods. Therefore, our aims were to conduct a cross sectional study to assess the psychological capital, quality of life, sleep hygiene and physical activity in a cohort of women during pregnancy and lactation periods. Women were recruited from Spanish maternity and lactation non-profit associations and social networks through an online platform with the following inclusion criteria: pregnancy (in any period of gestation) or breastfeeding period (≤6 months postpartum). The cohort was categorized into ≤12 weeks of gestation (n = 32), >12 weeks of gestation (n = 119) and lactation (n = 60). The women self-reported the sociodemographic data, obstetric complications and full breastfeeding or mixed practices. In addition, women responded to the psychological capital instrument, the health survey form, the Pittsburg sleep quality index and the pregnancy physical activity questionnaire. Overall, the groups were similar in sociodemographic variables. Women in the lactation period perceived lower social support compared to the gestation period. No statistically differences were found between groups in the psychological capital nor in the general health survey form. However, the models adjusted by employment and civil and economic status and perceived social support, demonstrated that the sleep duration negatively associated with the lactation period (ß = 1.13 ± 0.56; p-Value = 0.016), and the household tasks were associated with this period (ß = 2147.3 ± 480.7; p-Value < 0.001). A decrease in physical daily activities were associated with both the end of gestation and the lactation periods. In addition, the decreasing total activity was associated with the lactation period (ß = 1683.67 ± 688.05; p-Value = 0.016). In conclusion, during lactation, the poorer sleep and physical activity, together with a lower social support of the woman, may lead to deficient mental health adjustment. Our data suggest that women are at higher risk of vulnerability in lactation compared to the gestation period.

Effect of Chronic Altitude Hypoxia on Redox Balance in Preadolescents and Adolescents / Efectos de la hipoxia altitudinal crónica sobre el balance Redox de preadolescentes y adolescentes / Efeito da exposição crônica à hipóxia de altitude no equilíbrio oxidante/antioxidante de crianças e adolescentes

Living at high altitude increases oxidative stress. Likewise, growth and maturation during adolescence can increase levels of reactive oxygen species (ros). Changes in redox profiles have been evaluated in adults living at high altitudes; however, there are no studies on these changes in peripubertal populations living at moderate altitudes, we determine how living at moderate altitude affects the oxidative and inflammatory status of healthy preadolescents and adolescents. Materials and Methods: A cross-sectional study was conducted in healthy male Colombian preadolescents and adoles-cents (9­18 years old, Tanner scale classification) who lived at low altitude (n = 26) or moderate altitude (n = 26). Plasma oxidative and inflammatory status was assessed via spectrophotometry. Oxidative markers included malondialdehyde, 4-hydroxy-trans-2-nonenal, and carbonyl groups. Antioxidant markers included total antioxidant status, glutathione, catalase, superoxide dismutase, uric acid, and thiols. Inflammatory markers included interleukins-1, -6, and -10 and tumor necrosis factor. Results:Only uric acid levels were higher in adolescents (5.34 and 5.66 mg/dl) compared to preadolescents (3.85 and 4.07 mg/dl) in both moderate and low altitude groups, respectively. Participants who lived at mod-erate altitude presented significantly higher levels of malondialdehyde (4.82 and 3.73 nM/mg protein) and lower level of glutathione and thiols (1.21 and 1.26 µmol/mg protein) than in those at low altitude. Their inflammatory profiles did not differ. Conclusion: Oxidant profiles increased in peripubertal popu-lations residing at moderate altitude; this could be owing to antioxidant consumption by ros and active metabolism during puberty.

