[Production and characterization of a novel aminopeptidase A from Lactococcus lactis].

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co2+, Mn2+, and Zn2+ but was strongly inhibited by Ni2+and Cu2+. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.

New Approaches Targeting the Renin-Angiotensin System: Inhibition of Brain Aminopeptidase A, ACE2 Ubiquitination, and Angiotensinogen.

Despite the availability of various therapeutic classes of antihypertensive drugs, hypertension remains poorly controlled, in part because of poor adherence. Hence, there is a need for the development of antihypertensive drugs acting on new targets to improve control of blood pressure. This review discusses novel insights (including the data of recent clinical trials) with regard to interference with the renin-angiotensin system, focusing on the enzymes aminopeptidase A and angiotensin-converting enzyme 2 (ACE2) in the brain, as well as the substrate of renin- angiotensinogen-in the liver. It raises the possibility that centrally acting amino peptidase A inhibitors (eg, firibastat), preventing the conversion of angiotensin II to angiotensin III in the brain, might be particularly useful in African Americans and patients with obesity. Firibastat additionally upregulates brain ACE2, allowing the conversion of angiotensin II to its protective metabolite angiotensin-(1-7). Furthermore, antisense oligonucleotides or small interfering ribonucleic acids suppress hepatic angiotensinogen for weeks to months after 1 injection and thus could potentially overcome adherence issues. Finally, interference with ACE2 ubiquitination is emerging as a future option for the treatment of neurogenic hypertension, given that ubiquitination resistance might upregulate ACE2 activity.

The Entomopathogenic Fungus Beauveria bassiana Employs Autophagy as a Persistence and Recovery Mechanism during Conidial Dormancy.

Many filamentous fungi develop a conidiation process as an essential mechanism for their dispersal and survival in natural ecosystems. However, the mechanisms underlying conidial persistence in environments are still not fully understood. Here, we report that autophagy is crucial for conidial lifespans (i.e., viability) and vitality (e.g., stress responses and virulence) in the filamentous mycopathogen Beauveria bassiana. Specifically, Atg11-mediated selective autophagy played an important, but not dominant, role in the total autophagic flux. Furthermore, the aspartyl aminopeptidase Ape4 was found to be involved in conidial vitality during dormancy. Notably, the vacuolar translocation of Ape4 was dependent on its physical interaction with autophagy-related protein 8 (Atg8) and associated with the autophagic role of Atg8, as determined through a truncation assay of a critical carboxyl-tripeptide. These observations revealed that autophagy acted as a subcellular mechanism for conidial recovery during dormancy in environments. In addition, a novel Atg8-dependent targeting route for vacuolar hydrolase was identified, which is essential for conidial exit from a long-term dormancy. These new insights improved our understanding of the roles of autophagy in the physiological ecology of filamentous fungi as well as the molecular mechanisms involved in selective autophagy. IMPORTANCE Conidial environmental persistence is essential for fungal dispersal in ecosystems while also serving as a determinant for the biocontrol efficacy of entomopathogenic fungi during integrated pest management. This study identified autophagy as a mechanism to safeguard conidial lifespans and vitality postmaturation. In this mechanism, the aspartyl aminopeptidase Ape4 translocates into vacuoles via its physical interaction with autophagy-related protein 8 (Atg8) and is involved in conidial vitality during survival. The study revealed that autophagy acted as a subcellular mechanism for maintaining conidial persistence during dormancy, while also documenting an Atg8-dependent targeting route for vacuolar hydrolase during conidial recovery from dormancy. Thus, these observations provided new insight into the roles of autophagy in the physiological ecology of filamentous fungi and documented novel molecular mechanisms involved in selective autophagy.

Characterization of an Aminopeptidase A from Tetragenococcus halophilus CY54 Isolated from Myeolchi-Jeotgal.

In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni- NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60°C, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

Cancer-Associated Fibroblasts Exert Proangiogenic Activity in Merkel Cell Carcinoma.

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention.

Production and characterization of a novel aminopeptidase A from Lactococcus lactis / 生物工程学报

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co2+, Mn2+, and Zn2+ but was strongly inhibited by Ni2+and Cu2+. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.

Activatable self-assembled organic nanotheranostics: Aspartyl aminopeptidase triggered NIR fluorescence imaging-guided photothermal/photodynamic synergistic therapy.

