Renin-Angiotensin System and Sex Differences in COVID-19: A Critical Assessment.

The current epidemic of corona virus disease (COVID-19) has resulted in an immense health burden that became the third leading cause of death and potentially contributed to a decline in life expectancy in the United States. The severe acute respiratory syndrome-related coronavirus-2 binds to the surface-bound peptidase angiotensin-converting enzyme 2 (ACE2, EC 3.4.17.23) leading to tissue infection and viral replication. ACE2 is an important enzymatic component of the renin-angiotensin system (RAS) expressed in the lung and other organs. The peptidase regulates the levels of the peptide hormones Ang II and Ang-(1-7), which have distinct and opposing actions to one another, as well as other cardiovascular peptides. A potential consequence of severe acute respiratory syndrome-related coronavirus-2 infection is reduced ACE2 activity by internalization of the viral-ACE2 complex and subsequent activation of the RAS (higher ratio of Ang II:Ang-[1-7]) that may exacerbate the acute inflammatory events in COVID-19 patients and possibly contribute to the effects of long COVID-19. Moreover, COVID-19 patients present with an array of autoantibodies to various components of the RAS including the peptide Ang II, the enzyme ACE2, and the AT1 AT2 and Mas receptors. Greater disease severity is also evident in male COVID-19 patients, which may reflect underlying sex differences in the regulation of the 2 distinct functional arms of the RAS. The current review provides a critical evaluation of the evidence for an activated RAS in COVID-19 subjects and whether this system contributes to the greater severity of severe acute respiratory syndrome-related coronavirus-2 infection in males as compared with females.

Association of Active Renin Content With Mortality in Critically Ill Patients: A Post hoc Analysis of the Vitamin C, Thiamine, and Steroids in Sepsis (VICTAS) Trial.

OBJECTIVE: Sepsis is a leading cause of mortality. Predicting outcomes is challenging and few biomarkers perform well. Defects in the renin-angiotensin system (RAS) can predict clinical outcomes in sepsis and may outperform traditional biomarkers. We postulated that RAS dysfunction (elevated active renin, angiotensin 1-7 [Ang-(1-7)], and angiotensin-converting enzyme 2 (ACE2) activity with depressed Ang-II and ACE activity) would be associated with mortality in a cohort of septic patients. DESIGN: Post hoc analysis of patients enrolled in the Vitamin C, Thiamine, and Steroids in Sepsis (VICTAS) randomized controlled trial. SETTING: Forty-three hospitals across the United States. PATIENTS: Biorepository samples of 103 patients. INTERVENTIONS: We analyzed day 0 (within 24 hr of respiratory failure, septic shock, or both) and day 3 samples ( n = 103 and 95, respectively) for assessment of the RAS. The association of RAS values with 30-day mortality was determined using Cox proportional hazards regression with multivariable adjustments for age, sex, VICTAS treatment arm, systolic blood pressure, Sequential Organ Failure Assessment Score, and vasopressor use. MEASUREMENTS AND MAIN RESULTS: High baseline active renin values were associated with higher 30-day mortality when dichotomized to the median of 188.7 pg/mL (hazard ratio [HR] = 2.84 [95% CI, 1.10-7.33], p = 0.031) or stratified into quartiles (Q1 = ref, HR Q2 = 2.01 [0.37-11.04], HR Q3 = 3.22 [0.64-16.28], HR Q4 = 5.58 [1.18-26.32], p for linear trend = 0.023). A 1- sd (593.6 pg/mL) increase in renin from day 0 to day 3 was associated with increased mortality (HR = 3.75 [95% CI, 1.94-7.22], p < 0.001), and patients whose renin decreased had improved survival compared with those whose renin increased (HR 0.22 [95% CI, 0.08-0.60], p = 0.003). Ang-(1-7), ACE2 activity, Ang-II and ACE activity did not show this association. Mortality was attenuated in patients with renin over the median on day 0 who received the VICTAS intervention, but not on day 3 ( p interaction 0.020 and 0.137, respectively). There were no additional consistent patterns of mortality on the RAS from the VICTAS intervention. CONCLUSIONS: Baseline serum active renin levels were strongly associated with mortality in critically ill patients with sepsis. Furthermore, a greater relative activation in circulating renin from day 0 to day 3 was associated with a higher risk of death.

