Julie L. Lavoie

Enzymatic Activity of Lysosomal Carboxypeptidase (Cathepsin) A Is Required for Proper Elastic Fiber Formation and Inactivation of Endothelin-1

Background— Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with &bgr;-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome. Methods and Results— We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalytically inactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiency of Neu1 and GAL. Conversely, the mice showed a reduced degradation rate of the vasoconstrictor peptide, endothelin-1, and significantly increased arterial blood pressure. CathA-deficient mice also displayed scarcity of elastic fibers in lungs, aortic adventitia, and skin. Conclusions— Our results provide the first evidence that CathA acts in vivo as an endothelin-1–inactivating enzyme and strongly confirm a crucial role of this enzyme in effective elastic fiber formation.

Evidence Supporting a Functional Role for Intracellular Renin in the Brain

The brain renin–angiotensin system is implicated in the regulation of blood pressure (BP) and fluid homeostasis. Recent studies reveal that 2 forms of renin are expressed in the brain of rodents and humans: secreted prorenin and a nonsecreted intracellular form of active renin (icREN). Although the intracellular action of renin has long been postulated, no data supporting its role in BP regulation has been reported. Therefore, we directly evaluated whether this form of renin has physiological implications for BP regulation by characterizing transgenic mice expressing human icREN driven by the glial fibrillary acidic protein (GFAP) promoter and comparing it with similar mice expressing the secreted form of renin. GFAP-icREN mice express hREN primarily in the brain and at the same level of expression as GFAP-secreted prorenin. Unlike the secreted form, which can be detected in cerebrospinal fluid, no human renin could be detected in the cerebrospinal fluid of GFAP-icREN mice. GFAP-icREN mice were then bred with transgenic mice expressing human angiotensinogen, also driven by the GFAP promoter. Double-transgenic mice expressing either the intracellular renin (2.0±0.12 mL/10 g/day) or secreted renin (2.8±0.3 mL/10 g/day) exhibited an increase in drinking volume compared with nontransgenic littermates (1.5±0.1 mL/10 g/day). Both models exhibited an increase in mean arterial pressure (137±5 and 133±8 mm Hg, respectively) compared with control littermates (115±3 mm Hg), which could be rapidly reduced after ICV injection of losartan. These data support the concept of an intracellular form of renin in the brain, which may provoke functional changes in fluid homeostasis and BP regulation.

Adjacent Expression of Renin and Angiotensinogen in the Rostral Ventrolateral Medulla Using a Dual-Reporter Transgenic Model

Abstract—All components of the renin-angiotensin system are localized in the brain. However, because renin is present in very low concentrations, the mechanism by which angiotensin II is formed in the brain remains unclear. We previously reported the development of 2 transgenic mouse models using sensitive reporters, enhanced green fluorescent protein (eGFP) and β-galactosidase (β-Gal), to examine the cellular localization of renin and angiotensinogen in the mouse brain. To determine whether renin and angiotensinogen are coexpressed or present in neighboring cells in the rostral ventrolateral medulla (RVLM) and other cardiovascular control regions of the brain, we produced and examined double-transgenic mice, which express eGFP driven by the renin promoter (REN-1c/eGFP) and β-gal driven by the human angiotensinogen promoter (hAGT/β-gal). Using these reporter transgenes as sensitive markers for renin and angiotensinogen expression, we conclude that both proteins are coexpressed in the parabrachial nucleus and central nucleus of the amygdala and are in adjacent cells in the RVLM, reticular formation, bed nucleus of the stria terminalis, subfornical organ, and CA1–3 region. These data suggests that, in these areas, both renin and angiotensinogen are in close proximity providing the potential for the local formation of angiotensin I either intracellularly, when there is colocalization, or in the interstitium, when they are in juxtaposed cells.

Impact of Exercise Training on Preeclampsia: Potential Preventive Mechanisms

Preeclampsia is characterized by hypertension and de novo proteinuria after 20 weeks of pregnancy. It is the leading cause of perinatal morbidity and mortality in the developed world, and to date, the only means of treating the disease is by inducing delivery. Many studies have shown the benefits of exercise training on normal pregnancy. Conversely, because the impact of exercise on reducing the risk of preeclampsia has long been debated, the American College of Obstetricians and Gynecologists has yet to support the prescription of exercise training to women at risk of developing the disease. There is, however, a significant body of evidence in support of the protective role of exercise training against preeclampsia. A recent animal study demonstrated that many preeclampsia features can be eliminated with prenatal followed by gestational exercise training. Hence, the present article reviews the literature on the impact of exercise training on preeclampsia risk, as well as the mechanisms that may be involved.

