Finerenone protects against progression of kidney and cardiovascular damage in a model of type 1 diabetes through modulation of proinflammatory and osteogenic factors.

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 TGFß) 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.

Role of PVAT in coronary atherosclerosis and vein graft patency: friend or foe?

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. LINKED ARTICLES: This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

Genetic predisposition to albuminuria is associated with increased arterial stiffness: role of elastin.

BACKGROUND AND PURPOSE: The Munich Wistar Frömter (MWF) rat strain represents an experimental model to study cardiovascular alterations under conditions of progressive albuminuria. The aim of this study was to evaluate the association between genetic predisposition to albuminuria and the development of arterial stiffness and/or vascular remodelling. EXPERIMENTAL APPROACH: Experiments were performed in mesenteric arteries from 12-week-old MWF, Wistar Kyoto (WKY) and consomic MWF-6(SHR) and MWF-8(SHR) rats in which chromosomes 6 or 8 associated with albuminuria from MWF were replaced by the respective chromosome from spontaneously hypertensive rats (SHR). KEY RESULTS: Incremental distensibility, wall stress and strain were reduced, and arterial stiffness was significantly increased in albuminuric MWF compared with WKY. Albuminuria suppression in both consomic strains was associated with lower ß-values in MWF-8(SHR) and MWF-6(SHR) compared with MWF. Moreover, elastin content was significantly lower in MWF external elastic lamina compared with WKY and both consomic strains. In addition, a reduction in arterial external and internal diameter and cross-sectional area was detected in MWF compared with WKY, thus exhibiting an inward hypotrophic remodelling. However, these alterations remained unchanged in both consomic strains. CONCLUSION AND IMPLICATIONS: These data demonstrate that albuminuria in MWF is associated with increased arterial stiffness due to a reduction of elastin content in the external elastic lamina. Moreover, inward hypotrophic remodelling in MWF is not directly associated with albuminuria. In contrast, we demonstrated that two major genetic loci affect both the development of albuminuria and arterial stiffness, thus linking albuminuria and impairment of mechanical properties of resistance arteries.

Vascular AMPK as an attractive target in the treatment of vascular complications of obesity.

The key for the survival of all organisms is the regulation and control of energy metabolism. Thus, several strategies have evolved in each tissue in order to balance nutrient supply with energy demand. Adenosine monophosphate-activated protein kinase (AMPK) is now recognized as a key participant in energy metabolism. It ensures an appropriate energetic supply by promoting energy conserving pathways in detriment of anabolic processes not essential for cell survival. Vascular AMPK plays a critical role in the regulation of blood flow and vascular tone through several mechanisms, including vasodilation by stimulating nitric oxide release in endothelial cells. Since obesity leads to endothelial damage and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. In the present review we focus on the role of vascular AMPK in both endothelial and smooth muscle cells, paying special attention to its dysregulation in obesity- and high-fat diet-related complications, as well as to the mechanisms and benefits of vascular AMPK activation.

Mild caloric restriction reduces blood pressure and activates endothelial AMPK-PI3K-Akt-eNOS pathway in obese Zucker rats.

Genetic obesity models exhibit endothelial dysfunction associated to adenosine monophosphate-activated protein kinase (AMPK) dysregulation. This study aims to assess if mild short-term caloric restriction (CR) restores endothelial AMPK activity leading to an improvement in endothelial function. Twelve-week old Zucker lean and obese (fa/fa) male rats had access to standard chow either ad libitum (AL, n=8) or 80% of AL (CR, n=8) for two weeks. Systolic blood pressure was significantly higher in fa/fa AL rats versus lean AL animals, but was normalized by CR. Endothelium-dependent relaxation to acetylcholine (ACh, 10(-9) to 10(-4) M) was reduced in fa/fa AL compared to control lean AL rats (p<0.001), and restored by CR. The AMPK activator AICAR (10(-5) to 8·10(-3) M) elicited a lower relaxation in fa/fa AL rings that was normalized by CR (p<0.001). Inhibition of PI3K (wortmannin, 10(-7) M), Akt (triciribine, 10(-5) M), or eNOS (L-NAME, 10(-4) M) markedly reduced AICAR-induced relaxation in lean AL, but not in fa/fa AL rats. These inhibitions were restored by CR in Zucker fa/fa rings. These data show that mild short-term CR improves endothelial function and lowers blood pressure in obesity due to the activation of the AMPK-PI3K-Akt-eNOS pathway.

Ex vivo microperfusion system of the adipose organ: a new approach to studying the mobilization of adipose cell populations.