Phototherapy has developed as a powerful method for remedial modalities. The conventional photosensitizers are "always on" state and lack tumor targeting, which contributed to poor therapeutic effect and high toxicity. Therefore, we developed an aspartyl aminopeptidase (DNPEP) activated self-assembled organic nanoparticles (NRh-Asp NPs) with sensitive external irradiation-induced photothermal therapy and photodynamic therapy (PTT/PDT). NRh-Asp NPs can be activated to NRh-NH2 NPs by DNPEP, demonstrating strong near-infrared (NIR) fluorescence, and efficiently generating heat and singlet oxygen under the near-infrared laser. NRh-Asp NPs was successfully used for visualizing DNPEP in vitro and in vivo in NIR region, and demonstrated good synergistic anti-cancer efficacy of PDT and PTT. These results suggest that DNPEP-mediated NRh-Asp NPs are promising candidates for image-guided phototherapeutic of tumor.

Characterization of aspartyl aminopeptidase from Schistosoma japonicum.

The tegument of schistosomes is the interface between the worm and the host environment. Some molecules distributed on the tegument participate in host-parasite interactions. Aspartyl aminopeptidase (AAP), identified on the tegument of Schistosoma japonicum (S. japonicum), facilitate protein turnover by acting in concert with other aminopeptidases. In this study, the gene encoding S. japonicum aspartyl aminopeptidase (SjAAP) was cloned, expressed and characterized. Quantitative real-time PCR analysis showed that SjAAP was expressed in all studied developmental stages. The transcript level was higher in 8, 14, 21, and 28 days old worms than the other detected stages. Moreover, the level of expression in 42-day-old male worms was significantly higher than that in females. The recombinant SjAAP (rSjAAP) was expressed as both supernatant and inclusion bodies in Escherichia coli BL21 cells. The enzymatic activity of rSjAAP was 4.45 U/mg. The Km and Vmax values for H-Asp-pNA hydrolysis were discovered to be 5.93 mM and 0.018 mM·min-1. Immunofluorescence analysis revealed that SjAAP is primarily distributed on the tegument and parenchyma of schistosomes. Western blot showed that rSjAAP possessed good immunogenicity. Although specific antibodies were produced in BALB/c mice vaccinated with rSjAAP emulsified with ISA 206 adjuvant, no significant reduction of worm burden and number of eggs in the liver was observed. Therefore, rSjAAP may not be suitable to act as a potential vaccine candidate against schistosomiasis japonica in mice. However, this study provides some foundation for further exploration of the biological function of this molecule.

QGC606: A Best-in-Class Orally Active Centrally Acting Aminopeptidase A Inhibitor Prodrug for Treating Heart Failure Following Myocardial Infarction.

BACKGROUND: Blockade of brain renin-angiotensin system (RAS) overactivity by firibastat, the first centrally acting aminopeptidase A (APA) inhibitor prodrug, has already demonstrated its effectiveness in improving cardiac function after myocardial infarction (MI). We developed QGC606, a more potent and more selective APA inhibitor prodrug and studied its effects after long-term oral administration in mice post-MI. METHODS: Two days after MI induced by the left anterior descending artery ligation, adult male mice were randomized into 4 groups to receive oral treatment during 4 weeks with vehicle; QGC606; firibastat; or the angiotensin-I converting enzyme inhibitor ramipril, used as positive control. RESULTS: Four weeks post-MI, brain APA was overactivated in vehicle-treated MI mice. QGC606 treatment normalized brain APA hyperactivity to control values measured in sham-operated mice. Four weeks post-MI, QGC606-treated mice had higher left ventricular (LV) ejection fractions, significantly smaller LV end-systolic diameter and volume, significantly lower HF biomarkers mRNA expression (Myh7 and Anf) and plasma N-terminal pro B-type natriuretic peptide (NT-pro-BNP) and noradrenaline levels than saline-treated mice. QGC606 treatment significantly improved the dP/dt max and min, LV end-diastolic pressure without affecting blood pressure (BP), whereas we observed a decrease in BP in ramipril-treated mice. We observed also a reduction of cardiac fibrosis, highlighted by lower connective tissue growth factor mRNA levels and a reduction of both the fibrotic area and MI size in QGC606-treated mice. CONCLUSIONS: Chronic oral QGC606 administration in post-MI mice showed beneficial effects in improving cardiac function and reducing cardiac remodeling and fibrosis but, unlike ramipril, without lowering BP.

Firibastat: An Oral First-in-Class Brain Aminopeptidase A Inhibitor for Systemic Hypertension.