Development of Hypertension and Kidney Hypertrophy in Transgenic Mice Overexpressing ARAP1 Gene in the Kidney

Angiotensin II regulates blood pressure via activation of the type 1 receptor. We previously identified a novel angiotensin II type 1 receptor–associated protein and demonstrated that it promotes receptor recycling to the plasma membrane. To delineate the pathophysiological function of the ARAP1 in the kidneys, we generated transgenic mice that overexpress rat ARAP1 cDNA specifically in proximal tubules and tested the hypothesis that proximal tubule-specific overexpression of ARAP1 causes hypertension. Two lines of male transgenic mice, 650 and 670, displayed kidney-specific transgene expression. Systolic blood pressure was significantly elevated by ≈20 to 25 mm Hg in these lines of mice at 20 weeks of age compared with their nontransgenic litter mates. Urine volume, but not water intake, was significantly decreased in both lines compared with nontransgenic controls. The kidney/body weight ratio was significantly increased in both lines compared with their nontransgenic litter mates at 12 and 20 weeks of age. In contrast, no difference was observed in the ratio of brain, spleen, heart, and testis to body weight between male transgenic and nontransgenic animals. Inhibitions of the renin–angiotensin system completely normalized the systolic blood pressure of transgenic mice. Moreover, low salt intake prevented the development of hypertension, whereas high salt intake exacerbated the increase in blood pressure in transgenic mice. Therefore, our data show that proximal tubule-specific overexpression of ARAP1 leads to hypertension, suggesting that renal ARAP1 plays an important role in the regulation of blood pressure and renal function via activation of the intrarenal renin–angiotensin system.

The Two fACEs of the Tissue Renin-Angiotensin Systems: Implication in Cardiovascular Diseases

The implication of the renin-angiotensin system (RAS) in the regulation of the cardiovascular system has been well known for many years. Accordingly, many pharmaceutical inhibitors have been developed to treat several pathologies, like hypertension and heart failure, and angiotensin converting enzyme (ACE) became one of the major target in the treatment of these cardiovascular diseases. In the last decade however, it has become apparent that the classical view of the RAS was not quite accurate. For instance, ACE has been shown to work not only by generating angiotensin-II but also by interacting with receptors outside the renin-angiotensin system. Moreover, it has been shown that many local RAS are present in different tissues, such as the heart, brain, kidney and vasculature. However, in the past, it was impossible to determine the role of these local systems as they were pharmacologically indistinguishable from the systemic RAS. Hence, in recent years, the development of transgenic animals has allowed us to determine that these local systems are implicated in the roles that had been originally attributed exclusively to the systemic action of the RAS. However, with almost 30% of the medicated hypertensive patients harboring an uncontrolled blood pressure, a need for new drugs and new targets appears necessary. With the new century came the discovery of a new homolog of ACE, called ACE2, and early studies suggest that it may play a pivotal role in the RAS by controlling the balance between the vasoconstrictor effects of angiotensin-II and the vasodilatory properties of the angiotensin(1-7) peptide. Like ACE, ACE2 appears to hydrolyze peptides not related with the RAS and the enzyme has also been identified as a receptor for the severe acute respiratory syndrome (SARS) coronavirus. Although the tissue localization of ACE2 was originally though to be very restricted, new studies have emerged showing a more widespread distribution. Therefore, the whole dynamics of the RAS has to be re-evaluated in light of this new information. In this review, we will compare the structures, distributions and properties of ACE and its new homologue in the context of cardiovascular function, focusing on the autocrine/paracrine cardiac and brain renin-angiotensin systems and we will present recent data from the literature and our laboratory offering a new perspective on this potential target for the treatment of cardiovascular diseases.

