Angiotensin-converting Enzyme Substrate Research Articles

Uremic Apelin and Leucocytic Angiotensin-Converting Enzyme 2 in CKD Patients.

Apelin peptides (APLN) serve as second substrates for angiotensin-converting enzyme 2 (ACE2) and, in contrast to angiotensin II (AngII), exert blood-pressure lowering and vasodilatation effects through binding to G-coupled APLN receptor (APLNR). ACE2-mediated cleavage of the APLN may reduce its vasodilatory effects, but decreased ACE2 may potentiate the hypotensive properties of APLN. The role of APLN in uremia is unclear. We investigated the correlations between serum-APLN, leucocytic APLNR, and ACE2 in 32 healthy controls (NP), 66 HD, and 24 CKD3–5 patients, and the impact of APLN peptides on monocytic behavior and ACE2 expression under uremic conditions in vitro. We observed that serum APLN and leucocytic APLNR or SLCO2B1 were significantly elevated in uremic patients and correlated with decreased ACE2 on uremic leucocytes. APLN-treated THP-1 monocytes revealed significantly increased APLNR and ACE2, and reduced TNFa, IL-6, and MCSF. Uremic toxins induced a dramatic increase of miR-421 followed by significant reduction of ACE2 transcripts, partially counteracted with APLN-13 and -36. APLN-36 triggered the most potent transmigration and reduction of endothelial adhesion. These results suggest that although APLN peptides may partly protect against the decay of monocytic ACE2 transcripts, uremic milieu is the most dominant modulator of local ACE2, and likely to contribute to the progression of atherosclerosis.

Фенотипирование ангиотензинпревращающего фермента предстательной железы при раке простаты и доброкачественной гиперплазии

Angiotensin-converting enzyme (ACE) is expressed by all epithelial cells of the human body. Although the main proportion of ACE is synthesized by the lungs, in men, ACE is also secreted by the testes (testicular form), seminal vesicles and the prostate. In semen, the level of ACE is up to 50 times higher than in blood plasma. The substitution of highly specific epithelial cells of the prostate by tumor cells causes a dramatic decrease in ACE production by the prostate cells. To assess the possibility of using prostatic ACE as a new marker of prostate cancer (PCa). ACE phenotyping in prostate of patients with PCa and benign prostatic hyperplasia (BPH) included measurement of the activity of two ACE substrates (HHL and ZPHL); calculation of the ratio of their hydrolysis rates (ZPHL/HHL ratio); quantitative assessment of the ACE immunoreactive protein, the ratio of the immunoreactive protein to the ACE activity, as well as the conformation of ACE using a panel of monoclonal antibodies (mAb) to different epitopes of ACE. ACE activity in tumor cells was markedly reduced and the ratio of immunoreactive ACE to its activity increased. The ratio of the hydrolysis rates of two substrates (ZPHL/HHL ratio) in patients with PCa increased compared to control group, while it was not observed in the vast majority of patients with BPH. There were several tissue samples with a histological diagnosis of BPH, but ACE phenotype was typical for PCa. Since a decrease in ACE activity was found in all patients with PCa, we suggest that it may serve as a reliable and early marker of the tumor development. Changes in the ACE phenotype, which are typical for PCa, but found in patients with BPH, may indicate earlier malignant changes in prostate cells, which are not visible on routine prostate biopsy. ACE activity and its conformation in prostatic biopsies has the potential to be an early biomarker or a differential criterion for PCa. In PCa, the ACE activity in the prostate is significantly reduced, and the ZPHL/HHL ratio is markedly increased in comparison to control group. However, there were no such changes in patients with BPH. In hyperplastic processes of the prostate (BPH, PCa), there is a change in ACE sialylation, which is accompanied by an increase in the binding of ACE to mAb 3F10 compared to the control group. Patients with negative biopsy result, but properties of prostate ACE, which are typical for PCa, require close follow-up, since they may have an increased risk of subsequent developing PCa. However, due to a small sample of patients, the diagnostic potential of prostate ACE for PCa and BPH requires to be validated in a larger number of patients to confirm its predictive accuracy.