Systemic hypertension is the leading cause of death and disability worldwide. The management of hypertension is challenging in the high-risk patient population with high salt-sensitivity and low serum renin levels. The renin-angiotensin system (RAS) plays a central role in blood pressure (BP) regulation. While we have effective medications to act on peripheral RAS, our understanding of brain RAS and its effect on BP regulation is still in an evolving stage. Brain RAS hyperactivity is associated with the development and maintenance of hypertension. In comparison to peripheral RAS, where angiotensin II is the most crucial component responsible for BP regulation, angiotensin III is likely the main active peptide in the brain RAS. Angiotensin II is metabolized by aminopeptidase A into angiotensin III in the brain. EC33 is a potent inhibitor of brain aminopeptidase A tested in animal models. The use of EC33 in conscious spontaneously hypertensive rats, hypertensive deoxycorticosterone acetate-salt rats, and conscious normotensive rat models leads to a reduction in BP. In order to facilitate the passage of EC33 through the blood-brain barrier, the 2 molecules of EC33 were linked by a disulfide bridge to form a prodrug called RB150. RB150, later renamed as QGC001 or firibastat, was found to be effective in animal models and well-tolerated when used in healthy participants. Firibastat was found to be safe and effective in phase 2 trials, and is now planned to undergo a phase 3 trial. Firibastat has the potential to be groundbreaking in the management of resistant hypertension.

Firibastat: a Novel Treatment for Hypertension.

PURPOSE OF REVIEW: The purpose of this review is to discuss the unique mechanism of firibastat, a new antihypertension medication. Hypertension continues to be a highly prevalent public health issue. RECENT FINDINGS: Firibastat is a novel agent developed to treat hypertension. As the first member in the class of centrally acting agents to target the brain renin angiotensin system, firibastat offers new pathways to consider and enhances the regimen of agents currently available to treat hypertension. Recent clinical trials have demonstrated effectiveness and safety in mild hypertension as well as resistant hypertension. This review introduces firibastat as a new therapeutic class of treatment for hypertension focused on the renin angiotensin system in the brain. Early studies have shown a significant reduction in blood pressure with minimal side effects particularly in patients who are difficult to treat.

The Level and Significance of Circulating Angiotensin-III in Patients with Coronary Atherosclerosis.

OBJECTIVE: Angiotensin-III (Ang-III) is the downstream product of angiotensin-II (Ang-II) metabolized by aminopeptidase A (APA). At present, the research of Ang-III mainly concentrates on hypertension and the central renin-angiotensin system (RAS). However, few studies have focused on the relationship between Ang-III and coronary atherosclerosis (CAS). METHODS AND RESULTS: Plasma Ang-III and APA levels were measured by the enzyme-linked immunosorbent assay (ELISA) in 44 normal subjects and 84 patients confirmed as having CAS by coronary angiography. Circulating Ang-III levels were significantly lower in patients with CAS than in normal controls (P = 0.013). APA levels were slightly lower in the CAS group (P = 0.324). According to the severity of atherosclerosis, CAS patients were divided into two groups. Compared with the controls, the APA and Ang-III levels were lower in the high scoring group and APA decreased significantly. CONCLUSIONS: Circulating Ang-III levels were reduced in patients with CAS, and the possible reason may be related to the decrease in the APA level.

Molecular cloning and characterization of a novel aspartyl aminopeptidase from Trichinella spiralis.

Trichinellosis is an important zoonotic parasitic disease worldwide and is principally caused by ingesting animal meat containing Trichinella infective larvae. Aspartyl aminopeptidase is an intracytoplasmic metalloproteinase that specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids (aspartic acid and glutamate), and plays an important role in the metabolism, growth and development of organisms. In this study, a novel T. spiralis aspartyl aminopeptidase (TsAAP) was cloned and expressed, and its biological properties and roles in worm growth and development were investigated. The results revealed that TsAAP transcription and expression in diverse T. spiralis stages were detected by RT-PCR and Western blotting, and primarily localized at cuticle, stichosome and intrauterine embryos of this nematode by immunofluorescence test. rTsAAP has the enzymatic activity of native AAP to hydrolyze the substrate H-Glu-pNA. There was a specific binding between rTsAAP and murine erythrocyte, and the binding site was localized in erythrocyte membrane proteins. Silencing of TsAAP gene by specific dsRNA significantly reduced the TsAAP expression, enzymatic activity, intestinal worm burdens and female fecundity. The results demonstrated that TsAAP participates in the growth, development and fecundity of T. spiralis and it might be a potential target molecule for anti-Trichinella vaccines.

The Long-Term Study of Urinary Biomarkers of Renal Injury in Spontaneously Hypertensive Rats.