BACKGROUND/OBJECTIVES: Adipose tissue (AT) is a dynamic organ that expands and contracts rapidly. It is composed of adipocytes and of cell populations among which immune cells and mesenchymal progenitors known as adipose stromal cells (ASCs). The AT cell turnover has been extensively studied. Surprisingly it has only been viewed as the result of both cell proliferation/death and cell infiltration. Nevertheless, both immune cells and ASCs exhibit migration abilities; therefore their egress from AT in response to physiological/pathophysiological stimuli has to be considered. To do so, the aim of the present work was to develop a model allowing the study of cell release from the adipose organ. SUBJECTS/METHODS: Mesenteric (Mes) ATs were isolated from 9-week-old C57BL/6 male mice and were catheterized via the superior mesenteric artery and were perfused with a saline solution. After an equilibration period, the mesenteric fat pad was perfused with CXCL12 (stromal-derived factor-1, SDF-1) or sphingosine 1-phosphate (S1P) to trigger cell mobilization and perfusates were collected every 30 min for subsequent flow cytometry analyses. RESULTS: We report here that CXCL12 induces the specific release of ASCs from MesAT thus demonstrating that ASCs are specifically mobilized from fat depots by a CXCL12-dependent pathway. Moreover, we showed that leukocyte mobilization can be triggered via a S1P-dependent pathway. CONCLUSIONS: We have developed a microperfusion model of an intact fat depot allowing the study of AT cell release in response to various molecules. The perfusion system described here demonstrates that ASCs and leukocytes can be pharmacologically mobilized from AT. Therefore, AT microperfusion might constitute an appropriate and reliable approach for evaluating the mobilization of different cell populations from AT in various physiological and pathophysiological contexts. Such a model might help in identifying factors and drugs controlling AT cell release, impacting the medical fields of regenerative medicine and of obesity or its associated comorbidities.

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.

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.

The upregulation of angiotensin-converting enzyme during neointima formation does not have functional consequences.

The aim of this study was to analyze if the upregulation of angiotensin-converting enzyme and AT1 receptors observed in a model of neointima formation results in increased contractions to angiotensin I (AI) and AII. Endothelial denudation was performed in left common carotid arteries of 3-month-old male Sprague-Dawley rats. Rats were killed at days 0, 4, 8 and 14 after injury and vascular reactivity was assessed in an organ bath. Responses were always compared with their contralateral vessels as a control. Contractile responses to 75 mM KCl were similar between groups. Noradrenaline (0.1 microM) induced significantly higher contractions at days 0 and 4. Relaxation to acetylcholine (Ach) (1 nM to 0.1 mM) was suppressed at day 0 and increased with time after injury. Relaxations to sodium nitroprusside (0.1 nM to 0.1 mM) were similar at all time points studied. Responses to AI and AII were increased at early steps of neointima formation and decreased with time after injury correlating with increased responses to Ach. Concentration-response curves to AI and AII had similar EC50 or Emax values at the same time points. These results indicate that in the rat i) neointima formation does not impair contractile responses to KCl nor relaxation to SNP, ii) a functional endothelium seems to regenerate with time after injury, and iii) the increase in ACE activity and AT1 receptor number does not have functional consequences.

Differential effect of chronic antihypertensive treatment on vascular smooth muscle cell phenotype in spontaneously hypertensive rats.

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

Effect of removal of adventitia on vascular smooth muscle contraction and relaxation.

The aim of the present study was to determine whether the adventitia of large arteries modulates vascular function. We developed a method to obtain functional vascular rings devoid of adventitia. Carotid and iliac arteries from 3-mo-old Sprague-Dawley rats were denuded from adventitia after treatment with collagenase followed by gentle peeling. Adventitia removal and integrity of the media was demonstrated by optical and confocal microscopy. Arterial rings with or without adventitia and with or without endothelium were mounted in an organ bath for isometric tension recording. Responses to 75 mM KCl or norepinephrine (0.1 nM-1 microM) were significantly reduced in segments without adventitia. Acetylcholine-induced relaxation (0.1 microM-0.1 mM) was enhanced in arteries without adventitia, whereas sodium nitroprusside-induced responses were not modified. These results demonstrate that the combination of stripping with a previous collagenase treatment allows us to obtain functional rings devoid of adventitia and that this layer plays a role in contractile capacity and in endothelium-modulated responses.

Interrelaciones sistema renina-angiotensina/sistema no-GMPC

El óxido nítrico (NO) y la angiotensina 11 (Ang 11) tienen acciones opuestas sobre la contracción y sobre la proliferación y la apoptosis de las células del músculo liso vascular. Existen, además, datos que apuntan a una interrelación más profunda entre ambos factores. El NO modula de forma negativa el sistema renina-angiotensina a varios niveles, mientras que la Ang Il estimula positivamente la síntesis y la liberación de NO. En esta revisión se analizan los datos que sugieren la existencia de una regulación mutua entre ambos sistemas y se discute su posible relevancia fisiológica como un mecanismo homeostático encaminado a mantener la función cardiovascular (AU)

Chronic losartan treatment decreases angiotensin II-mediated facilitation of noradrenaline release in the caudal artery of spontaneously hypertensive rats.

