Our Publications

Check out the articles published by members of our group in the last 5 years.

Publicaciones MET-VASC

2023

  • Finerenone protects against progression of kidney and cardiovascular damage in a model of type 1 diabetes through modulation of proinflammatory and osteogenic factors.
    Sanz-Gómez M, Manzano-Lista FJ, Vega-Martín E, González-Moreno D, Alcalá M, Gil-Ortega M, Somoza B, Pizzamiglio C, Ruilope LM, Aránguez I, Kolkhof P, Kreutz R, Fernández-Alfonso MS.Biomed Pharmacother. 2023 Oct 11;168:115661. doi: 10.1016/j.biopha.2023.115661
The non-steroidal mineralocorticoid receptor antagonist (MRA) finerenone (FIN) improves kidney and cardiovascular outcomes in patients with chronic kidney disease (CKD) in type 2 diabetes (T2D). We explored the effect of FIN in a novel model of type 1 diabetic Munich Wistar Frömter (MWF) rat (D) induced by injection of streptozotocin (15 mg/kg) and additional exposure to a high-fat/high-sucrose diet. Oral treatment with FIN (10 mg/kg/day in rat chow) in diabetic animals (D-FIN) was compared to a group of D rats receiving no treatment and a group of non-diabetic untreated MWF rats (C) (n = 7-10 animals per group). After 6 weeks, D and D-FIN exhibited significantly elevated blood glucose levels (271.7 ± 67.1 mg/dl and 266.3 ± 46.8 mg/dl) as compared to C (110.3 ± 4.4 mg/dl; p < 0.05). D showed a 10-fold increase of kidney damage markers Kim-1 and Ngal which was significantly suppressed in D-FIN. Blood pressure, pulse wave velocity (PWV) and arterial collagen deposition were lower in D-FIN, associated to an improvement in endothelial function due to a reduction in pro-contractile prostaglandins, as well as reactive oxygen species (ROS) and inflammatory cytokines (IL-1, IL-6, TNFα and TGFβ) in perivascular and perirenal adipose tissue (PVAT and PRAT, respectively). In addition, FIN restored the imbalance observed in CKD between the procalcifying BMP-2 and the nephroprotective BMP-7 in plasma, kidney, PVAT, and PRAT. Our data show that treatment with FIN improves kidney and vascular damage in a new rat model of DKD with T1D associated with a reduction in inflammation, fibrosis and osteogenic factors independently from changes in glucose homeostasis.
  • Short-term dietary intervention improves endothelial dysfunction induced by high-fat feeding in mice through upregulation of the AMPK-CREB signaling pathway.
    González-Blázquez R, Gil-Ortega M, Alcalá M, González-Moreno D, Viana M, Chowen JA, Sanz-Gómez M, Fernández-Alfonso MS, Somoza B. Acta Physiol (Oxf). 2023 Aug 8:e14023. doi: 10.1111/apha.14023.

Aim: In addition to functioning as an energy sensor switch, AMPK plays a key role in the maintenance of cardiovascular homeostasis. However, obesity disrupts AMPK signaling, contributing to endothelial dysfunction and cardiovascular disease. This study aimed to elucidate if a short-term dietary intervention consisting in replacing the high-fat diet with a standard diet for 2 weeks could reverse obesity-induced endothelial dysfunction via AMPK-CREB activation.

Methods: For this, 5-week-old male C57BL6J mice were fed a standard (Chow) or a high-fat (HF) diet for 8 weeks. The HF diet was replaced by the chow diet for the last 2 weeks in half of HF mice, generating 3 groups: Chow, HF and HF-Chow. Vascular reactivity and western-blot assays were performed in the thoracic aorta.

Results: Returning to a chow diet significantly reduced body weight and glucose intolerance. Relaxant responses to acetylcholine and the AMPK activator (AICAR) were significantly impaired in HF mice but improved in HF-Chow mice. The protein levels of AMPKα, p-CREB and antioxidant systems (heme oxygenase-1 (HO-1) and catalase) were significantly reduced in HF but normalized in HF-Chow mice.

Conclusion: Improving dietary intake by replacing a HF diet with a standard diet improves AMPK-mediated responses due to the upregulation of the AMPK/CREB/HO-1 signaling pathway.

  • Angiotensin II type 2 receptor as a novel activator of brown adipose tissue in obesity. 
    Alvarez-Gallego F, González-Blázquez R, Gil-Ortega M, Somoza B, Calderón-Dominguez M, Moratinos J, Garcia-Garcia V, Fernández P, González-Moreno D, Viana M, Alcalá M. Biofactors. 2023 Jun 7. doi: 10.1002/biof.1981. Epub ahead of print.

The angiotensin II type 2 receptor (AT2R) exerts vasorelaxant, anti-inflammatory, and antioxidant properties. In obesity, its activation counterbalances the adverse cardiovascular effects of angiotensin II mediated by the AT1R. Preliminary results indicate that it also promotes brown adipocyte differentiation in vitro. Our hypothesis is that AT2R activation could increase BAT mass and activity in obesity. Five-week-old male C57BL/6J mice were fed a standard or a high-fat (HF) diet for 6 weeks. Half of the animals were treated with compound 21 (C21), a selective AT2R agonist, (1 mg/kg/day) in the drinking water. Electron transport chain (ETC), oxidative phosphorylation, and UCP1 proteins were measured in the interscapular BAT (iBAT) and thoracic perivascular adipose tissue (tPVAT) as well as inflammatory and oxidative parameters. Differentiation and oxygen consumption rate (OCR) in the presence of C21 was tested in brown preadipocytes. In vitro, C21-differentiated brown adipocytes showed an AT2R-dependent increase of differentiation markers (Ucp1, Cidea, Pparg) and increased basal and H+ leak-linked OCR. In vivo, HF-C21 mice showed increased iBAT mass compared to HF animals. Both their iBAT and tPVAT showed higher protein levels of the ETC protein complexes and UCP1, together with a reduction of inflammatory and oxidative markers. The activation of the AT2R increases BAT mass, mitochondrial activity, and reduces markers of tissue inflammation and oxidative stress in obesity. Therefore, insulin reduction and better vascular responses are achieved. Thus, the activation of the protective arm of the renin-angiotensin system arises as a promising tool in the treatment of obesity.

  • Generation of Highly Antioxidant Submicron Particles from Myrtus communis Leaf Extract by Supercritical Antisolvent Extraction Process. 
    Valor D, Montes A, Calderón-Domínguez M, Aghziel I, Sánchez-Gomar I, Alcalá M, Durán-Ruiz MC, Pereyra C. Antioxidants (Basel). 2023 Feb 20;12(2):530.

Submicron particles have been produced from an ethanolic extract of Myrtus communnis leaves using supercritical carbon dioxide technology, hereinafter referred to as Supercritical Antisolvent Extraction (SAE). The influence of pressure (9-20 MPa), temperature (308 and 328 K) and injection rate (3 and 8 mL/min) on the particles’ precipitation has been investigated, and it has been confirmed that increases in pressure and temperature led to smaller particle sizes. The obtained particles had a quasi-spherical shape with sizes ranging from 0.42 to 1.32 μm. Moreover, the bioactivity of the generated particles was assessed and large contents of phenolic compounds with a high antioxidant activity were measured. The particles were also subjected to in vitro studies against oxidative stress. The myrtle particles demonstrated cytoprotective properties when applied at low concentrations (1 μM) to macrophage cell lines.

  • Sex-Specific Relationships of Physical Activity and Sedentary Behaviour with Oxidative Stress and Inflammatory Markers in Young Adults. 
    Corral-Pérez J, Alcala M, Velázquez-Díaz D, Perez-Bey A, Vázquez-Sánchez MÁ, Calderon-Dominguez M, Casals C, Ponce-González JG. Int J Environ Res Public Health. 2023 Jan 4;20(2):899.

This study aims to analyse sex-specific associations of physical activity and sedentary behaviour with oxidative stress and inflammatory markers in a young-adult population. Sixty participants (21 women, 22.63 ± 4.62 years old) wore a hip accelerometer for 7 consecutive days to estimate their physical activity and sedentarism. Oxidative stress (catalase, superoxide dismutase, glutathione peroxidase, glutathione, malondialdehyde, and advanced oxidation protein products) and inflammatory (tumour necrosis factor-alpha and interleukin-6) markers were measured. Student t-tests and single linear regressions were applied. The women presented higher catalase activity and glutathione concentrations, and lower levels of advanced protein-oxidation products, tumour necrosis factor-alpha, and interleukin-6 than the men (p &lt; 0.05). In the men, longer sedentary time was associated with lower catalase activity (β = -0.315, p = 0.04), and longer sedentary breaks and higher physical-activity expenditures were associated with malondialdehyde (β = -0.308, p = 0.04). Vigorous physical activity was related to inflammatory markers in the women (tumour necrosis factor-alpha, β = 0.437, p = 0.02) and men (interleukin-6, β = 0.528, p &lt; 0.01). In conclusion, the women presented a better redox and inflammatory status than the men; however, oxidative-stress markers were associated with physical activity and sedentary behaviours only in the men. In light of this, women could have better protection against the deleterious effect of sedentarism but a worse adaptation to daily physical activity.

  • Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury. 
    Sastourné-Arrey Q, Mathieu M, Contreras X, Monferran S, Bourlier V, Gil-Ortega M, Murphy E, Laurens C, Varin A, Guissard C, Barreau C, André M, Juin N, Marquès M, Chaput B, Moro C, O’Gorman D, Casteilla L, Girousse A, Sengenès C. Nat Commun. 2023 Jan 5;14(1):80

Fibro-adipogenic progenitors (FAPs) play a crucial role in skeletal muscle regeneration, as they generate a favorable niche that allows satellite cells to perform efficient muscle regeneration. After muscle injury, FAP content increases rapidly within the injured muscle, the origin of which has been attributed to their proliferation within the muscle itself. However, recent single-cell RNAseq approaches have revealed phenotype and functional heterogeneity in FAPs, raising the question of how this differentiation of regenerative subtypes occurs. Here we report that FAP-like cells residing in subcutaneous adipose tissue (ScAT), the adipose stromal cells (ASCs), are rapidly released from ScAT in response to muscle injury. Additionally, we find that released ASCs infiltrate the damaged muscle, via a platelet-dependent mechanism and thus contribute to the FAP heterogeneity. Moreover, we show that either blocking ASCs infiltration or removing ASCs tissue source impair muscle regeneration. Collectively, our data reveal that ScAT is an unsuspected physiological reservoir of regenerative cells that support skeletal muscle regeneration, underlining a beneficial relationship between muscle and fat.

2022

  • Imbalance in Bone Morphogenic Proteins 2 and 7 Is Associated with Renal and Cardiovascular Damage in Chronic Kidney Disease. 
    Manzano-Lista FJ, Sanz-Gómez M, González-Moreno D, Vega-Martín E, Gil-Ortega M, Schulz A, Rubio MÁ, Ruiz-Hurtado G, Ruilope LM, Aránguez I, Kreutz R, Fernández-Alfonso MS. Int J Mol Sci. 2022 Dec 20;24(1):40. 

Arterial stiffness is a major vascular complication of chronic kidney disease (CKD). The development of renal damage, hypertension, and increased pulse wave velocity (PWV) in CKD might be associated with an imbalance in bone morphogenetic proteins (BMP)-2 and BMP-7. Plasma BMP-2 and BMP-7 were determined by ELISA in CKD patients (stages I-III; n = 95) and Munich Wistar Frömter (MWF) rats. Age-matched Wistar rats were used as a control. The expression of BMP-2, BMP-7, and profibrotic and calcification factors was determined in kidney and perivascular adipose tissues (PVAT). BMP-2 was higher in stage III CKD patients compared to control subjects. BMP-7 was lower at any CKD stage compared to controls, with a significant further reduction in stage III patients. A similar imbalance was observed in MWF rats together with the increase in systolic (SBP) and diastolic blood pressure (DBP), or pulse wave velocity (PWV). MWF exhibited elevated urinary albumin excretion (UAE) and renal expression of BMP-2 or kidney damage markers, Kim-1 and Ngal, whereas renal BMP-7 was significantly lower than in Wistar rats. SBP, DBP, PWV, UAE, and plasma creatinine positively correlated with the plasma BMP-2/BMP-7 ratio. Periaortic and mesenteric PVAT from MWF rats showed an increased expression of BMP-2 and profibrotic and calcification markers compared to Wistar rats, together with a reduced BMP-7 expression. BMP-2 and BMP-7 imbalance in plasma, kidney, and PVATs is associated with vascular damage, suggesting a profibrotic/pro-calcifying propensity associated with progressive CKD. Thus, their combined analysis stratified by CKD stages might be of clinical interest to provide information about the degree of renal and vascular damage in CKD.

  • Role of long non-coding RNAs in adipose tissue metabolism and associated pathologies. 
    Corral A, Alcala M, Carmen Duran-Ruiz M, I Arroba A, Ponce-Gonzalez JG, Todorčević M, Serra D, Calderon-Dominguez M, Herrero L.  Biochem Pharmacol. 2022 Oct 19:115305.

The incidence of obesity and its related disorders has increased dramatically in recent years and has become a pandemic. Adipose tissue is a crucial regulator of these diseases due to its endocrine capacity. Thus, understanding adipose tissue metabolism is essential to finding new effective therapeutic approaches. The “omic” revolution has identified new concepts about the complexity of the signaling pathways involved in the pathophysiology of adipose tissue-associated disorders. Specifically, advances in transcriptomics have allowed its application in clinical practice and primary or secondary prevention. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of adipose tissue since they can modulate gene expression at the epigenetic, transcriptional, and post-transcriptional levels. They interact with DNA, RNA, protein complexes, other non-coding RNAs, and microRNAs to regulate a wide range of physiological and pathological processes. Here, we review the emerging field of lncRNAs, including how they regulate adipose tissue biology, and discuss circulating lncRNAs, which may represent a turning point in the diagnosis and treatment of adipose tissue-associated disorders. We also highlight potential biomarkers of obesity and diabetes that could be considered as therapeutic targets

  • Unravelling the Inflammatory Processes in the Early Stages of Diabetic Nephropathy and the Potential Effect of (Ss)-DS-ONJ.
    Gómez-Jaramillo L, Cano-Cano F, Sánchez-Fernández EM, Ortiz Mellet C, García-Fernández JM, Alcalá M, Álvarez-Gallego F, Iturregui M, González-Montelongo MDC, Campos-Caro A, Arroba AI, Aguilar-Diosdado M.  Int J Mol Sci. 2022 Jul 30;23(15):8450.

Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) in the early stages of the disease. The authors demonstrate that the glycolipid mimetic (Ss)-DS-ONJ is able to abolish inflammation via the induction of autophagy flux and provokes the inhibition of inflammasome complex in ex vivo and in vitro models, using adult kidney explants from BB rats. The contribution of (Ss)-DS-ONJ to reducing inflammatory events is mediated by the inhibition of classical stress kinase pathways and the blocking of inflammasome complex activation. The (Ss)-DS-ONJ treatment is able to inhibit the epithelial-to-mesenchymal transition (EMT) progression, but only when the IL18 levels are reduced by the treatment. These findings suggest that (Ss)-DS-ONJ could be a novel, and multifactorial treatment for DN.

  • Adult kidney explants is a physiologic model for studying diabetic nephropathy.
    Gómez-Jaramillo L, Cano-Cano F, Campos-Caro A, Álcala M, Álvarez-Gallego F, Arroba AI, Aguilar-Diosdado M. Life Sci. 2022 Jul 1;300:120575.

Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) in the early stages of the disease. In vitro approach using cell lines help to understand the mechanisms involves and allow the molecular and biochemical processes. Adult kidney (AK) explants remain an essential instrument for advancing our understanding of the molecular and cellular regulation of signalling pathways from an organotipic view with physiological system interaction integrated. AK explants from T1DM animal model (BB rat) are obtained by slicing central kidney area preserving the organ’s cytoarchitecture and reproduce the classical events detected during the DN in an in vivo model such as inflammation, epithelial-mesenchymal transition (EMT) processes by the modulation of a-SMA and e-Cadherin among others which have been determined by qRT-PCR, western-blot and immunohistochemistry. In this regard, AK explants reproduce the signalling pathways involve in DN progression (proinflammatory NFkB and inflammasome complex). This work demonstrates AK explants is a physiological experimental approach for studying the development and progression of DN. Furthermore, the inflammatory processes in AK explants under a diabetic environment and/or BB rats could be modulated by potential treatments for DN.

  • Effects of saturated versus unsaturated fatty acids on metabolism, gliosis, and hypothalamic leptin sensitivity in male mice.
    Fernández-Felipe J, Valencia-Avezuela M, Merino B, Somoza B, Cano V, Sanz-Martos AB, Frago LM, Fernández-Alfonso MS, Ruiz-Gayo M, Chowen JA.  Nutr Neurosci. 2022 Feb 6:1-14.

Background: Development of obesity and its comorbidities is not only the result of excess energy intake, but also of dietary composition. Understanding how hypothalamic metabolic circuits interpret nutritional signals is fundamental to advance towards effective dietary interventions.

Objective: We aimed to determine the metabolic response to diets enriched in specific fatty acids.