BACKGROUND: The age-related increase in blood pressure in spontaneously hypertensive rats (SHRs) is associated to cardiac hypertrophy, heart failure, and renal injury. Here, we investigated for the first time the urinary enzymatic activities of glutamil aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), and Klotho urinary levels, proteins that are strongly expressed in the kidney, as early biomarkers of renal injury in SHRs. METHODS: Male SHR and Wistar Kyoto (WKY) rats were studied from 2 to 8 months old. Systolic blood pressure (SBP), the heart rate (HR), metabolic variables, and urinary markers were measured monthly. At the end of the study, a histopathological evaluation of the kidney was performed. RESULTS: Kidneys of SHR did not develop signs of relevant histopathological changes, but showed increased glomerular area and cellularity. Plasma creatinine was decreased, and creatinine clearance was augmented in SHR at the end of the study. Urinary excretion of Klotho was higher in SHR at 5 and 8 months old, whereas plasma Klotho levels were similar to WKY. GluAp, AlaAp, and DPP4 urinary activities were increased in SHR throughout the time-course study. A positive correlation between glomerular area and cellularity with creatinine clearance was observed. Urinary GluAp, AlaAp, DPP4, and Klotho showed positive correlations with SBP. CONCLUSIONS: GluAp, AlaAp, DPP4, and Klotho in the urine are useful tools for the evaluation of renal damage at early stages, before the whole histopathological and biochemical manifestations of renal disease are established. Moreover, these observations may represent a novel and noninvasive diagnostic approach to assess the evolution of kidney function in hypertension and other chronic diseases.

Effects of firibastat in combination with enalapril and hydrochlorothiazide on blood pressure and vasopressin release in hypertensive DOCA-salt rats.

In the brain, aminopeptidase A (APA) generates angiotensin III, one of the effector peptides of the brain renin-angiotensin system (RAS), exerting tonic stimulatory control over blood pressure (BP) in hypertensive rats. Oral administration of firibastat, an APA inhibitor prodrug, in hypertensive rats, inhibits brain APA activity, blocks brain angiotensin III formation and decreases BP. In this study, we evaluated the efficacy of firibastat in combination with enalapril, an angiotensin I-converting enzyme inhibitor, and hydrochlorothiazide (HCTZ), in conscious hypertensive deoxycorticosterone acetate (DOCA)-salt rats, which display high plasma arginine-vasopressin levels, low circulating renin levels and resistance to treatment by systemic RAS blockers. We determined mean arterial BP, heart rate, plasma arginine-vasopressin levels and renin activity in DOCA-salt rats orally treated with firibastat, enalapril or HCTZ administered alone or in combination. Acute oral firibastat administration (30 mg/kg) induced a significant decrease in BP, whereas enalapril (10 mg/kg) or HCTZ (10 mg/kg) administered alone induced no significant change in BP in conscious DOCA-salt rats. The BP decrease induced by acute and nine-day chronic tritherapy [Firibastat+Enalapril+HCTZ] was significantly greater than that observed after bitherapy [Enalapril+HCTZ]. Interestingly, the chronic administration of a combination of firibastat with [Enalapril+HCTZ] reduced plasma arginine-vasopressin levels by 62% relative to those measured in DOCA-salt rats receiving bitherapy. Our data show that tritherapy with firibastat, enalapril and HCTZ improves BP control and arginine-vasopressin release in an experimental salt-dependent model of hypertension, paving the way for the development of new treatments for patients with currently difficult-to-treat or resistant hypertension.

Aminopeptidase A contributes to biochemical, anatomical and cognitive defects in Alzheimer's disease (AD) mouse model and is increased at early stage in sporadic AD brain.

One of the main components of senile plaques in Alzheimer's disease (AD)-affected brain is the Aß peptide species harboring a pyroglutamate at position three pE3-Aß. Several studies indicated that pE3-Aß is toxic, prone to aggregation and serves as a seed of Aß aggregation. The cyclisation of the glutamate residue is produced by glutaminyl cyclase, the pharmacological and genetic reductions of which significantly alleviate AD-related anatomical lesions and cognitive defects in mice models. The cyclisation of the glutamate in position 3 requires prior removal of the Aß N-terminal aspartyl residue to allow subsequent biotransformation. The enzyme responsible for this rate-limiting catalytic step and its relevance as a putative trigger of AD pathology remained yet to be established. Here, we identify aminopeptidase A as the main exopeptidase involved in the N-terminal truncation of Aß and document its key contribution to AD-related anatomical and behavioral defects. First, we show by mass spectrometry that human recombinant aminopeptidase A (APA) truncates synthetic Aß1-40 to yield Aß2-40. We demonstrate that the pharmacological blockade of APA with its selective inhibitor RB150 restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aß-precursor protein (ßAPP). Pharmacological reduction of APA activity and lowering of its expression by shRNA affect pE3-42Aß- and Aß1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA inhibitors and shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of pE3-42Aß-positive plaques, APA activity is augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA is a key enzyme involved in Aß N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with AD pathology.