ACE-domain selectivity extends beyond direct interacting residues at the active site

Angiotensin-converting enzyme (ACE) is best known for its formation of the vasopressor angiotensin II that controls blood pressure but is also involved in other physiological functions through the hydrolysis of a variety of peptide substrates. The enzyme contains two catalytic domains (nACE and cACE) that have different affinities for ACE substrates and inhibitors. We investigated whether nACE inhibitor backbones contain a unique property which allows them to take advantage of the hinging of nACE. Kinetic analysis showed that mutation of unique nACE residues, in both the S2 pocket and around the prime subsites (S′) to their C-domain counterparts, each resulted in a decrease in the affinity of nACE specific inhibitors (SG6, 33RE and ketoACE-13) but it required the combined S2_S′ mutant to abrogate nACE-selectivity. However, this was not observed with the non-domain-selective inhibitors enalaprilat and omapatrilat. High-resolution structures were determined for the minimally glycosylated nACE with the combined S2_S′ mutations in complex with the ACE inhibitors 33RE (1.8 Å), omapatrilat (1.8 Å) and SG6 (1.7 Å). These confirmed that the affinities of the nACE-selective SG6, 33RE and ketoACE-13 are not only affected by direct interactions with the immediate environment of the binding site, but also by more distal residues. This study provides evidence for a more general mechanism of ACE inhibition involving synergistic effects of not only the S2, S1′ and S2′ subsites, but also residues involved in the sub-domain interface that effect the unique ways in which the two domains stabilize active site loops to favour inhibitor binding.

Neuropeptide reporter assay for serum, capillary blood and blood cards

In the associated main paper (JPBA 2019), substance P was shown to be a valuable neuropeptide reporter substance to monitor the protease activity of serum. The assay was developed based on the predecessor assay using bradykinin (JPBA 2017). Both neuropeptides are of interest in inflammation and pain research and were thus explored for use with capillary blood and blood cards. Here, we present the protocols and set them in perspective to above neuropeptide assays for serum.•Neuropeptide reporter substance protease activity assay for use with fresh and dried blood.•Dabsylated Substance P and bradykinin are substrates of angiotensin-converting enzyme and other proteases.•Neuropeptides of interest in inflammation and pain.

Labeled substance P as a neuropeptide reporter substance for enzyme activity

Recently, we developed a bradykinin reporter assay and demonstrated the differing protease activity in Complex Regional Pain Syndrome patients vs. controls. In order to further characterize CRPS pathophysiology, the neuropeptide substance P was evaluated as possible reporter substance, here. It was labeled with a chromophore at the lysine residue and generated two major fragments following incubation with serum (amino acid residues 3–8 and 3–11) which were reproducibly separated by thin-layer chromatography. Dabsylated substance P was shown to be a substrate of angiotensin-converting enzyme. The combination of both bradykinin and substance P reporter substances with specific enzyme inhibitors will shed more light on biochemical pathways in inflammatory processes and pain. Comparative clinical studies are now needed to define the application range of both assays in more detail.

The Plethora of Angiotensin-Converting Enzyme-Processed Peptides in Mouse Plasma.

Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2' position and Gly in the P6 position. Pro in P1' and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.

Activated Protein C has No Effect on Pulmonary Capillary Endothelial Function in Septic Patients with Acute Respiratory Distress Syndrome: Association of Endothelial Dysfunction with Mortality.

