María S. Fernández-Alfonso

Comparative expression analysis of the renin–angiotensin system components between white and brown perivascular adipose tissue

Recent studies have demonstrated that the rat adipose tissue expresses some of the components necessary for the production of angiotensin II (Ang II) and the receptors mediating its actions. The aim of this work is to characterize the expression of the renin-angiotensin system (RAS) components in perivascular adipose tissue and to assess differences in the expression pattern depending on the vascular bed and type of adipose tissue. We analyzed Ang I and Ang II levels as well as mRNA levels of RAS components by a quantitative RT-PCR method in periaortic (PAT) and mesenteric adipose tissue (MAT) of 3-month-old male Wistar-Kyoto rats. PAT was identified as brown adipose tissue expressing uncoupling protein-1 (UCP-1). It had smaller adipocytes than those from MAT, which was identified as white adipose tissue. All RAS components, except renin, were detected in both PAT and MAT. Levels of expression of angiotensinogen, Ang-converting enzyme (ACE), and ACE2 were similar between PAT and MAT. Renin receptor expression was five times higher, whereas expression of chymase, AT(1a), and AT(2) receptors were significantly lower in PAT compared with MAT respectively. In addition, three isoforms of the AT(1a) receptor were found in perivascular adipose tissue. The AT(1b) receptor was found at very a low expression level. Ang II levels were higher in MAT with no differences between tissues in Ang I. The results show that the RAS is differentially expressed in white and brown perivascular adipose tissues implicating a different role for the system depending on the vascular bed and the type of adipose tissue.

High-fat diets impair spatial learning in the radial-arm maze in mice

It has been suggested that hyperglycemia and insulin resistance triggered by energy-dense diets can account for hippocampal damage and deficits of cognitive behaviour. We wonder if the impairment of learning and memory processes detected in diet-induced obese (DIO) mice is linked to diet composition itself. With this purpose we have evaluated learning performance in mice undergoing a short-term high-fat (HF) treatment, which leads to a pre-obese state characterized by increased adiposity without significant changes of glucose and insulin plasma levels. C57BL/6J mice were fed either a HF (45 kcal% from fat) or control diet (10 kcal% from fat) during 8 weeks. Learning performance was evaluated by using the four-arm baited version of the eight-arm radial maze test (RAM). Mice were trained to learn the RAM protocol and then memory was tested at different time-points. Time spent to consume food placed in baited arms and errors committed to find them were measured in all sessions. DIO mice significantly spent more time in learning the task and made a greater number of errors than controls. Moreover, retention tests revealed that both working and total memory errors were also more numerous in DIO mice. The current results show that short-term DIO impairs spatial learning and suggest that impairment of hippocampal learning elicited by HF diets might be perceptible before metabolic alterations linked to obesity develop.

Modulation of angiotensin-converting enzyme by nitric oxide

The aim of the present study was to determine the effect of nitric oxide (NO) on angiotensin‐converting enzyme (ACE) activity. A biochemical study was performed in order to analyse the effect of the NO‐donors, SIN‐1 and diethylamine/NO (DEA/NO), and of an aqueous solution of nitric oxide on the ACE activity in plasma from 3‐month old male Sprague‐Dawley rats and on ACE purified from rabbit lung. SIN‐1 significantly inhibited the activity of both enzymes in a concentration‐dependent way between 1 and 100 μM. DEA/NO inhibited the activity of purified ACE from 0.1 μM to 10 μM and plasma ACE, with a lower potency, between 1 and 100 μM. An aqueous solution of NO (100 and 150 μM) also inhibited significantly the activity of both enzymes. Lineweaver‐Burk plots indicated an apparent competitive inhibition of Hip‐His‐Leu hydrolysis by NO‐donors. Modulation of ACE activity by NO was also assessed in the rat carotid artery by comparing contractions elicited by angiotensin I (AI) and AII. Concentration‐response curves to both peptides were performed in arteries with endothelium in the presence of the guanylyl cyclase inhibitor, ODQ (10 μM), and the inhibitor of NO formation, L‐NAME (0.1 mM). NO, which is still released from endothelium in the presence of 10 μM ODQ, elicited a significant inhibition of AI contractions at low concentrations (1 and 5 nM). In the absence of endothelium, 1 μM SIN‐1 plus 10 μM ODQ, as well as 10 μM DEA/NO plus 10 μM ODQ induced a significant inhibition on AI‐induced contractions at 1 and 5 nM and at 1–100 nM, respectively. In conclusion, we demonstrated that (i) NO and NO‐releasing compounds inhibit ACE activity in a concentration‐dependent and competitive way and that (ii) NO release from endothelium physiologically reduces conversion of AI to AII.