vivir en altura es un factor que se asocia con el estrés oxidativo. El crecimiento y la maduración pueden ser un estresor adicional. Es insuficiente la evidencia sobre alteraciones del perfil redox en peripúberes residentes a altitudes moderadas. El propósito fue establecer el efecto de vivir en una altitud moderada sobre el perfil redox e inflamatorio en preadolescentes y adolescentes sanos. Materiales y métodos: estudio transversal en varones preadolescentes y adolescentes sanos (9-18 años) que viven en altitud baja (n = 26) o altitud moderada (n = 26). El estado oxidativo plasmático se evaluó mediante espectrofotometría a través de marcadores de oxidación (malondialdehído e hidroxinonenal y grupos carbonilo) y antioxidantes (estado antioxidante total, glutatión, catalasa, superóxido dismutasa, ácido úrico y tioles). El perfil inflamatorio se midió con interleucinas 1, 6, 10 y factor de necrosis tumoral α. Resultados: solo el ácido úrico fue diferente entre adolescentes (5.34 y 5.66 mg/dl para moderada y baja altitud, respectivamente) y preadolescentes (3.85 y 4.07 mg/dl para moderada y baja altitud, res-pectivamente). El grupo de preadolescentes y adolescentes de moderada altitud presentó niveles más altos de malondialdehído (4.82 y 3.73 nM/mg de proteína, respectivamente) y menor glutatión y tioles (1.21 y 1.26 µmol/mg de proteína), en comparación con sus contrapartes de baja altitud. Conclusión: las poblaciones peripúberes que residen en una altitud moderada presentan un perfil oxidante más alto, lo que puede estar relacionado con la depleción de antioxidantes, por una mayor producción de especies reactivas de oxígeno relacionada con la hipoxia y el metabolismo activo de la pubertad.

viver em grandes altitudes é um fator de estresse associado ao estresse oxidativo. Durante a adolescência, os processos de crescimento e maturação podem aumentar as espécies reativas de oxi-gênio. Alterações no perfil redox foram estudadas em adultos expostos a grandes altitudes, mas não em populações peripubertais vivendo em altitudes moderadas. Nosso objetivo é estabelecer o efeito de viver em uma altitude moderada sobre o estado oxidativo e inflamatório em pré-adolescentes e adolescentes saudáveis. Materiais and métodos: foi realizado um estudo transversal em pré-adolescentes e adolescen-tes colombianos saudáveis (9-18 anos, na escala de classificação de Tanner) que viviam em baixa altitude (n = 26) ou altitude moderada (n = 26). O estado oxidativo e inflamatório do plasma foi avaliado por espectrofotometria: 1) Marcadores de oxidação: grupos Malondialdeído + 4-hidroxi-trans-2-nonenal e carbonila; 2) antioxidantes: estado antioxidante total, glutationa, catalase, superóxido dismutase, ácido úrico e tióis; 3) Marcadores de inflamação: interleucinas 1, 6, 10 e fator de necrose tumoral α. Resultados:apenas o ácido úrico foi maior em adolescentes (5,34 e 5,66 mg/dl) em comparação com pré-adolescentes (3,85 e 4,07 mg/dl) dos grupos de altitude moderada e baixa, respectivamente. A altitude moderada apre-sentou níveis significativamente maiores de Malondialdeído (4,82 e 3,73 nM/mg de proteína), e menores níveis de Glutationa e tióis (1,21 e 1,26 µmol/mg de proteína), em comparação com a baixa altitude. Nenhuma diferença foi detectada no perfil inflamatório. Conclusão: as populações peripubertais que residem em altitude moderada apresentam maior perfil oxidante, o que pode estar relacionado ao con-sumo de antioxidantes devido à maior produção de ros relacionada à hipóxia e ao metabolismo ativo por volta da puberdade.

Elevated Vascular Sympathetic Neurotransmission and Remodelling Is a Common Feature in a Rat Model of Foetal Programming of Hypertension and SHR.