Stronger association of intact angiotensinogen with mortality than lactate or renin in critical illness: post-hoc analysis from the VICTAS trial.

Sepsis and septic shock remain global healthcare problems associated with high mortality rates despite best therapy efforts. Circulating biomarkers may identify those patients at risk for poor outcomes, however, current biomarkers, most prominently lactate, are non-specific and have an inconsistent impact on prognosis and/or disease management. Activation of the renin-angiotensin- system (RAS) is an early event in sepsis patients and elevated levels of circulating renin are more predictive of worse outcomes than lactate. The precursor protein Angiotensinogen is another key component of the circulating RAS; it is the only known substrate for renin and the ultimate source of the vasopressor Angiotensin II (Ang II). We postulate that lower Angiotensinogen concentrations may reflect a dysfunctional RAS characterized by high renin concentrations but attenuated Ang II generation, which is disproportionate to the high renin response and may compromise adequate support of blood pressure and tissue perfusion in septic patients. The current study compared the association between serum Angiotensinogen with mortality to that of lactate and renin in the VICTAS cohort of sepsis patients at baseline (day 0) by receiver operating characteristic (ROC) and Kaplan-Meier curve analyses. Serum concentration of Angiotensinogen was more strongly associated with 30-day mortality than either the serum concentrations of renin or lactate in sepsis patients. Moreover, the clinical assessment of Angiotensinogen may have distinct advantages over the typical measures of renin. The assessment of intact Angiotensinogen may potentially facilitate more precise therapeutic approaches (including exogenous angiotensin II) to restore a dysfunctional RAS and improve patient outcomes. Additional prospective validation studies are clearly required for this biomarker in the future.

ACE inhibitors and angiotensin receptor blockers differentially alter the response to angiotensin II treatment in vasodilatory shock.

BACKGROUND: Angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blockers (ARB) medications are widely prescribed. We sought to assess how pre-admission use of these medications might impact the response to angiotensin-II treatment during vasodilatory shock. METHODS: In a post-hoc subgroup analysis of the randomized, placebo-controlled, Angiotensin Therapy for High Output Shock (ATHOS-3) trial, we compared patients with chronic angiotensin-converting enzyme inhibitor (ACEi) use, and patients with angiotensin receptor blocker (ARB) use, to patients without exposure to either ACEi or ARB. The primary outcome was mean arterial pressure after 1-h of treatment. Additional clinical outcomes included mean arterial pressure and norepinephrine equivalent dose requirements over time, and study-drug dose over time. Biological outcomes included baseline RAS biomarkers (renin, angiotensin-I, angiotensin-II, and angiotensin-I/angiotensin-II ratio), and the change in renin from 0 to 3 h. RESULTS: We included n = 321 patients, of whom, 270 were ACEi and ARB-unexposed, 29 were ACEi-exposed and 22 ARB-exposed. In ACEi/ARB-unexposed patients, angiotensin-treated patients, compared to placebo, had higher hour-1 mean arterial pressure (9.1 mmHg [95% CI 7.6-10.1], p < 0.0001), lower norepinephrine equivalent dose over 48-h (p = 0.0037), and lower study-drug dose over 48-h (p < 0.0001). ACEi-exposed patients treated with angiotensin-II showed similarly higher hour-1 mean arterial pressure compared to ACEi/ARB-unexposed (difference in treatment-effect: - 2.2 mmHg [95% CI - 7.0-2.6], pinteraction = 0.38), but a greater reduction in norepinephrine equivalent dose (pinteraction = 0.0031) and study-drug dose (pinteraction < 0.0001) over 48-h. In contrast, ARB-exposed patients showed an attenuated effect of angiotensin-II on hour-1 mean arterial pressure versus ACEi/ARB-unexposed (difference in treatment-effect: - 6.0 mmHg [95% CI - 11.5 to - 0.6], pinteraction = 0.0299), norepinephrine equivalent dose (pinteraction < 0.0001), and study-drug dose (pinteraction = 0.0008). Baseline renin levels and angiotensin-I/angiotensin-II ratios were highest in ACEi-exposed patients. Finally, angiotensin-II treatment reduced hour-3 renin in ACEi/ARB-unexposed and ACEi-exposed patients but not in ARB-exposed patients. CONCLUSIONS: In vasodilatory shock patients, the cardiovascular and biological RAS response to angiotensin-II differed based upon prior exposure to ACEi and ARB medications. ACEi-exposure was associated with increased angiotensin II responsiveness, whereas ARB-exposure was associated with decreased responsiveness. These findings have clinical implications for patient selection and dosage of angiotensin II in vasodilatory shock. Trial Registration ClinicalTrials.Gov Identifier: NCT02338843 (Registered January 14th 2015).