Methods: Male mice received a diet enriched in unsaturated fatty acids (UOLF) or saturated fatty acids (SOLF) for 8 weeks.

Results: UOLF and SOLF mice gained more weight and adiposity, but with no difference between these two groups. Circulating leptin levels increased on both fatty acid-enriched diet, but were higher in UOLF mice, as were leptin mRNA levels in visceral adipose tissue. In contrast, serum non-esterified fatty acid levels only rose in SOLF mice. Hypothalamic mRNA levels of NPY decreased and of POMC increased in both UOLF and SOLF mice, but only SOLF mice showed signs of hypothalamic astrogliosis and affectation of central fatty acid metabolism. Exogenous leptin activated STAT3 in the hypothalamus of all groups, but the activation of AKT and mTOR and the decrease in AMPK activation in observed in controls and UOLF mice was not found in SOLF mice.

Conclusions: Diets rich in fatty acids increase body weight and adiposity even if energy intake is not increased, while increased intake of saturated and unsaturated fatty acids differentially modify metabolic parameters that could underlie more long-term comorbidities. Thus, more understanding of how specific nutrients affect metabolism, weight gain, and obesity associated complications is necessary.

  • miR-16-5p Suppression Protects Human Cardiomyocytes against Endoplasmic Reticulum and Oxidative Stress-Induced Injury.
    Toro R, Pérez-Serra A, Mangas A, Campuzano O, Sarquella-Brugada G, Quezada-Feijoo M, Ramos M, Alcalá M, Carrera E, García-Padilla C, Franco D, Bonet F. Int J Mol Sci. 2022 Jan 18;23(3):1036.
Oxidative stress, defined as the excess production of reactive oxygen species (ROS) relative to antioxidant defense, plays a significant role in the development of cardiovascular diseases. Endoplasmic reticulum (ER) stress has emerged as an important source of ROS and its modulation could be cardioprotective. Previously, we demonstrated that miR-16-5p is enriched in the plasma of ischemic dilated cardiomyopathy (ICM) patients and promotes ER stress-induced apoptosis in cardiomyocytes in vitro. Here, we hypothesize that miR-16-5p might contribute to oxidative stress through ER stress induction and that targeting miR-16-5p may exert a cardioprotective role in ER stress-mediated cardiac injury. Analysis of oxidative markers in the plasma of ICM patients demonstrates that oxidative stress is associated with ICM. Moreover, we confirm that miR-16-5p overexpression promotes oxidative stress in AC16 cardiomyoblasts. We also find that, in response to tunicamycin-induced ER stress, miR-16-5p suppression decreases apoptosis, inflammation and cardiac damage via activating the ATF6-mediated cytoprotective pathway. Finally, ATF6 is identified as a direct target gene of miR-16-5p by dual-luciferase reporter assays. Our results indicate that miR-16-5p promotes ER stress and oxidative stress in cardiac cells through regulating ATF6, suggesting that the inhibition of miR-16-5p has potential as a therapeutic approach to protect the heart against ER and oxidative stress-induced injury.
  • Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK-eNOS-NO pathway.
    Sanz-Gómez M, Aledavood E, Beroiz-Salaverri M, Lagartera L, Vega-Martín E, Gil-Ortega M, Cumella J, Pérez C, Luque FJ, Estarellas C, Fernández-Alfonso MS, Castro A. Sci Rep. 2022 Mar 10;12(1):4225
Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK-eNOS-NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity.

2021

  • AT2R stimulation with C21 prevents arterial stiffening and endothelial dysfunction in the abdominal aorta from mice fed a high-fat diet.
    González-Blázquez R, Alcalá M, Cárdenas-Rebollo JM, Viana M, Steckelings UM, Boisvert WA, Unger T, Fernández-Alfonso MS, Somoza B, Gil-Ortega M.  Clin Sci (Lond). 2021 Dec 22;135(24):2763-2780
The aim of the present study was to evaluate the effect of Compound 21 (C21), a selective AT2R agonist, on the prevention of endothelial dysfunction, extracellular matrix (ECM) remodeling and arterial stiffness associated with diet-induced obesity (DIO). Five-week-old male C57BL/6J mice were fed a standard (Chow) or high-fat diet (HF) for 6 weeks. Half of the animals of each group were simultaneously treated with C21 (1 mg/kg/day, in the drinking water), generating four groups: Chow C, Chow C21, HF C, and HF C21. Vascular function and mechanical properties were determined in the abdominal aorta. To evaluate ECM remodeling, collagen deposition and TGF-β1 concentrations were determined in the abdominal aorta and the activity of metalloproteinases (MMP) 2 and 9 was analyzed in the plasma. Abdominal aortas from HF C mice showed endothelial dysfunction as well as enhanced contractile but reduced relaxant responses to Ang II. This effect was abrogated with C21 treatment by preserving NO availability. A left-shift in the tension-stretch relationship, paralleled by an augmented β-index (marker of intrinsic arterial stiffness), and enhanced collagen deposition and MMP-2/-9 activities were also detected in HF mice. However, when treated with C21, HF mice exhibited lower TGF-β1 levels in abdominal aortas together with reduced MMP activities and collagen deposition compared with HF C mice. In conclusion, these data demonstrate that AT2R stimulation by C21 in obesity preserves NO availability and prevents unhealthy vascular remodeling, thus protecting the abdominal aorta in HF mice against the development of endothelial dysfunction, ECM remodeling and arterial stiffness.
  • Prevention of Teratogenesis in Pregnancies of Obese Rats by Vitamin E Supplementation.
    Alcala M, Bolado VE, Sánchez-Vera I, Clapés S, Dasí F, Sáez G, Carrera E, Alvarez-Gallego F, Loeken MR, Viana M.  Antioxidants (Basel). 2021 Jul 23;10(8):1173

Congenital malformations are a common adverse outcome in pregnancies complicated by pregestational obesity, although the underlying mechanisms are still unrevealed. Our aim was to study the effect of oxidative stress in obesity-induced teratogenesis. Wistar rats were fed a high-fat diet for 13 weeks, with (OE group) or without (O group) vitamin E supplementation. Then, rats were mated and sacrificed at day 11.5 of gestation. Embryos from O dams presented a 25.9 ± 3.5% rate of malformations (vs. 8.7 ± 3.4% in C rats), which was reduced in the OE group (11.5 ± 2.3%). Pregestational obesity induced hepatic protein and DNA oxidation and a decline in antioxidant enzymes. Importantly, glutathione content was also decreased, limiting the availability of this antioxidant in the embryos. Vitamin E supplementation efficiently maintained glutathione levels in the obese mothers, which could be used in their embryos to prevent oxidation-induced malformations. To test the effect of decreasing glutathione levels alone in a cell culture model of neuroepithelium, murine embryonic stem cells (ESC) were induced to form neuronal precursors and glutathione synthesis was inhibited with the gamma-glutamylcysteine synthesis inhibitor, buthionine sulfoximine (BSO). BSO inhibited the expression of Pax3, a gene required for neural tube closure that is also inhibited by oxidative stress. Taken together, our data indicate that obesity causes malformations through the depletion of maternal glutathione, thereby decreasing glutathione-dependent free radical scavenging in embryos, which can be prevented by vitamin E supplementation.

  • C21 preserves endothelial function in the thoracic aorta from DIO mice: role for AT2, Mas and B2 receptors.
    González-Blázquez R, Alcalá M, Fernández-Alfonso MS, Steckelings UM, Lorenzo MP, Viana M, Boisvert WA, Unger T, Gil-Ortega M, Somoza B.  Clin Sci (Lond). 2021 May 14;135(9):1145-1163
Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.
  • The real impact of COVID-19 on community pharmacy professionals as part of the primary health care frontier workforce in Spain.
    Manouchehri M, Bhamra SK, Fernández-Alfonso M, Gil-Ortega M.  J Pharm Pharmacogn Res. 2021. 9(6), 878-891

Context: Spain was at the epicenter of the pandemic. Health centers across Spanish territory were not able to respond to non-emergency enquiries, leaving community pharmacies as the first point of patient’s contact.

Aims: To investigate the impact of COVID-19 on the mental and physical health of community pharmacy teams across Spain.

Methods: A cross-sectional observational study was performed with community pharmacy professionals throughout Spain. A questionnaire designed by our collaborator from the United Kingdom was adapted to the Spanish population and launched between October 2020 and February 2021.

Results: A total of 98 participants responded to the questionnaire. The survey showed an 80% increase in workload. The pandemic had negatively impacted the well-being of community pharmacy professionals. The survey indicated a national shortage of medicines and personal protective equipment across Spain, particularly during the first peak. To adapt to this pandora’s box of COVID-19, 96% of the pharmacies changed their settings to improve patients and staff’s safety. Most of these changes were self-financed by the pharmacy owner. The pharmacists kept up to date with information released from the pharmacists’ college, General Pharmaceutical Council and the Spanish Society of Community Pharmacists. The Public domain purchased more (79%) immune booster supplements.