Hypertension is of unknown aetiology, with sympathetic nervous system hyperactivation being one of the possible contributors. Hypertension may have a developmental origin, owing to the exposure to adverse factors during the intrauterine period. Our hypothesis is that sympathetic hyperinnervation may be implicated in hypertension of developmental origins, being this is a common feature with essential hypertension. Two-animal models were used: spontaneously hypertensive rats (SHR-model of essential hypertension) and offspring from dams exposed to undernutrition (MUN-model of developmental hypertension), with their respective controls. In adult males, we assessed systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), sympathetic nerve function (3H-tritium release), sympathetic innervation (immunohistochemistry) and vascular remodelling (histology). MUN showed higher SBP/DBP, but not HR, while SHR exhibited higher SBP/DBP/HR. Regarding the mesenteric arteries, MUN and SHR showed reduced lumen, increased media and adventitial thickness and increased wall/lumen and connective tissue compared to respective controls. Regarding sympathetic nerve activation, MUN and SHR showed higher tritium release compared to controls. Total tritium tissue/tyrosine hydroxylase detection was higher in SHR and MUN adventitia arteries compared to respective controls. In conclusion, sympathetic hyperinnervation may be one of the contributors to vascular remodelling and hypertension in rats exposed to undernutrition during intrauterine life, which is a common feature with spontaneous hypertension.

Vasoactive Properties of a Cocoa Shell Extract: Mechanism of Action and Effect on Endothelial Dysfunction in Aged Rats.

Cocoa has cardiovascular beneficial effects related to its content of antioxidant phytochemicals. Cocoa manufacturing produces large amounts of waste, but some by-products may be used as ingredients with health-promoting potential. We aimed to investigate the vasoactive actions of an extract from cocoa shell (CSE), a by-product containing theobromine (TH), caffeine (CAF) and protocatechuic acid (PCA) as major phytochemicals. In carotid and iliac arteries from 5-month and 15-month-old rats, we investigated CSE vasoactive properties, mechanism of action, and the capacity of CSE, TH, CAF and PCA to improve age-induced endothelial dysfunction. Vascular function was evaluated using isometric tension recording and superoxide anion production by dihydroethidium (DHE) staining and confocal microscopy. CSE caused endothelium-dependent vasorelaxation, blocked by L-NAME, but not indomethacin, regardless of sex, age, or vessel type. CSE maximal responses and EC50 were significantly lower compared to acetylcholine (ACh). Arterial preincubation with CSE, TH, CAF or PCA, significantly reduced the number of vascular DHE-positive cells. Compared to adult males, iliac arteries from aged males exhibited reduced ACh concentration-dependent vasodilatation but larger CSE responses. In iliac arteries from aged male and female rats, preincubation with 10-4 M CSE and PCA, but not TH or CAF, improved ACh-relaxations. In conclusion, CSE has vasodilatory properties associated with increased nitric oxide bioavailability, related to its antioxidant phytochemicals, being particularly relevant PCA. Therefore, CSE is a potential food ingredient for diseases related to endothelial dysfunction.

Fetal Undernutrition Modifies Vascular RAS Balance Enhancing Oxidative Damage and Contributing to Remodeling.

Fetal stress is known to increase susceptibility to cardiometabolic diseases and hypertension in adult age in a process known as fetal programming. This study investigated the relationship between vascular RAS, oxidative damage and remodeling in fetal programming. Six-month old Sprague-Dawley offspring from mothers that were fed ad libitum (CONTROL) or with 50% intake during the second half of gestation (maternal undernutrition, MUN) were used. qPCR or immunohistochemistry were used to obtain the expression of receptors and enzymes. Plasma levels of carbonyls were measured by spectrophotometry. In mesenteric arteries from MUN rats we detected an upregulation of ACE, ACE2, AT1 receptors and NADPH oxidase, and lower expression of AT2, Mas and MrgD receptors compared to CONTROL. Systolic and diastolic blood pressure and plasma levels of carbonyls were higher in MUN than in CONTROL. Vascular morphology evidenced an increased media/lumen ratio and adventitia/lumen ratio, and more connective tissue in MUN compared to CONTROL. In conclusion, fetal undernutrition indices RAS alterations and oxidative damage which may contribute to the remodeling of mesenteric arteries, and increase the risk of adverse cardiovascular events and hypertension.