Intensive Care Unit-Like Care of Nonhuman Primates with Ebola Virus Disease.

BACKGROUND: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model. METHODS: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (n = 7), intravenous fluids plus levofloxacin (n = 2), or a control group (n = 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values. RESULTS: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; P = .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury. CONCLUSIONS: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement.

A pilot study to assess the circulating renin-angiotensin system in COVID-19 acute respiratory failure.

The renin-angiotensin system (RAS) is fundamental to COVID-19 pathobiology, due to the interaction between the SARS-CoV-2 virus and the angiotensin-converting enzyme 2 (ACE2) coreceptor for cellular entry. The prevailing hypothesis is that SARS-CoV-2-ACE2 interactions lead to an imbalance of the RAS, favoring proinflammatory angiotensin II (ANG II)-related signaling at the expense of the anti-inflammatory ANG-(1-7)-mediated alternative pathway. Indeed, multiple clinical trials targeting this pathway in COVID-19 are underway. Therefore, precise measurement of circulating RAS components is critical to understand the interplay of the RAS on COVID-19 outcomes. Multiple challenges exist in measuring the RAS in COVID-19, including improper patient controls, ex vivo degradation and low concentrations of angiotensins, and unvalidated laboratory assays. Here, we conducted a prospective pilot study to enroll 33 patients with moderate and severe COVID-19 and physiologically matched COVID-19-negative controls to quantify the circulating RAS. Our enrollment strategy led to physiological matching of COVID-19-negative and COVID-19-positive moderate hypoxic respiratory failure cohorts, in contrast to the severe COVID-19 cohort, which had increased severity of illness, prolonged intensive care unit (ICU) stay, and increased mortality. Circulating ANG II and ANG-(1-7) levels were measured in the low picomolar (pM) range. We found no significant differences in circulating RAS peptides or peptidases between these three cohorts. The combined moderate and severe COVID-19-positive cohorts demonstrated a mild reduction in ACE activity compared with COVID-19-negative controls (2.2 ± 0.9 × 105 vs. 2.9 ± 0.8 × 105 RFU/mL, P = 0.03). These methods may be useful in designing larger studies to physiologically match patients and quantify the RAS in COVID-19 RAS augmenting clinical trials.

Metabolic rates of aggressive and submissive phenotypes are colour blind in the polymorphic Gouldian finch.

Evidence from a number of species suggests that behaviours associated with social rank are positively correlated with metabolic rate. These studies, however, are based on metabolic measurements of isolated individuals, thereby ignoring potential effects of social interactions on metabolic rates. Here, we characterised three pertinent metabolic indices in the two predominant genetic colour morphs of the Gouldian finch (Erythrura gouldiae): diurnal resting metabolic rate (RMR), nocturnal basal metabolic rate (BMR) and exercise-induced maximal metabolic rate (MMR). Research reveals that red-headed morphs consistently dominate the less aggressive black-headed morphs and that the two morphs differ in other behavioural and physiological traits. We measured daytime RMR of intermorph naïve birds (first-year virgin males maintained in total isolation from opposite colour morphs) and their metabolic responses to viewing a socially unfamiliar bird of each colour. Subsequently, each bird was placed in a home cage with an opposite colour morph (intermorph exposed) and the series of measurements was repeated. Daytime RMR was indistinguishable between the two morphs, regardless of whether they were intermorph naïve or intermorph exposed. However, both red- and black-headed birds showed a greater short-term increase in metabolic rate when viewing an unfamiliar red-headed bird than when seeing a black-headed bird, but only when intermorph naïve. Measurements of BMR and exercise-induced MMR did not differ between the two morphs, and consequently, aerobic scope was indistinguishable between them. We propose that the behavioural differences between these two sympatric morphs are functionally complementary and represent evolutionary stable strategies permitting establishment of dominance status in the absence of metabolic costs.