IntroductionPulmonary capillary endothelium-bound (PCEB) angiotensin-converting enzyme (ACE) activity is a direct and quantifiable index of pulmonary endothelial function that decreases early in acute respiratory distress syndrome (ARDS) and correlates with its severity. Endothelial dysfunction is a major pathophysiology that underlies sepsis-related ARDS. Recombinant human activated protein C (rhAPC), now withdrawn from the market, has been used in the recent past as an endothelial-protective treatment in patients with septic organ dysfunction.MethodsWe investigated the effect of rhAPC on pulmonary endothelial function in 19 septic patients suffering from ARDS. Applying indicator-dilution type techniques, we measured single-pass transpulmonary percent metabolism (%M) and hydrolysis (v) of the synthetic, biologically inactive, and highly specific for ACE substrate, 3H-benzoyl-Phe-Ala-Pro (BPAP), under first-order reaction conditions, and calculated lung functional capillary surface area before and after treatment with rhAPC.ResultsPulmonary endothelium ACE activity was severely impaired in septic patients with ARDS, and was not affected by rhAPC treatment. Additionally, poor outcome was related to a more profound decrease in PCEB-ACE activity. Angiotensin-converting enzyme–substrate utilization was statistically significantly lower in non-survivors as compared to survivors, with no changes over time within each group: BPAP %M: 32.7 ± 3.4% at baseline to 25.6 ± 2.9% at day 7 in survivors versus 20.8 ± 2.8 to 15.5 ± 5%, respectively, in non-survivors (p = 0.044), while hydrolysis (v): 0.41 ± 0.06 at baseline to 0.30 ± 0.04 at day 7 in survivors compared to 0.24 ± 0.04 to 0.18 ± 0.06, respectively, in non-survivors (p = 0.049).ConclusionrhAPC administration in septic patients with ARDS did not improve PCEB-ACE activity indices. However, these indices might be useful in the early recognition of septic patients with ARDS at high risk of mortality.

The lack of association between angiotensin-converting enzyme gene insertion/deletion polymorphism and nicotine dependence in multiple sclerosis.

ObjectiveBlood‐borne angiotensin II is generated from angiotensinogen via cleavage by renin and angiotensin‐converting enzyme (ACE), an enzymatic cascade known as the renin–angiotensin system (RAS). Several lines of evidence indicate that ACE, beyond its classical role of mediating blood pressure regulation, might contribute to the etiology of substance addictions by influencing dopaminergic signaling. A functional insertion/deletion (I/D) polymorphism of the ACE gene was associated with risk for being a smoker among individuals with depression and with smoking severity in studies comprising patients with depression and healthy controls. Several reports have described significantly increased ACE activity in cerebrospinal fluid and serum among MS patients. Furthermore, in our previous work with MS patients from Croatian and Slovenian populations, we demonstrated that the ACE‐I/D polymorphism contributes to an elevated MS risk among male patients. Here we investigated whether the ACE‐I/D polymorphism might influence smoking behavior among patients with MS.Patients and MethodsGenotyping was performed in 521 patients (males/females: 139/382) using polymerase chain reaction.ResultsWe revealed no significant differences in ACE genotype and allele frequencies between smokers and nonsmokers and no significant association between the ACE‐I/D polymorphism and either pack‐year smoking history or number of cigarettes smoked daily (p > .05, respectively).ConclusionThe ACE‐I/D polymorphism does not contribute either to risk for nicotine dependence or to smoking severity among MS patients. In the context of reports on the ACE‐I/D polymorphism and nicotine dependence among healthy controls and patients with depression, we may speculate that the mechanism by which this polymorphism influences nicotine dependence risk differs in MS compared to depression, although not compared to a healthy population. In addition to angiotensin II, other potential ACE substrates, such as substance P and neurotensin, which also influence dopaminergic neurotransmission (and are proposed to be associated with MS), may deserve study in future.

Unraveling the Pivotal Role of Bradykinin in ACE Inhibitor Activity.