Sympathetic activity is modulated by angiotensin II (AII), both at pre- and postsynaptic level in the rat caudal artery. In the spontaneously hypertensive rat (SHR), this artery receives more dense sympathetic innervation than blood vessels of normotensive strains. This fact seems to be linked to the enhanced pressor responses elicited by noradrenaline in SHR. In this work we describe, in the SHR, the effect of a chronic treatment with the angiotensin II AT1-receptor antagonist, losartan, in modulating noradrenergic mechanisms involved in caudal artery contraction. The effect of losartan is compared to that of captopril, given at doses leading to a similar decrease of both arterial blood pressure and left ventricular hypertrophy. The contractile response of caudal artery rings induced by endogenous noradrenaline released by low frequency transmural nerve stimulation (TNS) has been studied. Under our conditions, TNS (0.5-1 Hz) induced higher contractile responses in SHR treated with losartan than in the control and captopril-treated groups. This difference seems to be due to an increase of the postsynaptic effect of noradrenaline (NA) rather than to an increase of noradrenaline release from sympathetic endings, since i) DE50 value for NA was lower in losartan-treated SHR than in the other groups, and ii) AII induced a dose-dependent increase of TNS-evoked release of radioactivity from caudal artery segments loaded with [3H]-NA, in both control and captopril-treated groups but had no effect in the losartan-treated group. These results show that chronic treatment with losartan, although slightly enhancing the pressor effect of NA at postsynaptic level, fully supresses the facilitatory role of AII on NA release.

Nitric oxide and the renin-angiotensin system. Is there a physiological interplay between the systems?

Opposed actions for nitric oxide (NO) and angiotensin II (Ang II) in vascular contraction and vascular smooth muscle cell proliferation and apoptosis are well documented. In addition, various experimental approaches have shown that NO negatively modulates the renin-angiotensin system by inhibiting angiotensin-converting enzyme (ACE) activity and down-regulating AT1 receptors. On the other hand, Ang II and Ang-(1-7) positively stimulate NO synthesis and release. In this review, we analyse the data suggesting a mutual regulation between the renin-angiotensin and the nitric oxide-generating systems, and we propose a homeostatic interplay between both factors aimed at regulating cardiovascular function.

Modulation of angiotensin-converting enzyme by nitric oxide.

1. The aim of the present study was to determine the effect of nitric oxide (NO) on angiotensin-converting enzyme (ACE) activity. 2. 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 microM. DEA/NO inhibited the activity of purified ACE from 0.1 microM to 10 microM and plasma ACE, with a lower potency, between 1 and 100 microM. An aqueous solution of NO (100 and 150 microM) also inhibited significantly the activity of both enzymes. Lineweaver-Burk plots indicated an apparent competitive inhibition of Hip-His-Leu hydrolysis by NO-donors. 3. 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 microM), and the inhibitor of NO formation, L-NAME (0.1 mM). NO, which is still released from endothelium in the presence of 10 microM ODQ, elicited a significant inhibition of AI contractions at low concentrations (1 and 5 nM). In the absence of endothelium, 1 microM SIN-1 plus 10 microM ODQ, as well as 10 microM DEA/NO plus 10 microM ODQ induced a significant inhibition on AI-induced contractions at 1 and 5 nM and at 1-100 nM, respectively. 4. 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.

Differential development of early hypertension in heterozygous transgenic TGR(mREN2)27 rats.

The aim of the study was to determine blood pressures in young heterozygous transgenic rats harbouring the Ren-2 mouse gene, TGR(mREN2)27, and to examine the association between blood pressure and plasma renin-angiotensin parameters during the conscious state. Two litters of heterozygous TGR(mREN2)27 rats and their normotensive Sprague-Dawley reference animals were bred in our laboratory. Cardiovascular phenotypes and blood, for the evaluation of plasma renin-angiotensin-system parameters, were obtained via arterial catheters in conscious rats 4 weeks after birth. Blood pressure values showed a high degree of interindividual variability and significant litter differences in the group of transgenic animals ranging from normotensive tohypertensive values. Plasma prorenin levels were markedly increased in TGR(mREN2)27, whereas both active renin and angiotensin II plasma concentrations were reduced independently from hypertension. Plasma prorenin levels in litter 2 of transgenic animals which exhibited significantly elevated blood pressures were significantly higher as compared to those of litter 1 which showed blood pressures that were not significantly different from SD. These data demonstrate that the development of hypertension in heterozygous TGR(mREN2)27 takes place in the fourth week of postnatal life and exhibits large interindividual variability. Moreover, development of early hypertension in young heterozygous TGR(mREN2)27 is associated with higher plasma prorenin levels.