A test of altitude-related variation in aerobic metabolism of Andean birds.

Endotherms at high altitude face the combined challenges of cold and hypoxia. Cold increases thermoregulatory costs, and hypoxia may limit both thermogenesis and aerobic exercise capacity. Consequently, in comparisons between closely related highland and lowland taxa, we might expect to observe consistent differences in basal metabolic rate (BMR), maximal metabolic rate (MMR) and aerobic scope. Broad-scale comparative studies of birds reveal no association between BMR and native elevation, and altitude effects on MMR have not been investigated. We tested for altitude-related variation in aerobic metabolism in 10 Andean passerines representing five pairs of closely related species with contrasting elevational ranges. Mass-corrected BMR and MMR were significantly higher in most highland species relative to their lowland counterparts, but there was no uniform elevational trend across all pairs of species. Our results suggest that there is no simple explanation regarding the ecological and physiological causes of elevational variation in aerobic metabolism.

COVID-19, ACE2, and the cardiovascular consequences.

The novel SARS coronavirus SARS-CoV-2 pandemic may be particularly deleterious to patients with underlying cardiovascular disease (CVD). The mechanism for SARS-CoV-2 infection is the requisite binding of the virus to the membrane-bound form of angiotensin-converting enzyme 2 (ACE2) and internalization of the complex by the host cell. Recognition that ACE2 is the coreceptor for the coronavirus has prompted new therapeutic approaches to block the enzyme or reduce its expression to prevent the cellular entry and SARS-CoV-2 infection in tissues that express ACE2 including lung, heart, kidney, brain, and gut. ACE2, however, is a key enzymatic component of the renin-angiotensin-aldosterone system (RAAS); ACE2 degrades ANG II, a peptide with multiple actions that promote CVD, and generates Ang-(1-7), which antagonizes the effects of ANG II. Moreover, experimental evidence suggests that RAAS blockade by ACE inhibitors, ANG II type 1 receptor antagonists, and mineralocorticoid antagonists, as well as statins, enhance ACE2 which, in part, contributes to the benefit of these regimens. In lieu of the fact that many older patients with hypertension or other CVDs are routinely treated with RAAS blockers and statins, new clinical concerns have developed regarding whether these patients are at greater risk for SARS-CoV-2 infection, whether RAAS and statin therapy should be discontinued, and the potential consequences of RAAS blockade to COVID-19-related pathologies such as acute and chronic respiratory disease. The current perspective critically examines the evidence for ACE2 regulation by RAAS blockade and statins, the cardiovascular benefits of ACE2, and whether ACE2 blockade is a viable approach to attenuate COVID-19.

Central ANG-(1-7) infusion improves blood pressure regulation in antenatal betamethasone-exposed sheep and reveals sex-dependent effects on oxidative stress.