Implication of RAS in Postnatal Cardiac Remodeling, Fibrosis and Dysfunction Induced by Fetal Undernutrition.

Fetal undernutrition is a risk factor for cardiovascular diseases. Male offspring from rats exposed to undernutrition during gestation (MUN) exhibit oxidative stress during perinatal life and develop cardiac dysfunction in ageing. Angiotensin-II is implicated in oxidative stress-mediated cardiovascular fibrosis and remodeling, and lactation is a key developmental window. We aimed to assess if alterations in RAS during lactation participate in cardiac dysfunction associated with fetal undernutrition. Control dams received food ad libitum, and MUN had 50% nutrient restriction during the second half of gestation. Both dams were fed ad libitum during lactation, and male offspring were studied at weaning. We assessed: ventricular structure and function (echocardiography); blood pressure (intra-arterially, anesthetized rats); collagen content and intramyocardial artery structure (Sirius red, Masson Trichromic); myocardial and intramyocardial artery RAS receptors (immunohistochemistry); plasma angiotensin-II (ELISA) and TGF-ß1 protein expression (Western Blot). Compared to Control, MUN offspring exhibited significantly higher plasma Angiotensin-II and a larger left ventricular mass, as well as larger intramyocardial artery media/lumen, interstitial collagen and perivascular collagen. In MUN hearts, TGF-ß1 tended to be higher, and the end-diastolic diameter and E/A ratio were significantly lower with no differences in ejection fraction or blood pressure. In the myocardium, no differences between groups were detected in AT1, AT2 or Mas receptors, with MrgD being significantly lower in the MUN group. In intramyocardial arteries from MUN rats, AT1 and Mas receptors were significantly elevated, while AT2 and MrgD were lower compared to Control. Conclusions. In rats exposed to fetal undernutrition, RAS disbalance and associated cardiac remodeling during lactation may set the basis for later heart dysfunction.

Sex Differences in Placental Protein Expression and Efficiency in a Rat Model of Fetal Programming Induced by Maternal Undernutrition.

Fetal undernutrition programs cardiometabolic diseases, with higher susceptibility in males. The mechanisms implicated are not fully understood and may be related to sex differences in placental adaptation. To evaluate this hypothesis, we investigated placental oxidative balance, vascularization, glucocorticoid barrier, and fetal growth in rats exposed to 50% global nutrient restriction from gestation day 11 (MUN, n = 8) and controls (n = 8). At gestation day 20 (G20), we analyzed maternal, placental, and fetal weights; oxidative damage, antioxidants, corticosterone, and PlGF (placental growth factor, spectrophotometry); and VEGF (vascular endothelial growth factor), 11ß-HSD2, p22phox, XO, SOD1, SOD2, SOD3, catalase, and UCP2 expression (Western blot). Compared with controls, MUN dams exhibited lower weight and plasma proteins and higher corticosterone and catalase without oxidative damage. Control male fetuses were larger than female fetuses. MUN males had higher plasma corticosterone and were smaller than control males, but had similar weight than MUN females. MUN male placenta showed higher XO and lower 11ß-HSD2, VEGF, SOD2, catalase, UCP2, and feto-placental ratio than controls. MUN females had similar feto-placental ratio and plasma corticosterone than controls. Female placenta expressed lower XO, 11ß-HSD2, and SOD3; similar VEGF, SOD1, SOD2, and UCP2; and higher catalase than controls, being 11ß-HSD2 and VEGF higher compared to MUN males. Male placenta has worse adaptation to undernutrition with lower efficiency, associated with oxidative disbalance and reduced vascularization and glucocorticoid barrier. Glucocorticoids and low nutrients may both contribute to programming in MUN males.

Fetal Undernutrition Induces Resistance Artery Remodeling and Stiffness in Male and Female Rats Independent of Hypertension.

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.

First trimester elevations of hematocrit, lipid peroxidation and nitrates in women with twin pregnancies who develop preeclampsia.