Early induction of angiotensin I-converting enzyme in rat carotid artery after balloon injury.

Several studies have demonstrated the effectiveness of angiotensin I-converting enzyme (ACE) inhibitors in preventing the neointima formation found after denudation of the rat carotid artery by balloon injury. The aim of the present study was to determine the role of ACE in this model and to compare the treatment with the ACE inhibitor ramiprilat with that with the angiotensin II antagonist HR 720. The endothelial layer of the left carotid artery was removed using an inflated balloon catheter. Injured and control vessels were both submitted to histomorphological analysis and DNA content quantification at 2, 4, 6, 8, 12, and 14 days after injury. Evaluation of neointima thickening demonstrated a slow but steady increase of neointima that was significant after day 6 and reached 30% of the lumen in 2 weeks. This was paralleled by an increase in DNA content, which was significant 4 days after injury. ACE mRNA levels were quantified by polymerase chain reaction after reverse transcription. Measurement of ACE mRNA levels revealed a significant upregulation 2 and 8 days after injury, with no significant difference when compared with control tissue at later time points. ACE activity was also significantly enhanced at 2 and 8 days after injury, with no significant difference when compared with control tissue at later time points. In addition, the treatment with ramiprilat was more efficient in reducing neointima formation than that with HR 720. These data underlie the role of ACE in this model of restenosis. The early induction of ACE expression after endothelial injury but before significant changes in the vessel structure suggests that ACE activity might be one of the mechanisms that trigger neointima formation in the rat.

Effect of losartan on right ventricular hypertrophy and cardiac angiotensin I-converting enzyme activity in pulmonary hypertensive rats.

The aim of this study was to investigate the effect of prophylactic treatment with the angiotensin type 1 (AT1) receptor antagonist losartan on right ventricular hypertrophy and cardiac angiotensin 1-converting enzyme (ACE) activity in a rat model of monocrotaline-induced pulmonary hypertension. Losartan failed to prevent either pulmonary hypertension or right ventricular hypertrophy. Right ventricular ACE in untreated pulmonary hypertensive rats did not differ from control rats. Losartan treatment in pulmonary hypertensive rats caused a significant 2-fold increase of ACE activity in the hypertrophied right (p < 0.005) but not in the left ventricle. Thus, cardiac ACE activity is not stimulated in rats with monocrotaline-induced right ventricular hypertrophy. Prophylactic losartan treatment in this model of progressive pulmonary hypertension failed to prevent or reduce the increase in ventricular afterload. The relevance of the increase in right ventricular ACE activity during pulmonary hypertension after losartan treatment is unknown and needs to be evaluated in further studies.

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.

Induction of cardiac angiotensin I-converting enzyme with dietary NaCl-loading in genetically hypertensive and normotensive rats.

We have recently shown that the angiotensin I converting enzyme (ACE) gene is linked to NaCl-loaded blood pressure in the stroke-prone spontaneously hypertensive rat (SHRSP), and that high-NaCl loading selectively stimulates ACE in the aorta of SHRSP but not in normotensive Wistar-Kyoto (WKY) rats. We therefore investigated the relationship between cardiac ACE and the development of hypertension and left ventricular hypertrophy in response to normal- and high-NaCl diet in these rats. ACE mRNA and ACE activity were measured in left ventricular tissue after completion of hemodynamic characterization of the animals. While SHRSP rats increased blood pressure (P < 0.0001) and heart rate (P < 0.005) in response to high NaCl, blood pressure remained unchanged in WKY. Similarly, relative left ventricular weight increased only in SHRSP after high NaCl (P < 0.002). A significant two- to threefold increase of cardiac ACE mRNA and fourfold stimulation of ACE enzyme activity in response to high NaCl was found in both WKY and SHRSP rats (P < 0.005). The induction of ACE gene expression was significantly more pronounced in SHRSP compared to WKY (P < 0.02), whereas no significant strain differences in left ventricular ACE activity were found after either normal- or high-NaCl diet. Thus, arterial blood pressure and left ventricular weight remained unchanged in the WKY rats despite the activation of left ventricular ACE activity after high-NaCl exposure. These results demonstrate that left ventricular ACE activity is equally upregulated in response to high-NaCl in the normotensive and hypertensive strain, independently from the development of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)