Conclusions: Community pharmacy professionals have faced tremendous mental, physical and professional pressure in providing adequate personal protective equipment and medication supply to their communities. They have provided more pharmaceutical services during the crisis, although they have not been recognized as essential workers by the healthcare system.

  • Differential Deleterious Impact of Highly Saturated Versus Monounsaturated Fat Intake on Vascular Function, Structure, and Mechanics in Mice.
    Vega-Martín E, Gil-Ortega M, González-Blázquez R, Benedito S, Fernández-Felipe J, Ruiz-Gayo M, Del Olmo N, Chowen JA, Frago LM, Somoza B, Fernández-Alfonso MS.  Nutrients. 2021 Mar 19;13(3):1003.

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.

  • Antiphospholipid Antibodies From Women With Pregnancy Morbidity and Vascular Thrombosis Induce Endothelial Mitochondrial Dysfunction, mTOR Activation, and Autophagy.
    Rodríguez CM, Velásquez-Berrío M, Rúa C, Viana M, Abrahams VM, Cadavid AP, Alvarez AM. Front Physiol. 2021 Nov 29;12:706743.
Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and pregnancy morbidity (PM) obstetric events together with persistent high titers of circulating antiphospholipid antibodies (aPL). Several mechanisms that explain the development of thrombosis and PM in APS include the association of aPL with alterations in the coagulation cascade and inflammatory events. Other mechanisms disturbing cellular homeostases, such as mitochondrial dysfunction, autophagy, and cell proliferation, have been described in other autoimmune diseases. Therefore, the objective of this study was to investigate the impact of aPL from different patient populations on endothelial cell mitochondrial function, activation of the mammalian target of rapamycin (mTOR) and autophagy pathways, and cellular growth. Using an in vitro model, human umbilical vein endothelial cells (HUVECs) were treated with polyclonal immunoglobulin G (IgG) purified from the serum of women with both PM and vascular thrombosis (PM/VT), with VT only (VT), or with PM and non-criteria aPL (seronegative-obstetric APS, SN-OAPS). We included IgG from women with PM without aPL (PM/aPL-) and healthy women with previous uncomplicated pregnancies (normal human serum, NHS) as control groups. Mitochondrial function, mTOR activation, autophagy, and cell proliferation were evaluated by Western blotting, flow cytometry, and functional assays. IgG from women with PM/VT increased HUVEC mitochondrial hyperpolarization and activation of the mTOR and autophagic pathways, while IgG from patients with VT induced endothelial autophagy and cell proliferation in the absence of elevated mTOR activity or mitochondrial dysfunction. IgG from the SN-OAPS patient group had no effect on any of these HUVEC responses. In conclusion, aPL from women with PM and vascular events induce cellular stress evidenced by mitochondrial hyperpolarization and increased activation of the mTOR and autophagic pathways which may play a role in the pathogenesis of obstetric APS.

2020

  • High levels of maternal total tri-iodothyronine, and low levels of fetal free L-thyroxine and total tri-iodothyronine, are associated with altered deiodinase expression and activity in placenta with gestational diabetes mellitus.
    Gutiérrez-Vega S, Armella A, Mennickent D, Loyola M, Covarrubias A, Ortega-Contreras B, Escudero C, Gonzalez M, Alcalá M, Ramos MDP, Viana M, Castro E, Leiva A, Guzmán-Gutiérrez E. PLoS One. 2020 Nov 24;15(11):e0242743
Gestational Diabetes Mellitus (GDM) is characterized by abnormal maternal D-glucose metabolism and altered insulin signaling. Dysregulation of thyroid hormones (TH) tri-iodethyronine (T3) and L-thyroxine (T4) Hormones had been associated with GDM, but the physiopathological meaning of these alterations is still unclear. Maternal TH cross the placenta through TH Transporters and their Deiodinases metabolize them to regulate fetal TH levels. Currently, the metabolism of TH in placentas with GDM is unknown, and there are no other studies that evaluate the fetal TH from pregnancies with GDM. Therefore, we evaluated the levels of maternal TH during pregnancy, and fetal TH at delivery, and the expression and activity of placental deiodinases from GDM pregnancies. Pregnant women were followed through pregnancy until delivery. We collected blood samples during 10-14, 24-28, and 36-40 weeks of gestation for measure Thyroid-stimulating hormone (TSH), Free T4 (FT4), Total T4 (TT4), and Total T3 (TT3) concentrations from Normal Glucose Tolerance (NGT) and GDM mothers. Moreover, we measure fetal TSH, FT4, TT4, and TT3 in total blood cord at the delivery. Also, we measured the placental expression of Deiodinases by RT-PCR, western-blotting, and immunohistochemistry. The activity of Deiodinases was estimated quantified rT3 and T3 using T4 as a substrate. Mothers with GDM showed higher levels of TT3 during all pregnancy, and an increased in TSH during second and third trimester, while lower concentrations of neonatal TT4, FT4, and TT3; and an increased TSH level in umbilical cord blood from GDM. Placentae from GDM mothers have a higher expression and activity of Deiodinase 3, but lower Deiodinase 2, than NGT mothers. In conclusion, GDM favors high levels of TT3 during all gestation in the mother, low levels in TT4, FT4 and TT3 at the delivery in neonates, and increases deiodinase 3, but reduce deiodinase 2 expression and activity in the placenta.
  • Expression of Chitotriosidase in Macrophages Modulates Atherosclerotic Plaque Formation in Hyperlipidemic Mice.
    Yap J, McCurdy S, Alcala M, Irei J, Garo J, Regan W, Lee BH, Kitamoto S, Boisvert WA.  Front Physiol. 2020 Jun 23;11:714

Objective: To determine whether overexpression of the chitin degrading enzyme, chitotriosidase (CHIT1), modulates macrophage function and ameliorates atherosclerosis.

Approach and results: Using a mouse model that conditionally overexpresses CHIT1 in macrophages (CHIT1-Tg) crossbred with the Ldlr -/- mouse provided us with a means to investigate the effects of CHIT1 overexpression in the context of atherosclerosis. In vitro, CHIT1 overexpression by murine macrophages enhanced protein expression of IL-4, IL-8, and G-CSF by BMDM upon stimulation with a combination of lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Phosphorylation of ERK1/2 and Akt was also down regulated when exposed to the same inflammatory stimuli. Hyperlipidemic, Ldlr -/--CHIT1-Tg (CHIT1-OE) mice were fed a high-fat diet for 12 weeks in order to study CHIT1 overexpression in atherosclerosis. Although plaque size and lesion area were not affected by CHIT1 overexpression in vivo, the content of hyaluronic acid (HA) and collagen within atherosclerotic plaques of CHIT1-OE mice was significantly greater. Localization of both ECM components was markedly different between groups.

Conclusions: These data demonstrate that CHIT1 alters cytokine expression and signaling pathways of classically activated macrophages. In vivo, CHIT1 modifies ECM distribution and content in atherosclerotic plaques, both of which are important therapeutic targets.