Brain ACE2 activation following brain aminopeptidase A blockade by firibastat in salt-dependent hypertension.

In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats without inducing angiotensin II accumulation. We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. We found that the intracerebroventricular (icv) administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.

Hypothalamic Renin-Angiotensin System and Lipid Metabolism: Effects of Virgin Olive Oil versus Butter in the Diet.

The brain renin-angiotensin system (RAS) has been recently involved in the homeostatic regulation of energy. Our goal was to analyse the influence of a diet rich in saturated fatty acids (butter) against one enriched in monounsaturated fatty acids (olive oil) on hypothalamic RAS, and their relationship with the metabolism of fatty acids. Increases in body weight and visceral fat, together with an increase in aminopeptidase A expression and reductions in AngII and AngIV were observed in the hypothalamus of animals fed with the butter diet. In this group, a marked reduction in the expression of genes related to lipid metabolism (LPL, CD36, and CPT-1) was observed in liver and muscle. No changes were found in terms of body weight, total visceral fat and the expression of hepatic genes related to fatty acid metabolism in the olive oil diet. The expressions of LPL and CD36 were reduced in the muscles, although the decrease was lower than in the butter diet. At the same time, the fasting levels of leptin were reduced, no changes were observed in the hypothalamic expression of aminopeptidase A and decreases were noted in the levels of AngII, AngIV and AngIII. These results support that the type of dietary fat is able to modify the hypothalamic profile of RAS and the body energy balance, related to changes in lipid metabolism.

Aminopeptidase A (ENPEP) gene polymorphisms and preeclampsia: Descriptive analysis.

OBJECTIVE: The etiology of preeclampsia (PE) remains elusive. Recent genome-wide association studies have identified a number of genetic variants associated with blood pressure variations in east Asians. One of the genetic variants is the aminopeptidase A (ENPEP) gene, which converts angiotensin II to angiotensin III. The C allele of rs6825911 is a risk for hypertension. The current study investigated whether genetic variants of ENPEP play a role in the pathogenesis of preeclampsia. STUDY DESIGN: The study was a descriptive analysis of gene polymorphisms of ENPEP; 602 pregnant women of African ancestry [normotensive (n = 245) and PE (n = 357)] were recruited. The two groups were divided according to their HIV status. The PE group consisted of early- and late-onset sub-categories. A single nucleotide polymorphism of rs6825911 was analyzed using the TaqMan® Probe mix and by means of real time polymerase chain reaction. RESULTS: The risk of C allele for PE was 1.07 (95 % CI 0.83-1.38, P = 0.589) for allele comparison and the risk for preeclampsia CC to CT/TT was 1.33 (95 % CI 0.96-1.85, P = 0.086). The sub analysis for the PE group without HIV infection the risk of C allele was 1.25 (95 % CI 0.838-1.78, P = 0.199) and the risk of PE of CC to CT/TT was 1.51 (95 %CI: 0.96-2.35, P = 0.071). CONCLUSION: This is the first study in a homogenous South African population of African ancestry to show that the variant of ENPEP gene does not play a role in pathogenesis of preeclampsia.

Knockout of aminopeptidase A in mice causes functional alterations and morphological glomerular basement membrane changes in the kidneys.

Aminopeptidase A is one of the most potent enzymes within the renin-angiotensin system in terms of angiotensin II degradation. Here, we examined whether there is a kidney phenotype and any compensatory changes in other renin angiotensin system enzymes involved in the metabolism of angiotensin II associated with aminopeptidase A deficiency. Kidneys harvested from aminopeptidase A knockout mice were examined by light and electron microscopy, immunohistochemistry and immunofluorescence. Kidney angiotensin II levels and the ability of renin angiotensin system enzymes in the glomerulus to degrade angiotensin II ex vivo, their activities, protein and mRNA levels in kidney lysates were evaluated. Knockout mice had increased blood pressure and mild glomerular mesangial expansion without significant albuminuria. By electron microscopy, knockout mice exhibited a mild increase of the mesangial matrix, moderate thickening of the glomerular basement membrane but a striking appearance of knob-like structures. These knobs were seen in both male and female mice and persisted after the treatment of hypertension. In isolated glomeruli from knockout mice, the level of angiotensin II was more than three-fold higher as compared to wild type control mice. In kidney lysates from knockout mice angiotensin converting enzyme activity, protein and mRNA levels were markedly decreased possibly as a compensatory mechanism to reduce angiotensin II formation. Thus, our findings support a role for aminopeptidase A in the maintenance of glomerular structure and intra-kidney homeostasis of angiotensin peptides.