Twin pregnancies are considered a risk factor for preeclampsia, an obstetric complication with high maternal and infant morbi-mortality. We hypothesize that alterations in maternal hematocrit, plasma lipid peroxidation and nitrates in the first trimester of pregnancy are associated with preeclampsia development in twin pregnancies. Blood samples were extracted from 102 healthy women with twin pregnancies at tenth week of gestation to assess hematological parameters and plasma levels of malondialdehyde and nitrates. Logistic regression model showed an association between red blood cells (OR = 38.8; p-value = 0.009), hematocrit (OR = 1.6; p-value = 0.017), malondialdehyde (OR = 1.5; p-value = 0.002), and nitrates (OR = 1.1; p-value = 0.045) and preeclampsia development. These parameters are potential biomarkers for early preeclampsia detection in twin pregnancies. Future research is needed to assess their value in predictive algorithms.

Male fetal sex is associated with low maternal plasma anti-inflammatory cytokine profile in the first trimester of healthy pregnancies.

Male fetal sex associates with higher rates of materno-fetal complications. Inflammation and inadequate vasoactive responses are mechanisms implicated in obstetric complications, and alterations in maternal plasma cytokine profile and nitric oxide (NO) metabolites are potential predictive biomarkers. We aimed to assess if these parameters are influenced by fetal sex. A prospective, observational study was carried out in 85 healthy pregnant women with singleton pregnancies in the first trimester of gestation. A blood sample was extracted at the tenth week of gestation. In plasma, we assessed: 1) cytokines (micro-array): pro-inflammatory (IL1α, IL1 ß, IL6, TNFα), anti-inflammatory (IL4, IL10, IL13), and chemoattractant (IL8, MCP1, IFNγ), and 2) NO metabolites (liquid chromatography-tandem mass spectrometry and Griess reaction): L-arginine, ADMA, SDMA, nitrates (NOx). Women with a male fetus (n = 50) exhibited, compared with those with a female (n = 35): higher IL1ß (OR = 1.09 with 95% CI: 0.97-1.28), and lower IL13 (OR = 0.93 with 95% CI: 0.87-0.99), and higher plasma NOx (OR = 1.14 with 95% CI: 1.03-1.31). Our data suggest that fetal sex influences maternal plasma cytokine profile and NO in early pregnancy. Women with a male fetus may have a worse capacity to counteract an inflammatory response. They may have better vasodilator capacity, but in the presence of an oxidative environment, a higher nitrosative damage may occur. These data reinforce the need to include sex as variable in predictive models.

Influence of Maternal Age and Gestational Age on Breast Milk Antioxidants During the First Month of Lactation.

Breast milk (BM) is beneficial due to its content in a wide range of different antioxidants, particularly relevant for preterm infants, who are at higher risk of oxidative stress. We hypothesize that BM antioxidants are adapted to gestational age and are negatively influenced by maternal age. Fifty breastfeeding women from two hospitals (Madrid, Spain) provided BM samples at days 7, 14 and 28 of lactation to assess total antioxidant capacity (ABTS), thiol groups, reduced glutathione (GSH), superoxide dismutase (SOD) and catalase activities, lipid peroxidation (malondialdehyde, MDA + 4-Hydroxy-Trans-2-Nonenal, HNE), protein oxidation (carbonyl groups) (spectrophotometry) and melatonin (ELISA). Mixed random-effects linear regression models were used to study the influence of maternal and gestational ages on BM antioxidants, adjusted by days of lactation. Regression models evidenced a negative association between maternal age and BM melatonin levels (ß = -7.4 ± 2.5; p-value = 0.005); and a negative association between gestational age and BM total antioxidant capacity (ß = -0.008 ± 0.003; p-value = 0.006), SOD activity (ß = -0.002 ± 0.001; p-value = 0.043) and protein oxidation (ß = -0.22 ± 0.07; p-value = 0.001). In conclusion, BM antioxidants are adapted to gestational age providing higher levels to infants with lower degree of maturation; maternal ageing has a negative influence on melatonin, a key antioxidant hormone.