Spontaneous Stroke in a Genetic Model of Hypertension in Mice

Background and Purpose— Hypertension is the most common risk factor for hemorrhagic stroke. An experimental model of stroke, the stroke-prone spontaneously hypertensive rat (SHRSP), which has been enormously useful in studies of cerebral circulation, has been used in >1000 papers. However, SHRSP usually have an ischemic or less commonly hemorrhagic stroke in the cortex, not in the brain stem, cerebellum, or basal ganglia, as in patients with hypertension. The goal of this study was to develop a model of hemorrhagic stroke in hypertensive mice. Methods— A genetic model of hypertensive mice, double transgenic mice (R+/A+) that overexpress both human renin (R+) and human angiotensinogen (A+), and nonhypertensive control mice were divided into 3 groups: (1) high-salt diet; (2) N&ohgr;-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthases, in drinking water; and (3) high-salt diet and l-NAME. Results— All R+/A+ mice on high-salt diet and l-NAME died within 10 weeks, with hemorrhage in the brain stem, and several of the mice had hemorrhages in brain stem, cerebellum, and basal ganglia. No control mice on high-salt diet and l-NAME had hemorrhagic stroke. Arterial pressure in R+/A+ mice increased progressively during high-salt diet and l-NAME. In R+/A+ and control mice, high-salt diet or l-NAME alone did not increase arterial pressure. Conclusions— We now describe the first model of spontaneous hemorrhagic strokes in hypertensive mice. The type and locations of stroke are reasonably similar to those observed in patients with hypertension.

Mice Overexpressing Both Human Angiotensinogen and Human Renin as a Model of Superimposed Preeclampsia on Chronic Hypertension

Preeclampsia is the major cause of maternal and fetal mortality/morbidity. Because hypertension is an important risk factor for preeclampsia, we investigated whether hypertensive mice that overexpress human renin and angiotensinogen develop superimposed preeclampsia. Given that the mechanisms underlying this disease are still poorly understood, animal models are of great use for elucidatation. Blood pressure and proteinuria were measured by telemetry and ELISA, respectively. Heart function was evaluated by echocardiography, whereas pathological cardiac hypertrophy–related genes were assessed by real-time PCR. Soluble fms-like tyrosine kinase 1 plasma concentrations were quantitated by ELISA and placental expression by real-time PCR. Transgenic mice develop de novo proteinuria during gestation and marked blood pressure elevation, which are hallmarks of superimposed preeclampsia on chronic hypertension. Abnormal placentation present in these mothers produced a significant decrease in pup and placental weight and was associated with an increased placental expression of soluble fms-like tyrosine kinase 1. We also found heightened circulating levels of this receptor, when adjusted for placental mass, as has been observed in women who suffer from preeclampsia. Cardiac hypertrophy could be observed in the transgenic mice and was exacerbated by gestation. As a result, heart function was significantly decreased, and markers of pathological hypertrophy were increased. Our data, thus, confirm the characterization of a new model of superimposed preeclampsia on chronic hypertension. Because chronically hypertensive women are at risk of developing the pathology, our model reflects a clinical reality and is, thus, an excellent tool to elucidate the molecular mechanisms triggering this disease.

Novel Role of the Renin–Angiotensin System in Preeclampsia Superimposed on Chronic Hypertension and the Effects of Exercise in a Mouse Model

Gestational hypertensive disorders, such as preeclampsia, affect 6% to 8% of all pregnancies in North America, and they are the leading cause of maternal mortality in industrialized countries, accounting for 16% of deaths. Women with hypertension have an increased risk (15% to 25%) of developing preeclampsia. Our aim was to investigate the mechanisms implicated in preeclampsia superimposed on chronic hypertension and in the protective effects of exercise in a mouse model. Female mice overexpressing human angiotensinogen and human renin were used as a model of preeclampsia superimposed on chronic hypertension. In the trained group, mothers were placed in cages with access to a wheel before mating, and they remained within these throughout gestation. Blood pressure was measured by telemetry. We found that angiotensin II type I receptor was increased, whereas the Mas receptor was decreased in the placenta and the aorta of pregnant sedentary transgenic mice. This would produce a decrease in angiotensin-(1–7) effects in favor of angiotensin II. Supporting the functional contribution of this modulation, we found that the prevention of most pathological features in trained transgenic mice was associated with a normalization of placental angiotensin II type 1 and Mas receptors and an increase in aortic Mas receptor. We also found reduced circulating and placental soluble Fms-like tyrosine kinase-1 in trained transgenic mice compared with sedentary mice. This study demonstrates that modulation of the renin–angiotensin system is a key mechanism in the development of preeclampsia superimposed on chronic hypertension, which can be altered by exercise training to prevent disease features in an animal model.

Receptor tyrosine kinase Ephb6 regulates vascular smooth muscle contractility and modulates blood pressure in concert with sex hormones.

Background: Eph kinases constitute the largest receptor tyrosine kinase family, and there is no knowledge about their function in blood pressure regulation. Results: Ephb6 regulates vascular smooth muscle cell contraction, and its knock-out resulted in increased blood pressure in castrated male mice. Conclusion: Ephb6 and its ligands can regulate vessel tone and blood pressure. Significance: We have identified a new group of molecules capable of regulating blood pressure. Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.