Historically, the first described effect of an angiotensin converting enzyme (ACE) inhibitor was an increased activity of bradykinin, one of the substrates of ACE. However, in the subsequent years, molecular models describing the mechanism of action of ACE inhibitors in decreasing blood pressure and cardiovascular risk have focused mostly on the renin-angiotensin system. Nonetheless, over the last 20years, the importance of bradykinin in regulating vasodilation, natriuresis, oxidative stress, fibrinolysis, inflammation, and apoptosis has become clearer. The affinity of ACE appears to be higher for bradykinin than for angiotensin I, thereby suggesting that ACE inhibitors may be more effective inhibitors of bradykinin degradation than of angiotensin II production. Data describing the effect of ACE inhibition on bradykinin signaling support the hypothesis that the most cardioprotective benefits attributed to ACE inhibition may be due to increased bradykinin signaling rather than to decreased angiotensin II signaling, especially when high dosages of ACE inhibitors are considered. In particular, modulation of bradykinin in the endothelium appears to be a major target of ACE inhibition. These new mechanistic concepts may lead to further development of strategies enhancing the bradykinin signaling.

In Vitro Study on Antihypertensive and Antihypercholesterolemic Effects of a Curcumin Nanoemulsion.

Atherosclerosis and hypertension can potentially progess into dangerous cardiovascular diseases such as myocardial infarction and stroke. Statins are widely used to lower cholesterol levels while antihypertensive agents such as captopril are widely prescribed to treat high blood pressure. Curcumin, a phenolic compound isolated from Curcuma domestica, has been proven effective for a broad spectrum of diseases, including hypertension and hypercholesterolemia. Therefore, curcumin is quite promising as an alternative therapeutic compound. Our previous studies have proven a significant increase in physical properties, bioavailability, and stability of curcumin when encapsulated in a nanoemulsion. The purpose of this study was to assess the ability of the nanoemulsion in enhancing curcumin activity as a antihypertensive and antihypercholesterolemic agent. The formulation and preparation method of the curcumin nanoemulsion have been developed in our previous study. Physical characterization was performed, including measurement of droplet size, polidispersity index, zeta potential, entrapment efficiency, and loading capacity. Antihypertensive activity of curcumin was evaluated by determining Angiotensin Converting Enzyme (ACE) inhibition in vitro. A substrate for ACE, hippuryl-L-histidyl-L-leucine was allowed to react with ACE, resulting in hippuric acid formation as the product. The degree of ACE inhibition by curcumin was represented by the amount of hippuric acid formed. Antihypercholesterolemic activity of curcumin was studied using the HMG-CoA reductase assay equipped with a 96-well UV plate. This assay was based on the spectrophotometric measurement of the decrease in absorbance which represents the oxidation of NADPH by the catalytic subunit of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in the presence of the substrate HMG-CoA. Curcumin is known to have no significant difference in inhibiting ACE compared to Captopril, but when it was incorporated in the self-nanoemulsifying carrier, it slightly increased the inhibitory effect on ACE. In contrast, the effect of curcumin in reducing cholesterol based on the HMGR assay was more pronounced. Curcumin encapsulated in a nanoemulsion showed significant cholesterol-lowering activity compared to a standard drug, pravastatin. Therefore, we conclude that curcumin does not show ACE inhibitory effects, but has potential use as an alternative therapeutic compound to treat hyperlipidaemia. Curcumin encapsulated in a nanoemulsion increased not only the HMGR inhibition, but also ACE inhibition of curcumin. These effects are suggested to be the result of improved solubility in the nanoemulsion system.

Oral Administration of N-Acetyl-seryl-aspartyl-lysyl-proline Ameliorates Kidney Disease in Both Type 1 and Type 2 Diabetic Mice via a Therapeutic Regimen.

Kidney fibrosis is the final common pathway of progressive kidney diseases including diabetic nephropathy. Here, we report that the endogenous antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), the substrate of angiotensin-converting enzyme (ACE), is an orally available peptide drug used to cure kidney fibrosis in diabetic mice. We utilized two mouse models of diabetic nephropathy, streptozotocin- (STZ-) induced type 1 diabetic CD-1 mice and type 2 diabetic nephropathy model db/db mice. Intervention with the ACE inhibitor imidapril, oral AcSDKP, or imidapril + oral AcSDKP combination therapy increased urine AcSDKP levels. AcSDKP levels were significantly higher in the combination group compared to those of the other groups. AcSDKP oral administration, either AcSDKP alone or in addition to imidapril, ameliorated glomerulosclerosis and tubulointerstitial fibrosis. Plasma cystatin C levels were higher in both models, at euthanasia, and were restored by all the treatment groups. The levels of antifibrotic miRs, such as miR-29 or let-7, were suppressed in the kidneys of both models; all treatments, especially the combination of imidapril + oral AcSDKP, restored the antifibrotic miR levels to a normal value or even higher. AcSDKP may be an oral antifibrotic peptide drug that would be relevant to combating fibroproliferative kidney diseases such as diabetic nephropathy.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP): Potential target molecule in research of heart, kidney and brain.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a ubiquitous molecule generated in all mammalian tissues from the N-terminal sequence of thymosin β4 (Tβ4) by the action of propyl oligopeptidase. Ac-SDKP is an alternative substrate for angiotensin converting enzyme (ACE). There are several indications that Ac-SDKP may be protective in the cardiovascular system. First, the level of Ac- SDKP in plasma and tissues is reduced in some cardiovascular pathologies such as hypertension. Second, an administration of Ac-SDKP to rodents attenuates inflammation, cell differentiation, proliferation, and migration resulting in a reduction of fibrosis in the heart, vessels and kidneys in conditions of their disorders. Third, the treatment with ACE-inhibitors is associated with a reduced degradation and hence increased levels of Ac-SDKP, while a simultaneous treatment with monoclonal antibodies against Ac- SDKP partly counteracts the benefit of ACE-inhibition. Since Ac-SDKP fails to reduce blood pressure and left ventricular hypertrophy (LVH), its potential structural benefit is obviously mediated by direct action on tissue in preventing or reversing excessive fibrosis. The protection by ACE-inhibition seems to be partly mediated by increased availability of Ac-SDKP. Thus, it is to suppose that harvesting the knowledge on the role of Ac-SDKP in cardiovascular physiology and pathology could deepen our insight into the mechanisms of action of the renin-angiotensin system (RAS) as well as agents interfering with this system. The exciting protective potential of Ac-SDKP suggests that this compound could be a focused drug target not only in animal experiments but also in the clinical cardio-pharmacologic research in the near future.

Pharmacodynamic effects of C-domain-specific ACE inhibitors on the renin-angiotensin system in myocardial infarcted rats.

The renin-angiotensin system (RAS) is a dynamic network that plays a critical role in blood pressure regulation and fluid and electrolyte homeostasis. Modulators of the RAS, such as angiotensin-converting enzyme (ACE) inhibitors, are widely used to treat hypertension, heart failure and myocardial infarction. The effect of ACE inhibitors (lisinopril and C-domain-selective LisW-S) on the constituent peptides of the RAS following myocardial infarction was examined in rats. Ten angiotensin peptides were analysed using a sensitive LC-MS/MS-based assay to examine both the circulating and equilibrium levels of these peptides. Administration of lisinopril or LisW-S caused a significant decrease in Ang 1-8/Ang 1-10 ratios as determined by circulating and equilibrium peptide level analysis. Furthermore, Ang 1-7 levels were elevated by both ACE inhibitors, but only lisinopril decreased the Ang 1-5/Ang 1-7 ratio. This indicates LisW-S C-domain specificity as Ang 1-5 is generated by hydrolysis of Ang 1-7 by the N-domain. Further corroboration of LisW-S C-domain specificity is that only lisinopril increased the circulating levels of the N-domain ACE substrate Ac-SDKP. LisW-S is able to effectively block ACE in vivo by C-domain-selective inhibition. The LC-MS/MS-based assay allows the evaluation of the pharmacologic impact of RAS inhibitors in different pathophysiological conditions.

GW25-e1669 Apelin Treatment Enhances ACE2 expression and Attenuates Angiotensin II-Mediated Aortic Fibrosis in Apolipoprotein E-Deficient Mice

Apelin is a second catalytic substrate for angiotensin-converting enzyme 2 (ACE2). Apelin and its cognate receptor APJ are essential for diverse biological processes. However, the function of Apelin signaling in the vasculature remains to be identified. We evaluated the effectiveness of apelin

N-acetyl-seryl-aspartyl-lysyl-proline: a valuable endogenous anti-fibrotic peptide for combating kidney fibrosis in diabetes.

Fibroproliferative diseases are responsible for 45% of deaths in the developed world. Curing organ fibrosis is essential for fibroproliferative diseases. Diabetic nephropathy is a common fibroproliferative disease of the kidney and is associated with multiorgan dysfunction. However, therapy to combat diabetic nephropathy has not yet been established. In this review, we discuss the novel therapeutic possibilities for kidney fibrosis in diabetes focusing on the endogenous anti-fibrotic peptide, N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), which is the substrate for angiotensin-converting enzyme and exhibits meaningful anti-fibrotic effects in various experimental models of fibrotic disease.

Substance P Increases Sympathetic Activity During Combined Angiotensin-Converting Enzyme and Dipeptidyl Peptidase-4 Inhibition

Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce postprandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme (ACE). During ACE inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, whereas bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when ACE is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg by mouth or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat; however, substance P increased heart rate and vascular release of norepinephrine during combined ACE and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined ACE and dipeptidyl peptidase-4 inhibition. - URL: http://www.clinicaltrials.gov. Unique identifier: NCT01413542.

N-acetyl-seryl-aspartyl-lysyl-proline inhibits diabetes-associated kidney fibrosis and endothelial-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) emerges as an important source of fibroblasts. MicroRNA let-7 exhibits anti-EndMT effects and fibroblast growth factor (FGF) receptor has been shown to be an important in microRNA let-7 expression. The endogenous antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a substrate of angiotensin-converting enzyme (ACE). Here, we found that AcSDKP inhibited the EndMT and exhibited fibrotic effects that were associated with FGF receptor-mediated anti-fibrotic program. Conventional ACE inhibitor plus AcSDKP ameliorated kidney fibrosis and inhibited EndMT compared to therapy with the ACE inhibitor alone in diabetic CD-1 mice. The endogenous AcSDKP levels were suppressed in diabetic animals. Cytokines induced cultured endothelial cells into EndMT; coincubation with AcSDKP inhibited EndMT. Expression of microRNA let-7 family was suppressed in the diabetic kidney; antifibrotic and anti-EndMT effects of AcSDKP were associated with the restoration of microRNA let-7 levels. AcSDKP restored diabetes- or cytokines-suppressed FGF receptor expression/phosphorylation into normal levels both in vivo and in vitro. These results suggest that AcSDKP is an endogenous antifibrotic molecule that has the potential to cure diabetic kidney fibrosis via an inhibition of the EndMT associated with the restoration of FGF receptor and microRNA let-7.

Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa

Formation of spermatozoa of normal shape, number, and motility is insufficient for the male siring of pups. The spermatozoa must be accompanied by sound fertilizing ability. We found that males with disrupted testis-expressed gene 101 (Tex101) produce normal-looking but fertilization-incompetent spermatozoa, which were accompanied by a deficiency of a disintegrin and metallopeptidase domain 3 (ADAM3) on sperm plasma membrane. It was also found that the existence of TEX101 on spermatozoa was regulated by angiotensin-converting enzyme (ACE). The removal of GPI-anchored protein TEX101 by ACE was essential to produce fertile spermatozoa, and the function of ACE was not depending on its well-known peptidase activity. The finding of TEX101 as a unique specific substrate for ACE may provide a potential target for the production of an awaited contraceptive medicine for men.