  • Relevance of control diet choice in metabolic studies: impact in glucose homeostasis and vascular function.
    González-Blázquez R, Alcalá M, Fernández-Alfonso MS, Villa-Valverde P, Viana M, Gil-Ortega M, Somoza B.  Sci Rep. 2020 Feb 19;10(1):2902
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.
  • Impact of caloric restriction on AMPK and endoplasmic reticulum stress in peripheral tissues and circulating peripheral blood mononuclear cells from Zucker rats.
    Vega-Martín E, González-Blázquez R, Manzano-Lista FJ, Martín-Ramos M, García-Prieto CF, Viana M, Rubio MA, Calle-Pascual AL, Lionetti L, Somoza B, Fernández-Alfonso MS, Alcalá M, Gil-Ortega M.  J Nutr Biochem. 2020 Apr;78:108342.
The activation of endoplasmic reticulum (ER) stress and a reduction of AMP-dependent protein kinase (AMPK) phosphorylation have been described in obesity. We hypothesize that a moderate caloric restriction (CR) might contribute to reducing ER stress and increasing AMPK phosphorylation in peripheral tissues from genetically obese Zucker fa/fa rats and in peripheral blood mononuclear cells (PBMCs). Zucker Lean and Zucker fa/fa rats were fed with chow diet either ad libitum (AL) (C, as controls) or 80% of AL (CR) for 2 weeks, giving rise to four experimental groups: Lean C, Lean CR, fa/fa C and fa/fa CR. CR significantly increased AMPK phosphorylation in the liver, perirenal adipose tissue (PRAT) and PBMCs from fa/fa rats but not in the subcutaneous AT (SCAT), suggesting a reduced response of SCAT to CR. Liver samples of fa/fa rats exhibited an increased mRNA expression of PERK, EIF-2α, XBP-1(s), Chop and caspase 3, which was significantly reduced by CR. PRAT exhibited an overexpression of Edem and PDIA-4 in fa/fa rats, but only PDIA-4 expression was reduced by CR. eIF-2α phosphorylation was significantly increased in all studied tissues from fa/fa rats and reduced by CR. A negative correlation was detected between p-AMPK and p-eIF-2α in the liver, PRAT and PBMCs from fa/fa rats but not in SCAT. This study shows that a moderate CR reduces ER stress and improves AMPK phosphorylation in several peripheral tissues and in circulating PBMCs, suggesting that alterations observed in PBMCs could reflect metabolic alterations associated with obesity.
  • Sex Differences in Placental Protein Expression and Efficiency in a Rat Model of Fetal Programming Induced by Maternal Undernutrition.
    Phuthong S, Reyes-Hernández CG, Rodríguez-Rodríguez P, Ramiro-Cortijo D, Gil-Ortega M, González-Blázquez R, González MC, López de Pablo AL, Arribas SM.  Int J Mol Sci. 2020 Dec 28;22(1):237.
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), 11β-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 11β-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, 11β-HSD2, and SOD3; similar VEGF, SOD1, SOD2, and UCP2; and higher catalase than controls, being 11β-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.
    Gutiérrez-Arzapalo PY, Rodríguez-Rodríguez P, Ramiro-Cortijo D, Gil-Ortega M, Somoza B, de Pablo ÁLL, González MDC, Arribas SM.  Biomedicines. 2020 Oct 16;8(10):424.
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.
  • Finerenone Reduces Intrinsic Arterial Stiffness in Munich Wistar Frömter Rats, a Genetic Model of Chronic Kidney Disease.
    Gil-Ortega M, Vega-Martín E, Martín-Ramos M, González-Blázquez R, Pulido-Olmo H, Ruiz-Hurtado G, Schulz A, Ruilope LM, Kolkhof P, Somoza B, Kreutz R, Fernández-Alfonso MS.  Am J Nephrol. 2020;51(4):294-303

Background: Development of albuminuria and arterial stiffness in Munich Wistar Frömter (MWF) rats, a model of chronic kidney disease, is related to alterations in extracellular matrix, increased oxidative stress, and endothelial dysfunction. Finerenone (FIN), a novel, nonsteroidal, potent, and selective mineralocorticoid receptor antagonist, improves endothelial dysfunction through enhancing nitric oxide (NO) bioavailability and decreasing superoxide anion levels due to an upregulation in vascular and renal superoxide dismutase activity. We hypothesize that FIN reduces arterial stiffness in this model associated to the reduction in albuminuria and matrix metalloproteinase (MMP)-2/9 activity.

Methods: Twelve-week-old MWF rats with established albuminuria and age-matched normoalbuminuric Wistar (W) rats were treated with FIN (10 mg/kg/day, once-daily oral gavage) or with vehicle (control, C) for 4 weeks.

Results: Arterial stiffness was significantly higher in mesenteric arteries (MA) of MWF-C as compared to W-C. FIN treatment significantly lowered β-index, a measure of intrinsic stiffness independent of geometry, in MWF (βMWF-FIN = 7.7 ± 0.4 vs. βMWF-C = 9.2 ± 0.5, p < 0.05) positively correlating with urinary albumin excretion. Elastin fenestrae area in the internal elastic lamina of MA from MWF-FIN was significantly larger (+377%, p < 0.05). FIN increased plasma pro-MMP-2 and decreased plasma MMP-2 and MMP-9 activities, correlating with reductions in β-index. MA from MWF-FIN exhibited higher NO bioavailability and reduced superoxide anion levels compared to MWF-C.

Conclusion: FIN treatment reduces intrinsic arterial stiffness in MA from MWF rats associated with changes in elastin organization, normalization of MMP-2 and MMP-9 activities, and reduction of oxidative stress. Moreover, reduction of arterial stiffness correlates with reduction in albuminuria.

  • Estudio del comsumo de antibióticos en pacientes de farmacia comunitaria.
    Gómez Martínez-Sagrera, P., Cárdenas, J. M., Martín, A., Gil Ortega, M., & Somoza, B. (2020). Pharmaceutical Care España, 22(1), 3–24.

Introducción: España presenta una de las cifras más elevadas de resistencias bacterianas de Europa y paralelamente se sitúa entre los países que más antibióticos consume. La participación del farmacéutico comunitario educando al paciente sobre el buen uso de los antibióticos es importante para conseguir los objetivos marcados en el PRAN 2019-21.

Objetivos: El objetivo de este estudio fue describir en pacientes de Farmacia Comunitaria: i) el tipo de antibiótico que el paciente retiraba, ii) el perfil de prescripción en Atención Primaria y iii) el conocimiento que los pacientes tenían sobre el tratamiento.

Métodos: Se realizó un estudio observacional descriptivo en 86 pacientes de 5 oficinas de farmacia. Para ello, se elaboró un cuestionario basado en el de Molinero y cols. (2018), al que se han incluido nuevas preguntas para evaluar el conocimiento y el tipo de infección/antibiótico que tenía el paciente

Resultados: El 90% de los pacientes encuestados mostró un buen conocimiento del tratamiento prescrito (duración y/o pauta); sin embargo, sólo un 56% retiraba el antibiótico sobrante en el punto SIGRE. El tratamiento antibiótico más utilizado en las infecciones más prevalentes en nuestro estudio, infecciones respiratorias y urinarias, siguió las recomendaciones de las guías terapéuticas, amoxicilina en el 39% y fosfomicina (75% mujeres)/ciprofloxacino (80% hombres), respectivamente.

Conclusiones: Las campañas dirigidas contra la resistencia a antibióticos están empezando a dar resultados positivos tanto en los pacientes como en los prescriptores de Atención Primaria. El farmacéutico comunitario constituye un pilar fundamental en la promoción del correcto uso de antibióticos.

2019

  • Caloric restriction induces H2O2 formation as a trigger of AMPK-eNOS-NO pathway in obese rats: Role for CAMKII.
    García-Prieto CF, Gil-Ortega M, Plaza A, Manzano-Lista FJ, González-Blázquez R, Alcalá M, Rodríguez-Rodríguez P, Viana M, Aránguez I, Gollasch M, Somoza B, Fernández-Alfonso MS. Free Radic Biol Med. 2019 Aug 1;139:35-45
Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H2O2) is upregulated in yeast subjected to CR. Our aim was to assess if mild short-term CR increases vascular H2O2 formation as a link with AMPK and eNOS activation. Twelve-week old Zucker obese (fa/fa) and control Zucker lean male rats were fed a standard chow either ad libitum (AL, n=10) or with a 20% CR (CR, n=10) for two weeks. CR significantly improved relaxation to ACh in fa/fa rats because of an enhanced endogenous production of H2O2 in aortic rings (H2O2 levels fa/faAL=0.5 ± 0.05 nmol/mg vs. H2O2 levels fa/faCR=0.76 ± 0.07 nmol/mg protein; p<0.05). Expression of mitochondrial superoxide dismutase (Mn-SOD) and total SOD activity were increased in aorta from fa/fa animals after CR. In cultured aortic endothelial cells, serum deprivation or 2-deoxy-d-glucose induced a significant increase in: i) superoxide anion and H2O2 levels, ii) p-AMPK/AMPK and p-eNOS/eNOS expression and iii) nitric oxide levels. This effect was reduced by catalase and strongly inhibited by Ca2+/calmodulin-dependent kinase II (CamkII) silencing. In conclusion, we propose that mild short-term CR might be a trigger of mechanisms aimed at protecting the vascular wall by the increase of H2O2, which then activates AMPK and nitric oxide release, thus improving endothelium-dependent relaxation. In addition, we demonstrate that CAMKII plays a key role in mediating CR-induced AMPK activation through H2O2 increase.
  • Mechanisms of Impaired Brown Adipose Tissue Recruitment in Obesity.
    Alcalá M, Calderon-Dominguez M, Serra D, Herrero L, Viana M.  Front Physiol. 2019 Feb 13;10:94.
Brown adipose tissue (BAT) dissipates energy to produce heat. Thus, it has the potential to regulate body temperature by thermogenesis. For the last decade, BAT has been in the spotlight due to its rediscovery in adult humans. This is evidenced by over a hundred clinical trials that are currently registered to target BAT as a therapeutic tool in the treatment of metabolic diseases, such as obesity or diabetes. The goal of most of these trials is to activate the BAT thermogenic program via several approaches such as adrenergic stimulation, natriuretic peptides, retinoids, capsinoids, thyroid hormones, or glucocorticoids. However, the impact of BAT activation on total body energy consumption and the potential effect on weight loss is still limited. Other studies have focused on increasing the mass of thermogenic BAT. This can be relevant in obesity, where the activity and abundance of BAT have been shown to be drastically reduced. The aim of this review is to describe pathological processes associated with obesity that may influence the correct differentiation of BAT, such as catecholamine resistance, inflammation, oxidative stress, and endoplasmic reticulum stress. This will shed light on the thermogenic potential of BAT as a therapeutic approach to target obesity-induced metabolic diseases.
  • Pleiotrophin deletion alters glucose homeostasis, energy metabolism and brown fat thermogenic function in mice.
    Sevillano J, Sánchez-Alonso MG, Zapatería B, Calderón M, Alcalá M, Limones M, Pita J, Gramage E, Vicente-Rodríguez M, Horrillo D, Medina-Gómez G, Obregón MJ, Viana M, Valladolid-Acebes I, Herradón G, Ramos-Álvarez MP. Diabetologia. 2019 Jan;62(1):123-135.
Aims/hypothesis: Pleiotrophin, a developmentally regulated and highly conserved cytokine, exerts different functions including regulation of cell growth and survival. Here, we hypothesise that this cytokine can play a regulatory role in glucose and lipid homeostasis.

Methods: To test this hypothesis, we performed a longitudinal study characterising the metabolic profile (circulating variables and tissue mRNA expression) of gene-targeted Ptn-deficient female mice and their corresponding wild-type counterparts at different ages from young adulthood (3 months) to older age (15 months). Metabolic cages were used to investigate the respiratory exchange ratio and energy expenditure, at both 24°C and 30°C. Undifferentiated immortalised mouse brown adipocytes (mBAs) were treated with 0.1 μg/ml pleiotrophin until day 6 of differentiation, and markers of mBA differentiation were analysed by quantitative real-time PCR (qPCR).

Results: Ptn deletion was associated with a reduction in total body fat (20.2% in Ptn+/+ vs 13.9% in Ptn-/- mice) and an enhanced lipolytic response to isoprenaline in isolated adipocytes from 15-month-old mice (189% in Ptn+/+ vs 273% in Ptn-/- mice). We found that Ptn-/- mice exhibited a significantly lower QUICKI value and an altered lipid profile; plasma triacylglycerols and NEFA did not increase with age, as happens in Ptn+/+ mice. Furthermore, the contribution of cold-induced thermogenesis to energy expenditure was greater in Ptn-/- than Ptn+/+ mice (42.6% and 33.6%, respectively). Body temperature and the activity and expression of deiodinase, T3 and mitochondrial uncoupling protein-1 in the brown adipose tissue of Ptn-/- mice were higher than in wild-type controls. Finally, supplementing brown pre-adipocytes with pleiotrophin decreased the expression of the brown adipocyte markers Cidea (20% reduction), Prdm16 (21% reduction), and Pgc1-α (also known as Ppargc1a, 11% reduction).

Conclusions/interpretation: Our results reveal for the first time that pleiotrophin is a key player in preserving insulin sensitivity, driving the dynamics of adipose tissue lipid turnover and plasticity, and regulating energy metabolism and thermogenesis. These findings open therapeutic avenues for the treatment of metabolic disorders by targeting pleiotrophin in the crosstalk between white and brown adipose tissue.

  • Beneficial effects of murtilla extract and madecassic acid on insulin sensitivity and endothelial function in a model of diet-induced obesity.
    Arancibia-Radich J, González-Blázquez R, Alcalá M, Martín-Ramos M, Viana M, Arribas S, Delporte C, Fernández-Alfonso MS, Somoza B, Gil-Ortega M.  Sci Rep. 2019 Jan 24;9(1):599.
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.
  • Effect of Bariatric Surgery on Abnormal MMP-9 and AMPK Activities: Potential Markers of Obesity-Related CV Risk.
    García-Prieto CF, Gil-Ortega M, Vega-Martín E, Ramiro-Cortijo D, Martín-Ramos M, Bordiú E, Sanchez-Pernaute A, Torres A, Aránguez I, Fernández-Alfonso M, Rubio MA, Somoza B. Beneficial  Front Physiol. 2019 May 8;10:553.
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/m2) and 7 non-obese subjects (7 women; mean age 45 years and BMI = 22.7 ± 0.6 kg/m2). 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-AMPKThr-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.
  • The Release of Adipose Stromal Cells from Subcutaneous Adipose Tissue Regulates Ectopic Intramuscular Adipocyte Deposition.
    Girousse A, Gil-Ortega M, Bourlier V, Bergeaud C, Sastourné-Arrey Q, Moro C, Barreau C, Guissard C, Vion J, Arnaud E, Pradère JP, Juin N, Casteilla L, Sengenès C.  Cell Rep. 2019 Apr 9;27(2):323-333.e5
Ectopic lipid deposition (ELD) is defined by excess fat storage in locations not classically associated with adipose tissue (AT) storage. ELD is positively correlated with insulin resistance and increased risk of metabolic disorders. ELD appears as lipid droplets or adipocytes, whose cell origin is unknown. We previously showed that subcutaneous AT (ScAT) releases adipocyte progenitors into the circulation. Here, we demonstrate that triggering or preventing the release of adipocyte precursors from ScAT directly promoted or limited ectopic adipocyte formation in skeletal muscle in mice. Importantly, obesity-associated metabolic disorders could be mimicked by causing adipocyte precursor release without a high-fat diet. Finally, during nutrient overload, adipocyte progenitors exited ScAT, where their retention signals (CXCR4/CXCL12 axis) were greatly decreased, and further infiltrated skeletal muscles. These data provide insights into the formation of ELD associated with calorie overload and highlight adipocyte progenitor trafficking as a potential target in the treatment of metabolic diseases.
  • Inflammatory stress and altered angiogenesis evoked by very high-fat diets in mouse liver.
    Plaza A, Naranjo V, Blonda AM, Cano V, González-Martín C, Gil-Ortega M, Ruiz-Gayo M, Merino B. Endocrinol Diabetes Nutr (Engl Ed). 2019 Aug-Sep;66(7):434-442

Background: Non-alcoholic fatty liver disease (NAFLD), a condition that leads to fibrosis, is caused by intake of very high-fat diets (HFDs). However, while the negative impact on the liver of these diets has been an issue of interest, systematic research on the effect of HFDs are lacking.

Objective: To characterize the overall impact of HFDs on both molecular and morphological signs of liver remodeling.

Methods: A study was conducted on male C57BL/6J mice to assess the effect of 4- and 8-week HFDs (60% kcal from fat) on (i) liver steatosis and fibrosis, and (ii) expression of factors involved in inflammation and angiogenesis.

Results: After an 8-week HFD, vascular endothelial growth factor type-2 receptor (VEGF-R2) and fatty acid translocase/trombospondin-1 receptor (CD36) were overexpressed in liver tissue of mice given HFDs. These changes suggest impaired liver angiogenesis and occurred together with (i) increased GPR78-BiP and EIF2α phosphorylation, suggesting endoplasmic reticulum stress, (ii) induction of Col1a1 gene expression, a marker of fibrosis, and (iii) increased CD31 immunolabeling, consistent with active angiogenesis and fibrosis.

Conclusion: Our data show that very HFDs promote a rapid inflammatory response, as well as deregulation of angiogenesis, both consistent with development of liver fibrosis.

  • Evaluación de la técnica inhalatoria y la adherencia al tratamiento con inhaladores en farmacia comunitaria.
    González Robayna, M. M., Montejo Rubio, C., & Gil Ortega, M. (2019).  Pharmaceutical Care España, 21(1), 41–64.

Introducción: La vía inhalatoria es de elección para la administración de la mayoría de los fármacos empleados en el tratamiento de las enfermedades pulmonares obstructivas. No obstante, una técnica inhalatoria incorrecta puede comprometer el control de dichas patologías.

Objetivos: El objetivo de este trabajo fue evaluar la técnica inhalatoria de los pacientes de una farmacia comunitaria y su adherencia al tratamiento.

Métodos: Se llevó a cabo un estudio observacional descriptivo transversal con 74 pacientes en tratamiento con inhaladores a los que se les encuestó sobre su técnica de inhalación y la adherencia al tratamiento. Además, se les solicitó una demostración práctica del manejo del inhalador.

