Sebastien Fuchs

Mice with Cardiac-Restricted Angiotensin-Converting Enzyme (ACE) Have Atrial Enlargement, Cardiac Arrhythmia, and Sudden Death

To investigate the local effects of angiotensin II on the heart, we created a mouse model with 100-fold normal cardiac angiotensin-converting enzyme (ACE), but no ACE expression in kidney or vascular endothelium. This was achieved by placing the endogenous ACE gene under the control of the alpha-myosin heavy chain promoter using targeted homologous recombination. These mice, called ACE 8/8, have cardiac angiotensin II levels that are 4.3-fold those of wild-type mice. Despite near normal blood pressure and a normal renal function, ACE 8/8 mice have a high incidence of sudden death. Both histological analysis and in vivo catheterization of the heart showed normal ventricular size and function. In contrast, both the left and right atria were three times normal size. ECG analysis showed atrial fibrillation and cardiac block. In conclusion, increased local production of angiotensin II in the heart is not sufficient to induce ventricular hypertrophy or fibrosis. Instead, it leads to atrial morphological changes, cardiac arrhythmia, and sudden death.

The absence of intrarenal ACE protects against hypertension.

Activation of the intrarenal renin-angiotensin system (RAS) can elicit hypertension independently from the systemic RAS. However, the precise mechanisms by which intrarenal Ang II increases blood pressure have never been identified. To this end, we studied the responses of mice specifically lacking kidney angiotensin-converting enzyme (ACE) to experimental hypertension. Here, we show that the absence of kidney ACE substantially blunts the hypertension induced by Ang II infusion (a model of high serum Ang II) or by nitric oxide synthesis inhibition (a model of low serum Ang II). Moreover, the renal responses to high serum Ang II observed in wild-type mice, including intrarenal Ang II accumulation, sodium and water retention, and activation of ion transporters in the loop of Henle (NKCC2) and distal nephron (NCC, ENaC, and pendrin) as well as the transporter activating kinases SPAK and OSR1, were effectively prevented in mice that lack kidney ACE. These findings demonstrate that ACE metabolism plays a fundamental role in the responses of the kidney to hypertensive stimuli. In particular, renal ACE activity is required to increase local Ang II, to stimulate sodium transport in loop of Henle and the distal nephron, and to induce hypertension.

Cardiac Specific Increase in Aldosterone Production Induces Coronary Dysfunction in Aldosterone Synthase–Transgenic Mice

Background—Elevated circulating aldosterone level is associated with impaired cardiovascular function. Although the mechanisms are not fully understood, aldosterone antagonists decrease total and cardiovascular mortality in heart failure and myocardial infarction. Aldosterone induces cardiac fibrosis in experimental models, and it is synthesized locally in rat heart. These observations suggest pathological effects of aldosterone in heart that remain unclear. Methods and Results—Transgenic mice (TG) that overexpress the terminal enzyme of aldosterone biosynthesis, aldosterone synthase (AS), in heart have been raised by gene targeting with the &agr;-myosin heavy chain promoter. AS mRNA increased 100-fold and aldosterone concentration 1.7-fold in hearts of male TG mice relative to wild-type. No structural or myocardial alterations were evidenced, because ventricle/body weight, AT1 and AT2 receptor binding, and collagen content were unchanged in TG. No alteration in cardiac function was evidenced by echocardiography, isolated perfused heart, or whole-cell patch clamp experiments. In contrast, coronary function was impaired, because basal coronary flow was decreased in isolated perfused heart (−55% of baseline values), and vasodilatation to acetylcholine, bradykinin, and sodium nitroprusside was decreased by 75%, 60%, and 75%, respectively, in TG mice compared with wild-type, showing that the defect was not related to NO production. Conclusions—Increased cardiac aldosterone production in male mice induces a major coronary endothelium-independent dysfunction with no detectable alterations in cardiac structure and function. However, coronary dysfunction may be harmful for coronary adaptation to increased flow demand.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

Angiotensin-converting enzyme (ACE) is a zinc-dependent peptidase responsible for converting angiotensin I into the vasoconstrictor angiotensin II. However, ACE is a relatively nonspecific peptidase that is capable of cleaving a wide range of substrates. Because of this, ACE and its peptide substrates and products affect many physiologic processes, including blood pressure control, hematopoiesis, reproduction, renal development, renal function, and the immune response. The defining feature of ACE is that it is composed of two homologous and independently catalytic domains, the result of an ancient gene duplication, and ACE-like genes are widely distributed in nature. The two ACE catalytic domains contribute to the wide substrate diversity of ACE and, by extension, the physiologic impact of the enzyme. Several studies suggest that the two catalytic domains have different biologic functions. Recently, the X-ray crystal structure of ACE has elucidated some of the structural differences between the two ACE domains. This is important now that ACE domain-specific inhibitors have been synthesized and characterized. Once widely available, these reagents will undoubtedly be powerful tools for probing the physiologic actions of each ACE domain. In turn, this knowledge should allow clinicians to envision new therapies for diseases not currently treated with ACE inhibitors.

Effect of Reduced Angiotensin-Converting Enzyme Gene Expression and Angiotensin-Converting Enzyme Inhibition on Angiotensin and Bradykinin Peptide Levels in Mice

Abstract—There is uncertainty about the contribution of angiotensin-converting enzyme (ACE) to angiotensin II formation, with recent studies suggesting that non-ACE enzymes may be the predominant pathway of angiotensin II formation in kidney, heart, and lung. To investigate the role of ACE in angiotensin II formation, we measured angiotensin I and II levels in blood, kidney, and heart of 2 mouse genetic models (ACE.1 and ACE.4) of reduced somatic ACE gene expression and in blood, kidney, heart, lung, adrenal, and brain of mice administered the ACE inhibitor lisinopril. We also measured the levels of bradykinin (1-9) and its ACE metabolite bradykinin (1-7). Reduced ACE gene expression and ACE inhibition had similar effects on angiotensin and bradykinin peptide levels. Angiotensin II levels were reduced by 70% to 97% in blood, 92% to 99% in kidney, 93% to 99% in heart, 97% in lung, and 85% in adrenal and brain. The marked reductions in angiotensin II/angiotensin I ratio indicated that ACE was responsible for at least 90% of angiotensin I conversion to angiotensin II in blood, kidney, heart, lung, and brain, and at least 77% in adrenal. Blood bradykinin (1-9) levels were increased 6.4-fold to 8.4-fold. Heart bradykinin (1-9) levels were increased in ACE.4 mice and the bradykinin (1-7)/bradykinin (1-9) ratio was reduced in kidney and heart of ACE.4 mice and heart of lisinopril-treated mice. These studies demonstrate that ACE is the predominant pathway of angiotensin II formation in blood and tissues of mice and plays a major role in bradykinin (1-9) metabolism in blood and, to a lesser extent, in kidney and heart.

Angiotensin-Converting Enzyme C-Terminal Catalytic Domain Is the Main Site of Angiotensin I Cleavage In Vivo

Angiotensin-converting enzyme (ACE) plays a central role in the production of the vasoconstrictor angiotensin II. ACE is a single polypeptide, but it contains 2 homologous and independent catalytic domains, each of which binds zinc. To understand the in vivo role of these 2 domains, we used gene targeting to create mice with point mutations in the ACE C-domain zinc-binding motif. Such mice, termed ACE13/13, produce a full-length ACE protein with tissue expression identical to wild-type mice. Analysis of ACE13/13 mice showed that they produce ACE having only N-domain catalytic activity, as determined by the hydrolysis of domain specific substrates and by chloride sensitivity. ACE13/13 mice have blood pressure and blood angiotensin II levels similar to wild-type mice. However, plasma renin concentration is increased 2.6-fold and blood angiotensin I levels are increased 7.5-fold. Bradykinin peptide levels are not different from wild-type levels. ACE13/13 mice have a reduced increase of blood pressure after intravenous infusion of angiotensin I. ACE13/13 mice have a normal renal structure, but they are not able to concentrate urine after dehydration as effectively as wild-type mice. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo. Although mice lacking C-domain activity have normal physiology under laboratory conditions, they respond less well to the stress of dehydration.

A Novel Distal Enhancer Confers Chorionic Expression on the Human Renin Gene

Renin catalyzes the rate-limiting step of the renin-angiotensin system, which regulates blood pressure and electrolyte homeostasis. To determine cell-specific human renin gene control elements, the transcriptional activity of promoter regions up to position −8876 was studied in renin-expressing cells. A positive regulatory region conferring ∼57-fold higher transcriptional activity to the human renin gene promoter in chorionic cells was identified between nucleotides −5777 and −5552. It had the orientation-independent activity typical of classical enhancers. It also conferred ∼59-fold higher transcriptional levels from the heterologous simian virus 40 (SV40) promoter in chorionic cells and ∼6-fold higher transcriptional levels in Calu-6 and As4.1 cells, whereas no effect was measured in non-renin-expressing cells. DNase I footprinting showed that this enhancer contains three binding sites for chorionic cell nuclear extracts. Functional analysis suggested that the activity of the enhancer is regulated by differential mechanisms in the three renin-expressing cells involving a complex arrangement of AP-1 motifs binding cell-specific members of the basic leucine zipper family of transcription factors. Thus, our results demonstrate that this enhancer plays a key role in the expression of the human renin gene in the chorion and may also be involved in its regulated expression in other tissues.

Male fertility is dependent on dipeptidase activity of testis ACE.

c Figure 2 ACE overexpression in CHO and HEK cells does not affect the shedding of multiple GPI-anchored proteins. (a) CHO cells were stably transfected with vector alone (mock), full-length wild-type (FL-ACE) or GPI-ACE10. Endogenous alkaline phosphatase activity shed into the media was determined using p-nitrophenylphosphate as substrate. Results are means ± s.d. (n = 3) and are expressed as a percentage of activity shed into media of mock-transfected cells. (b) HEK cells stably transfected with either doppel or prion protein were transiently transfected with vector alone (mock), FL-ACE or GPI-ACE. Endogenous alkaline phosphatase activity shed into the media was determined using p-nitrophenylphosphate as substrate. Doppel and prion protein shed into media were determined by immunoblotting followed by densitometric analysis. Results are means ± s.d. (n = 3 or 6 (alkaline phosphatase)) and are expressed as a percentage of protein shed into the media of mock-transfected cells. (c) ACE activity determined using BzGly-His-Leu as substrate in lysates from the HEK cells transiently transfected with vector alone (mock), FL-ACE or GPI-ACE as in b. Results are means ± s.d. (n = 3).

Functional characterization of three mutations of the endothelin B receptor gene in patients with Hirschsprung's disease: evidence for selective loss of Gi coupling.

BackgroundHirschsprung’s disease (HSCR) is one the most common congenital intestinal disease. It leads to aganglionic megacolon in the early childhood. Several susceptibility genes have been identified: RET protooncogene and its ligand, glial cell derived neutrophic factor (GDNF), Sox 10, Endothelin-3 (EDN3) and its receptor B (EDNRB). EDNRB mutations are found in 5% of familial or sporadic HSCR. Only few EDNRB mutations found in HSCR have been explored and some of them seem to be non fonctional variants.Materials and MethodsThe properties of three mutant human endothelin B receptor (hETB) (G57S, R319W and P383L) in isolated HSCR were analyzed. Stable recombinant cells expressing the three mutants and the wild-type (WT) were established. The hETB receptors were characterized for 125I ET-1 binding, ET-1 induced signaling: calcium transient, AP-1 transcriptional factor activation and cAMP accumulation.ResultsImmunofluorescence experiments showed normal cellular distributions of the mutant G57S, R319W and WT hETB receptors. In contrast, the P383L hETB mutant receptor was concentrated near the nucleus and essentially no ET-1 binding was detected. The two other mutants (G57S and R319W) bound ET-1 normally, induced calcium transients and activated the AP-1 pathway in the same way as wild type, but did not inhibit adenylate cyclase. The G57S hETB mutant even stimulated cAMP accumulation which was blocked by pertussis toxin.ConclusionThe absence of the P383L mutant receptor from the membrane clearly indicates that this mutation could be involved in HSCR. The G57S and R319W mutant receptors, despite their normal coupling to Gαq, have a defect in the Gαi signaling pathway and the G57s mutation couples to Gαs. These observations allow us to hypothesize that cAMP signaling might be involved in the differenciation of neural cells in the bowel.

Intrarenal Angiotensin-Converting Enzyme Induces Hypertension in Response to Angiotensin I Infusion

The contribution of the intrarenal renin-angiotensin system to the development of hypertension is incompletely understood. Here, we used targeted homologous recombination to generate mice that express angiotensin-converting enzyme (ACE) in the kidney tubules but not in other tissues. Mice homozygous for this genetic modification (ACE 9/9 mice) had low BP levels, impaired ability to concentrate urine, and variable medullary thinning. In accord with the ACE distribution, these mice also had reduced circulating angiotensin II and high plasma renin concentration but maintained normal kidney angiotensin II levels. In response to chronic angiotensin I infusions, ACE 9/9 mice displayed increased kidney angiotensin II, enhanced rate of urinary angiotensin II excretion, and development of hypertension. These findings suggest that intrarenal ACE-derived angiotensin II formation, even in the absence of systemic ACE, increases kidney angiotensin II levels and promotes the development of hypertension.