Fetal exposure to betamethasone (BMX) as a consequence of glucocorticoid administration to women threatening premature delivery may lead to long-term deleterious effects on the cardiovascular system and dysregulation of blood pressure in exposed adults. Indeed, adult offspring of BMX sheep exhibit increased mean arterial pressure (MAP) and attenuated baroreflex sensitivity (BRS) that are associated with lower medullary and cerebrospinal fluid (CSF) angiotensin-(1-7) [(ANG-(1-7)] content. Thus we determined the effects of ANG-(1-7) supplementation in the CSF on MAP, BRS, blood pressure (BPV) and heart rate variability (HRV) in conscious animals. The peptide or artificial CSF (aCSF) was infused continuously into the lateral ventricle (intracerebroventricular) of 4-mo-old male and female BMX sheep for 2 wk. Analysis of data from males and females combined revealed that intracerebroventricular ANG-(1-7) significantly lowered MAP and heart rate and improved BRS as compared with baseline; intracerebroventricular aCSF did not change these indexes. Similar patterns were observed for altered hemodynamics and autonomic function produced by intracerebroventricular ANG-(1-7) in both sexes. Oxidative stress and MAP kinase (MAPK) activation were lower in tissues from the dorsomedial medulla (DMM) of ANG-(1-7)-treated males but were unchanged in the treated females, when assessed at the end of the treatment period. We conclude that in the face of ANG-(1-7) deficiency in CSF and medullary tissue in BMX sheep intracerebroventricular supplementation of ANG-(1-7) lowers MAP and restores the impaired autonomic function to a similar degree in both males and females; however, the attenuation of MAPK and oxidative stress within the DMM was evident only in males. NEW & NOTEWORTHY We demonstrate that intracerebroventricular angiotensin-(1-7) [(ANG-(1-7)] treatment for 2 wk in antenatal betamethasone-exposed sheep provides beneficial effects on blood pressure and autonomic function. The physiological improvements are accompanied by an attenuation of oxidative stress in males but not females. The finding that ANG-(1-7) supplementation lowers blood pressure and restores the impaired autonomic function in a model of fetal programming previously shown to exhibit a deficiency in cerebrospinal fluid and brain tissue illustrates the potential for new therapeutic strategies for reducing cardiovascular dysfunction arising from prenatal events.

Obesity is Associated with Higher Blood Pressure and Higher Levels of Angiotensin II but Lower Angiotensin-(1-7) in Adolescents Born Preterm.

OBJECTIVES: To evaluate if obesity is associated with increased angiotensin II (Ang II) and decreased angiotensin-(1-7) or Ang-(1-7) in the circulation and urine among adolescents born prematurely. STUDY DESIGN: In a cross-sectional analysis of 175 14-year-olds born preterm with very low birth weight, we quantified plasma and urinary Ang II and Ang-(1-7) and compared their levels between subjects with overweight/obesity (body mass index ≥85th percentile, n = 61) and those with body mass index <85th percentile (n = 114) using generalized linear models, adjusted for race and antenatal corticosteroid exposure. RESULTS: Overweight/obesity was associated with higher systolic blood pressure and a greater proportion with high blood pressure. After adjustment for confounders, overweight/obesity was associated with an elevated ratio of plasma Ang II to Ang-(1-7) (ß: 0.57, 95% CI 0.23-0.91) and higher Ang II (ß: 0.21 pmol/L, 95% CI 0.03-0.39) but lower Ang-(1-7) (ß: -0.37 pmol/L, 95% CI -0.7 to -0.04). Overweight/obesity was associated with a higher ratio of urinary Ang II to Ang-(1-7) (ß: 0.21, 95% CI -0.02 to 0.44), an effect that approached statistical significance. CONCLUSIONS: Among preterm-born adolescents, overweight/obesity was associated with increased Ang II but reduced Ang-(1-7) in the circulation and the kidney as well as higher blood pressure. Obesity may compound the increased risk of hypertension and cardiovascular disease in individuals born prematurely by further augmenting the prematurity-associated imbalance in the renin-angiotensin system.

Fetal programming and the angiotensin-(1-7) axis: a review of the experimental and clinical data.