Resultados: El 47% de los encuestados cometieron errores críticos de uso del inhalador, aunque el 97.3% creían saber utilizarlo correctamente. El error más frecuente fue no esperar al menos un minuto entre inhalaciones cuando era necesario repetir la dosis. Aunque el 96% de los encuestados refirió haber recibido información sobre cómo utilizar el inhalador, solo en un 1% fue impartida por farmacéuticos. El 47% de los pacientes presentó una buena adherencia al tratamiento, pero el 60% presentó algún tipo de incumplimiento, siendo el incumplimiento errático el más frecuente.

Conclusiones:

Este estudio demuestra que un elevado porcentaje de pacientes utiliza una técnica inhalatoria incorrecta y presenta una mala adherencia terapéutica, lo que puede contribuir a una menor efectividad del tratamiento y a un peor control de sus problemas de salud. Es necesario un papel más activo de los farmacéuticos comunitarios para evitar esta situación.

2018

  • Antioxidants and Oxidative Stress: Focus in Obese Pregnancies.
    Alcala M, Gutierrez-Vega S, Castro E, Guzman-Gutiérrez E, Ramos-Álvarez MP, Viana M.  Front Physiol. 2018 Nov 6;9:1569.
The prevalence of obesity in women of childbearing age around the globe has dramatically increased in the last decades. Obesity is characterized by a low-state chronic inflammation, metabolism impairment and oxidative stress, among other pathological changes. Getting pregnant in this situation involves that gestation will occur in an unhealthy environment, that can potentially jeopardize both maternal and fetal health. In this review, we analyze the role of maternal obesity-induced oxidative stress as a risk factor to develop adverse outcomes during gestation, including reduced fertility, spontaneous abortion, teratogenesis, preeclampsia, and intrauterine growth restriction. Evidences of macromolecule oxidation increase in reactive oxygen species generation and antioxidant defense alterations are commonly described in maternal and fetal tissues. Thus, antioxidant supplementation become an interesting prophylactic and therapeutic tool, that yields positive results in cellular, and animal models. However, the results from most meta-analysis studying the effect of these therapies in complicated gestations in humans are not really encouraging. It is still to be analyzed whether these therapies could work if applied to cohorts of patients at a high risk, such as those with low concentration of antioxidants or obese pregnant women.
  • Finerenone Attenuates Endothelial Dysfunction and Albuminuria in a Chronic Kidney Disease Model by a Reduction in Oxidative Stress.
    González-Blázquez R, Somoza B, Gil-Ortega M, Martín Ramos M, Ramiro-Cortijo D, Vega-Martín E, Schulz A, Ruilope LM, Kolkhof P, Kreutz R, Fernández-Alfonso MS. Front Pharmacol. 2018 Oct 9;9:1131.
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.
  • Role of Perivascular Adipose Tissue in Health and Disease.
    Fernández-Alfonso MS, Somoza B, Tsvetkov D, Kuczmanski A, Dashwood M, Gil-Ortega M. Compr Physiol. 2017 Dec 12;8(1):23-59
Perivascular adipose tissue (PVAT) is cushion of fat tissue surrounding blood vessels, which is phenotypically different from other adipose tissue depots. PVAT is composed of adipocytes and stromal vascular fraction, constituted by different populations of immune cells, endothelial cells, and adipose-derived stromal cells. It expresses and releases an important number of vasoactive factors with paracrine effects on vascular structure and function. In healthy individuals, these factors elicit a net anticontractile and anti-inflammatory paracrine effect aimed at meeting hemodynamic and metabolic demands of specific organs and regions of the body. Pathophysiological situations, such as obesity, diabetes or hypertension, induce changes in its amount and in the expression pattern of vasoactive factors leading to a PVAT dysfunction in which the beneficial paracrine influence of PVAT is shifted to a pro-oxidant, proinflammatory, contractile, and trophic environment leading to functional and structural cardiovascular alterations and cardiovascular disease. Many different PVATs surrounding a variety of blood vessels have been described and exhibit regional differences. Both protective and deleterious influence of PVAT differs regionally depending on the specific vascular bed contributing to variations in the susceptibility of arteries and veins to vascular disease. PVAT therefore, might represent a novel target for pharmacological intervention in cardiovascular disease.
  • Diabetes in pregnancy: a new decade of challenges ahead.
    Schaefer-Graf U, Napoli A, Nolan CJ; Diabetic Pregnancy Study Group.  Diabetologia. 2018 May;61(5):1012-1021
Every 10 years, the Diabetic Pregnancy Study Group, a study group of the EASD, conducts an audit meeting to review the achievements of the preceding decade and to set the directions for research and clinical practice improvements for the next decade. The most recent meeting focused on the following areas: improving pregnancy outcomes for women with pregestational type 1 diabetes and type 2 diabetes; the influence of obesity and gestational diabetes on pregnancy outcomes; the determinants and assessment of fetal growth and development; and public health issues, including consideration of transgenerational consequences and economic burden. The audit meeting also considered the likely impact of ‘omics’ on research within the field and the potential of these technologies to enable precision-medicine approaches to management. Through sharing of the findings and ideas of audit meeting participants, the DPSG hopes to promote networking, research and advances in clinical care, to improve outcomes for all women and their offspring affected by diabetes and obesity in pregnancy.
  • Estudio observacional sobre el grado de conocimiento de los pacientes acerca del tratamiento con acenocumarol.
    Gil Ortega, M., Gil Ortega, V., Somoza Hernández, B., & Cano González, V. (2018).  Pharmaceutical Care España, 20(1), Pharm Care Esp. 2018; 20(1): 27–49

Introducción: El tratamiento con anticoagulantes orales derivados de cumarinas como el acenocumarol es bastante complejo pues, además de presentar un estrecho margen terapéutico, pueden interaccionar con numerosos fármacos, alimentos o productos de herbolario, favoreciendo la aparición de reacciones adversas y comprometiendo la seguridad del paciente anticoagulado.

Objetivos: El objetivo de este trabajo ha sido evaluar la relación entre el grado de conocimiento del paciente en tratamiento con acenocumarol y la aparición de efectos adversos derivados del mismo.

Métodos: Se realizó un estudio observacional descriptivo en 30 pacientes de 3 oficinas de farmacia y un centro de atención primaria de la Comunidad de Madrid. Para ello, se elaboró un cuestionario basado en el de Zeolla y cols. (2016), validado en EE.UU. para pacientes en tratamiento con warfarina y adaptado a nuestra zona geográfica y al tratamiento con acenocumarol.

Resultados: Del 67% de los pacientes encuestados que afirmaron haber solicitado y obtenido consejo sobre el tratamiento con acenocumarol, sólo el 56% adquirió un grado de conocimiento adecuado y únicamente el 29% demostró conocer los medicamentos y productos de herbolario/dietéticos que debe evitar el paciente anticoagulado. Además, se observó que dicho grado de conocimiento era significativamente mayor en los pacientes que no habían presentado efectos adversos asociados al tratamiento anticoagulante con anterioridad.

Conclusiones: Un adecuado grado de conocimiento acerca del tratamiento con acenocumarol y las posibles interacciones del mismo con otros medicamentos que no requieren prescripción médica o productos de herbolario/dietéticos reduce significativamente el riesgo de presentar efectos adversos asociados al tratamiento anticoagulante

2017

  • Increased inflammation, oxidative stress and mitochondrial respiration in brown adipose tissue from obese mice.
    Alcalá M, Calderon-Dominguez M, Bustos E, Ramos P, Casals N, Serra D, Viana M, Herrero L. Sci Rep. 2017 Nov 22;7(1):16082.
Obesity is associated with severe metabolic diseases such as type 2 diabetes, insulin resistance, cardiovascular disease and some forms of cancer. The pathophysiology of obesity-induced metabolic diseases has been strongly related to white adipose tissue (WAT) dysfunction through several mechanisms such as fibrosis, apoptosis, inflammation, ER and oxidative stress. However, little is known of whether these processes are also present in brown adipose tissue (BAT) during obesity, and the potential consequences on mitochondrial activity. Here we characterized the BAT of obese and hyperglycemic mice treated with a high-fat diet (HFD) for 20 weeks. The hypertrophic BAT from obese mice showed no signs of fibrosis nor apoptosis, but higher levels of inflammation, ER stress, ROS generation and antioxidant enzyme activity than the lean counterparts. The response was attenuated compared with obesity-induced WAT derangements, which suggests that BAT is more resistant to the obesity-induced insult. In fact, mitochondrial respiration in BAT from obese mice was enhanced, with a 2-fold increase in basal oxygen consumption, through the upregulation of complex III of the electron transport chain and UCP1. Altogether, our results show that obesity is accompanied by an increase in BAT mitochondrial activity, inflammation and oxidative damage.
  • Short-term vitamin E treatment impairs reactive oxygen species signaling required for adipose tissue expansion, resulting in fatty liver and insulin resistance in obese mice.
    Alcala M, Calderon-Dominguez M, Serra D, Herrero L, Ramos MP, Viana M. PLoS One. 2017 Oct 13;12(10):e0186579

Objectives: The use of antioxidant therapy in the treatment of oxidative stress-related diseases such as cardiovascular disease, diabetes or obesity remains controversial. Our aim is to demonstrate that antioxidant supplementation may promote negative effects if used before the establishment of oxidative stress due to a reduced ROS generation under physiological levels, in a mice model of obesity.