Preclinical Pulmonary Capillary Endothelial Dysfunction is Present in Brain Dead Subjects

Pulmonary endothelium is a major metabolic organ affecting pulmonary and systemic vascular homeostasis. Brain death (BD)-induced physiologic and metabolic derangements in donors’ lungs, in the absence of overt lung pathology, may cause pulmonary dysfunction and compromise post-transplant graft function. To explore the impact of BD on pulmonary endothelium, we estimated pulmonary capillary endothelium-bound (PCEB)-angiotensin converting enzyme (ACE) activity, a direct and quantifiable index of pulmonary endothelial function, in eight brain-dead patients and ten brain-injured mechanically ventilated controls. No subject suffered from acute lung injury or any other overt lung pathology. Applying indicator-dilution type techniques, we measured single-pass transpulmonary percent metabolism (%M) and hydrolysis (v) of the synthetic, biologically inactive, and highly specific for ACE substrate 3H-benzoyl-Phe-Ala-Pro, under first order reaction conditions, and calculated lung functional capillary surface area (FCSA). Substrate %M (35 ± 6.8%) and v (0.49 ± 0.13) in BD patients were decreased as compared to controls (55.9 ± 4.9, P = 0.033 and 0.9 ± 0.15, P = 0.033, respectively), denoting decreased pulmonary endothelial enzyme activity at the capillary level; FCSA, a reflection of endothelial enzyme activity per vascular bed, was also decreased (BD patients: 1,563 ± 562 mL/min vs 4,235 ± 559 in controls; P = 0.003). We conclude that BD is associated with subtle pulmonary endothelial injury, expressed by decreased PCEB-ACE activity. The applied indicator-dilution type technique provides direct and quantifiable indices of pulmonary endothelial function at the bedside that may reveal the existence of preclinical lung pathology in potential lung donors.

AMP-Activated Protein Kinase Regulates Endothelial Cell Angiotensin-Converting Enzyme Expression via p53 and the Post-Transcriptional Regulation of microRNA-143/145

High-angiotensin-converting enzyme (ACE)-levels are associated with cardiovascular disease, but little is known about the regulation of its expression. To assess the molecular mechanisms regulating endothelial ACE expression focusing on the role of the AMP-activated protein kinase (AMPK) and miR-143/145. Shear stress decreased ACE expression in cultured endothelial cells, an effect prevented by downregulating AMPKα2 but not AMPKα1. AMPKα2(-/-) mice expressed higher ACE levels than wild-type littermates resulting in impaired hindlimb vasodilatation to the ACE substrate, bradykinin. The latter response was also evident in animals lacking the AMPKα2 subunit only in endothelial cells. In cultured endothelial cells, miR-143/145 levels were increased by shear stress in an AMPKα2-dependent manner, and miR-143/145 overexpression decreased ACE expression. The effect of shear stress was unrelated to an increase in miR-143/145 promoter activity and transcription but could be attributed to post-transcriptional regulation of precursor-miR-143/145 by AMPKα2. The AMPK substrate, p53, can enhance the post-transcriptional processing of several microRNAs, including miR-143/145. We found that shear stress elicited the AMPKα2-dependent phosphorylation of p53 (on Ser15), and that p53 downregulation prevented the shear stress-induced decrease in ACE expression. Streptozotocin-induced diabetes mellitus in mice was studied as a pathophysiological model of altered AMPK activity. Diabetes mellitus increased tissue phosphorylation of the AMPK substrates, p53 and acetyl-coenzyme A carboxylase, changes that correlated with increased miR-143/145 levels and decreased ACE expression. AMPKα2 suppresses endothelial ACE expression via the phosphorylation of p53 and upregulation of miR-143/145. Post-transcriptional regulation of miR-143/145 may contribute to the vascular complications associated with diabetes mellitus.