The cellular basis of angiotensin converting enzyme mRNA expression in rat heart

Angiotensin converting enzyme (ACE) is a key factor in the regulation of two peptide systems: the renin angiotensin system (RAS) and the kinin-kallikrein system (KKS). Since it is involved in the biosynthesis of Angiotensin II (Ang II) as well as in the degradation of bradykinin (BK) it could play an important role in cardiovascular physiology and pathophysiology. ACE is widely expressed in the heart and upregulated in pathophysiological situations such as heart failure and cardiac hypertrophy. In addition, inhibition of ACE has beneficial effects in these conditions. Whereas the regulation of cardiac ACE has been studied extensively, little is known concerning the cellular expression of ACE in cardiac tissue. To define the cellular localization of ACE mRNA expression in the rat heart, we separated coronary microvascular endothelial cells from cardiac myocytes using differential centrifugation and growth on selective media. ACE mRNA expression was measured by a specific polymerase chain reaction assay after reverse transcription (RT-PCR) in different cardiac cells. The studies showed that ACE is differentially expressed in endothelial cells as well as in cardiac myocytes. This differential regulation of ACE in myocytes and non-myocytes may play a role for the diverse actions of the cardiac angiotensin system under physiological and pathological conditions.

Adaptative Nitric Oxide Overproduction in Perivascular Adipose Tissue during Early Diet-Induced Obesity

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

Aliskiren reduces body-weight gain, adiposity and plasma leptin during diet-induced obesity

Background and purpose:  Overfeeding increases adipose tissue mass and leptin production and up‐regulates the renin‐angiotensin system in adipose tissue in rodents. Here, we determined the effect of chronic treatment with the renin inhibitor, aliskiren, in a model of diet‐induced obesity in mice, on: (i) body weight, adipose tissue weight and plasma leptin; (ii) food intake and caloric efficiency; and (iii) angiotensin II (Ang II) in adipose tissue.

A reduction in the amount and anti-contractile effect of periadventitial mesenteric adipose tissue precedes hypertension development in spontaneously hypertensive rats.

The aim of this study was to determine whether alterations in periadventitial adipose tissue and its anti-contractile effect precede hypertension development. We used 4-week-old male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), which were pre-hypertensive. Vascular function was studied in the perfused mesenteric bed (MB, 1.5 mL/min). MB weight was lower in SHR (8.0±0.3 mg/g body weight) than in WKY (9.0±0.3 mg/g body weight) rats. Concentration-response curves to KCl (6 to 75 mmol/L) and to acetylcholine (10−9 to 10−5 mol/L) were similar between groups. Contractile responses to serotonin (10−9 to 10−5 mol/L) were significantly higher in SHR compared to WKY. 4-Aminopyridine (4-AP, 2 mmol/L), a blocker of Kv channels, induced a similar increase in perfusion pressure in both strains. However, 4-AP (2 mmol/L) significantly increased the contractile response to serotonin (10−9 to 10−5 mol/L) only in WKY. The anti-contractile effect of fat was confirmed by a comparison of (+) fat and (−) fat mesenteric arteries, which revealed that 4-AP significantly enhanced contractions only in (+) fat rings from WKY. These results show that alterations in visceral periadventitial fat mass and function in SHR precede hypertension, suggesting a constitutive mechanism independent of age and the hypertensive state.

Anticontractile Effect of Perivascular Adipose Tissue and Leptin are Reduced in Hypertension

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

Regional differences in perivascular adipose tissue impacting vascular homeostasis

Perivascular adipose tissue (PVAT) releases several important vasoactive factors with physiological and pathophysiological paracrine effects. A large body of evidence suggests regional phenotypic and functional differences among PVAT depots, depending on the specific vascular bed or different regions in the vascular bed where the PVAT is located. These non-uniform and separate PVATs exert various paracrine effects on vascular structure and function that largely impact disease states, such as endothelial dysfunction, atherosclerosis, or insulin resistance. This emerging view of PVAT function requires considering heterogeneous PVAT as a specialized organ that can differentially regulate vascular function depending on its anatomical location. In this context, the adipose-vascular axis may represent a novel target for pharmacological intervention in vasculopathy in cardiometabolic disorders.

Mechanisms of Perivascular Adipose Tissue Dysfunction in Obesity

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