Methods: C57BL/6J mice were fed with a high-fat diet for 14 weeks, with (OE group) or without (O group) vitamin E supplementation.

Results: O mice developed a mild degree of obesity, which was not enough to induce metabolic alterations or oxidative stress. These animals exhibited a healthy expansion of retroperitoneal white adipose tissue (rpWAT) and the liver showed no signs of lipotoxicity. Interestingly, despite achieving a similar body weight, OE mice were insulin resistant. In the rpWAT they presented a reduced generation of ROS, even below physiological levels (C: 1651.0 ± 212.0; O: 3113 ± 284.7; OE: 917.6 ±104.4 RFU/mg protein. C vs OE p< 0.01). ROS decay may impair their action as second messengers, which could account for the reduced adipocyte differentiation, lipid transport and adipogenesis compared to the O group. Together, these processes limited the expansion of this fat pad and as a consequence, lipid flux shifted towards the liver, causing steatosis and hepatomegaly, which may contribute to the marked insulin resistance.

Conclusions: This study provides in vivo evidence for the role of ROS as second messengers in adipogenesis, lipid metabolism and insulin signaling. Reducing ROS generation below physiological levels when the oxidative process has not yet been established may be the cause of the controversial results obtained by antioxidant therapy.

  • Vascular Dysfunction in Mother and Offspring During Preeclampsia: Contributions from Latin-American Countries.
    Giachini FR, Galaviz-Hernandez C, Damiano AE, Viana M, Cadavid A, Asturizaga P, Teran E, Clapes S, Alcala M, Bueno J, Calderón-Domínguez M, Ramos MP, Lima VV, Sosa-Macias M, Martinez N, Roberts JM, Escudero C; RIVA-TREM. Curr Hypertens Rep. 2017 Oct 6;19(10):83
Pregnancy is a physiologically stressful condition that generates a series of functional adaptations by the cardiovascular system. The impact of pregnancy on this system persists from conception beyond birth. Recent evidence suggests that vascular changes associated with pregnancy complications, such as preeclampsia, affect the function of the maternal and offspring vascular systems, after delivery and into adult life. Since the vascular system contributes to systemic homeostasis, defective development or function of blood vessels predisposes both mother and infant to future risk for chronic disease. These alterations in later life range from fertility problems to alterations in the central nervous system or immune system, among others. It is important to note that rates of morbi-mortality due to pregnancy complications including preeclampsia, as well as cardiovascular diseases, have a higher incidence in Latin-American countries than in more developed countries. Nonetheless, there is a lack both in the amount and impact of research conducted in Latin America. An impact, although smaller, can be seen when research in vascular disorders related to problems during pregnancy is analyzed. Therefore, in this review, information about preeclampsia and endothelial dysfunction generated from research groups based in Latin-American countries will be highlighted. We relate the need, as present in many other countries in the world, for increased effective regional and international collaboration to generate new data specific to our region on this topic..
  • Brown Adipose Tissue Bioenergetics: A New Methodological Approach.
    Calderon-Dominguez M, Alcalá M, Sebastián D, Zorzano A, Viana M, Serra D, Herrero L. Adv Sci (Weinh). 2017 Mar 13;4(4):1600274
The rediscovery of brown adipose tissue (BAT) in humans and its capacity to oxidize fat and dissipate energy as heat has put the spotlight on its potential as a therapeutic target in the treatment of several metabolic conditions including obesity and diabetes. To date the measurement of bioenergetics parameters has required the use of cultured cells or extracted mitochondria with the corresponding loss of information in the tissue context. Herein, we present a method to quantify mitochondrial bioenergetics directly in BAT. Based on XF Seahorse Technology, we assessed the appropriate weight of the explants, the exact concentration of each inhibitor in the reaction, and the specific incubation time to optimize bioenergetics measurements. Our results show that BAT basal oxygen consumption is mostly due to proton leak. In addition, BAT presents higher basal oxygen consumption than white adipose tissue and a positive response to b-adrenergic stimulation. Considering the whole tissue and not just subcellular populations is a direct approach that provides a realistic view of physiological respiration. In addition, it can be adapted to analyze the effect of potential activators of thermogenesis, or to assess the use of fatty acids or glucose as a source of energy.
  • CD98 regulates vascular smooth muscle cell proliferation in atherosclerosis.
    Baumer Y, McCurdy S, Alcala M, Mehta N, Lee BH, Ginsberg MH, Boisvert WA.  Atherosclerosis. 2017 Jan;256:105-114.

Background and aims: Vascular smooth muscle cells (VSMC) migrate and proliferate to form a stabilizing fibrous cap that encapsulates atherosclerotic plaques. CD98 is a transmembrane protein made of two subunits, CD98 heavy chain (CD98hc) and one of six light chains, and is known to be involved in cell proliferation and survival. Because the influence of CD98hc on atherosclerosis development is unknown, our aim was to determine if CD98hc expressed on VSMC plays a role in shaping the morphology of atherosclerotic plaques by regulating VSMC function.

Methods: In addition to determining the role of CD98hc in VSMC proliferation and apoptosis, we utilized mice with SMC-specific deletion of CD98hc (CD98hcfl/flSM22αCre+) to determine the effects of CD98hc deficiency on VSMC function in atherosclerotic plaque.

Results: After culturing for 5 days in vitro, CD98hc-/- VSMC displayed dramatically reduced cell counts, reduced proliferation, as well as reduced migration compared to control VSMC. Analysis of aortic VSCM after 8 weeks of HFD showed a reduction in CD98hc-/- VSMC proliferation as well as increased apoptosis compared to controls. A long-term atherosclerosis study using SMC-CD98hc-/-/ldlr-/- mice was performed. Although total plaque area was unchanged, CD98hc-/- mice showed reduced presence of VSMC within the plaque (2.1 ± 0.4% vs. 4.3 ± 0.4% SM22α-positive area per plaque area, p < 0.05), decreased collagen content, as well as increased necrotic core area (25.8 ± 1.9% vs. 10.9 ± 1.6%, p < 0.05) compared to control ldlr-/- mice.

Conclusions: We conclude that CD98hc is required for VSMC proliferation, and that its deficiency leads to significantly reduced presence of VSMC in the neointima. Thus, CD98hc expression in VSMC contributes to the formation of plaques that are morphologically more stable, and thereby protects against atherothrombosis.

  • Role of PVAT in coronary atherosclerosis and vein graft patency: friend or foe?
    Fernández-Alfonso MS, Gil-Ortega M, Aranguez I, Souza D, Dreifaldt M, Somoza B, Dashwood MR.Br J Pharmacol. 2017 Oct;174(20):3561-3572.

Perivascular adipose tissue (PVAT) releases numerous factors and adipokines with paracrine effects on both vascular structure and function. These effects are variable as they depend on regional differences in PVAT among blood vessels and vary with changes in adiposity. There is considerable evidence demonstrating an association between coronary PVAT and the development and progression of coronary artery disease, which is associated with inflammation, oxidative stress, angiogenesis, vascular remodelling and blood clotting. However, PVAT also has a protective role in vascular grafts, especially the no-touch saphenous vein, in patients undergoing coronary artery bypass. This beneficial influence of PVAT involves factors such as adipocyte-derived relaxing factor, nitric oxide (NO), leptin, adiponectin, prostanoids, hydrogen sulphide and neurotransmitters, as well as mechanical protection. This article aims to highlight and compare the dual role of PVAT in the development and progression of coronary atherosclerosis, as well as in increased graft patency. Different deleterious and protective mechanisms of PVAT are also discussed and the inside-outside signalling paradigm of atherosclerosis development re-evaluated. The bidirectional communication between the arterial and venous wall and their surrounding PVAT, where signals originating from the vascular wall or lumen can affect PVAT phenotype, has been shown to be very complex. Moreover, signals from PVAT also influence the structure and function of the vascular wall in a paracrine manner.

  • Long term effects of fetal undernutrition on rat heart. Role of hypertension and oxidative stress.
     Rodríguez-Rodríguez P, López de Pablo AL, García-Prieto CF, Somoza B, Quintana-Villamandos B, Gómez de Diego JJ, Gutierrez-Arzapalo PY, Ramiro-Cortijo D, González MC, Arribas SM. PLoS One. 2017 Feb 17;12(2):e0171544.

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.