Hypertension is the primary risk factor for cardiovascular disease that constitutes a serious worldwide health concern and a significant healthcare burden. As the majority of hypertension has an unknown etiology, considerable research efforts in both experimental models and human cohorts has focused on the premise that alterations in the fetal and perinatal environment are key factors in the development of hypertension in children and adults. The exact mechanisms of how fetal programming events increase the risk of hypertension and cardiovascular disease are not fully elaborated; however, the focus on alterations in the biochemical components and functional aspects of the renin-angiotensin (Ang) system (RAS) has predominated, particularly activation of the Ang-converting enzyme (ACE)-Ang II-Ang type 1 receptor (AT1R) axis. The emerging view of alternative pathways within the RAS that may functionally antagonize the Ang II axis raise the possibility that programming events also target the non-classical components of the RAS as an additional mechanism contributing to the development and progression of hypertension. In the current review, we evaluate the potential role of the ACE2-Ang-(1-7)-Mas receptor (MasR) axis of the RAS in fetal programming events and cardiovascular and renal dysfunction. Specifically, the review examines the impact of fetal programming on the Ang-(1-7) axis within the circulation, kidney, and brain such that the loss of Ang-(1-7) expression or tone, contributes to the chronic dysregulation of blood pressure (BP) and cardiometabolic disease in the offspring, as well as the influence of sex on potential programming of this pathway.

Comparison of Candesartan and Angiotensin-(1-7) Combination to Mito-TEMPO Treatment for Normalizing Blood Pressure and Sympathovagal Balance in (mREN2)27 Rats.

Hypertensive transgenic (mRen2)27 rats exhibit impaired baroreflex sensitivity (BRS) for control of heart rate (HR). Intracerebroventricular infusion of Ang-(1-7) improves indices of vagal BRS independent of lowering mean arterial pressure (MAP), whereas AT1 receptor blockade normalizes MAP and indices of sympathetic tone without correcting the vagal BRS. Scavenging cellular reactive oxygen species (ROS) with tempol in brain fails to correct either hypertension or sympathovagal balance in these animals, despite reports that mitochondrial ROS contributes to Ang II-infusion hypertension. To examine effects of a putative preferential mitochondrial ROS scavenger in the brain of (mRen2)27 rats, ICV infusions of Mito-TEMPO (3.2 µg/2.5 µL/h) were compared with artificial cerebrospinal fluid (aCSF; 2.5 µL/h) and combination AT1 receptor antagonist candesartan (CAN: 4 µg/2.5 µL/h) plus Ang-(1-7) (0.1 µg/2.5 µL/h) treatment. MAP was lower after CAN + Ang-(1-7) treatment, and both vagal and sympathetic components of BRS and sympathovagal balance were improved. By contrast, Mito-TEMPO improved sympathetic components of BRS and tended to improve overall sympathovagal balance but failed to alter MAP in this model of hypertension. Although further studies are required to determine whether Mito-TEMPO or CAN + Ang-(1-7) treatment at the doses used altered mitochondrial ROS, optimal therapeutic benefits are achieved by shifting the balance from Ang II toward Ang-(1-7) in this model of chronic RAS-dependent hypertension.

Renal function and blood pressure are altered in adolescents born preterm.

BACKGROUND: Preterm birth increases the risk of hypertension and kidney disease. However, it is unclear when changes in blood pressure (BP) and renal function become apparent and what role obesity and sex play. We hypothesized adolescents born preterm have higher BP and worse kidney function compared to term in an obesity- and sex-dependent manner. METHODS: Cross-sectional analysis of 14-year-olds born preterm with very low birth weight (n = 96) compared to term (n = 43). We used generalized linear models to estimate the associations among preterm birth and BP, estimated glomerular filtration rate (eGFR), and ln (x) urinary albumin-to-creatinine ratio (ACR), stratified by overweight/obesity (OWO, body mass index (BMI) ≥ 85th percentile) and sex. RESULTS: Compared to term, preterm-born adolescents had higher systolic blood pressure (SBP) and diastolic blood pressure (DBP) (adjusted ß (aß) 3.5 mmHg, 95% CI - 0.1 to 7.2 and 3.6 mmHg, 95% CI 0.1 to 7.0), lower eGFR (ß - 8.2 mL/min/1.73 m2, 95% CI - 15.9 to - 0.4), and higher ACR (aß 0.34, 95% CI - 0.04 to 0.72). OWO modified the preterm-term difference in DBP (BMI < 85th percentile aß 5.0 mmHg, 95% CI 0.7 to 9.2 vs. OWO 0.2 mmHg, 95% CI - 5.3 to 5.6) and ACR (OWO aß 0.72, 95% CI 0.15 to 1.29 vs. BMI < 85th percentile 0.17, 95% CI - 0.31 to 0.65). Sex modified the preterm-term ACR difference (female aß 0.52, 95% CI 0.001 to 1.04 vs. male 0.18, 95% CI - 0.36 to 0.72). CONCLUSIONS: Prematurity was associated with higher BP and reduced renal function that were detectable in adolescence. OWO and sex may modify the strength of these relationships.

Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg9 bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.

Sex-dependent expression of brain medullary MAP and PI3 kinases in adult sheep with antenatal betamethasone exposure.

Antenatal betamethasone (BM) therapy for women in jeopardy of premature delivery accelerates the lung development in preterm infants and reduces infant mortality rates, but may induce early programming events with chronic cardiovascular consequences. In a sheep model of fetal programming, in utero BM-exposed (BMX) offspring as adults exhibit elevated mean arterial pressure (MAP), decreased baroreflex sensitivity (BRS) for the control of heart rate and insulin resistance accompanied by dysregulation of the brain renin-angiotensin (Ang) system (RAS). However, the status of signaling mechanisms in the brain dorsomedial medulla (DMM) of the BMX sheep that comprise both the mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) cellular pathways is unknown. Given the importance of these signaling pathways in the actions of Ang peptides as well as baroreflex function and autonomic integration, we applied both a kinase signaling array and associated individual immunoblots of the dorsomedial brain medulla from adult female and male sheep with antenatal BMX. MAPK and PI3K pathways were altered significantly in the BMX sheep in a sex-dependent manner. A protein array for kinases (PathScan® Intracellular Signaling Array Kit, Cell Signaling) and subsequent verification by immunoblot revealed that within the DMM, female BMX sheep exhibit lower expression of proteins in the PI3K pathway, while male BMX sheep show greater expression of p-MAPK pathway proteins extracellular signal regulated kinase (ERK) 1/2. We conclude that maladaptive changes in these two kinase pathways in the DMM may contribute to the chronic dysregulation of blood pressure in this model of fetal programming.

Effects of a physical and energetic challenge on male California mice (Peromyscus californicus): modulation by reproductive condition.

Reproduction strongly influences metabolism, morphology and behavior in female mammals. In species in which males provide parental care, reproduction might have similar effects on fathers. We examined effects of an environmental challenge on metabolically important physiological, morphological and behavioral measures, and determined whether these effects differed between reproductive and non-reproductive males in the biparental California mouse (Peromyscus californicus). Males were paired with an ovary-intact female, an ovariectomized female treated with estrogen and progesterone to induce estrus, or an untreated ovariectomized female. Within each group, half of the animals were housed under standard laboratory conditions and half in cages requiring them to climb wire towers to obtain food and water; these latter animals were also fasted for 24 h every third day. We predicted that few differences would be observed between fathers and non-reproductive males under standard conditions, but that fathers would be in poorer condition than non-reproductive males under challenging conditions. Body and fat mass showed a housing condition×reproductive group interaction: the challenge condition increased body and fat mass in both groups of non-reproductive males, but breeding males were unaffected. Males housed under the physical and energetic challenge had higher blood lipid content, lower maximal aerobic capacity and related traits (hematocrit and relative triceps surae mass), increased pain sensitivity and increased number of fecal boli excreted during tail-suspension tests (a measure of anxiety), compared with controls. Thus, our physical and energetic challenge paradigm altered metabolism, morphology and behavior, but these effects were largely unaffected by reproductive condition.

Antenatal betamethasone attenuates the angiotensin-(1-7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells.

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1-7) [Ang-(1-7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1-7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1-7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1-7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1-7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1-7). We conclude that the tubular Ang-(1-7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

Angiotensinogen import in isolated proximal tubules: evidence for mitochondrial trafficking and uptake.

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min-1·mg protein-1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Stabilization of Angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid.

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer.