Gene-targeting studies in mice demonstrate that the renin-<em>angiotensin</em> system is required for the proper development of the renal medulla. In the absence of <em>angiotensin</em> II (ANG II) or the ANG II type <em>1</em> (AT<em>1</em>) receptor, mice exhibit poor papillary development and a severe urinary-concentrating defect. These findings imply that the ureteric bud (UB) and its branches are targets for ANG II actions during renal development. However, direct evidence linking ANG II with UB-branching morphogenesis does not exist. Using immunohistochemistry, we demonstrated that UB-derived epithelia express <em>angiotensin</em>ogen (Ao) and the AT<em>1</em> receptor during murine metanephrogenesis. Ao and AT<em>1</em> receptors are expressed in the UB branches and to a lesser extent in the stromal mesenchyme. AT<em>1</em> receptor expression in UB-derived epithelia increased from embryo day <em>1</em>2 to day <em>1</em>6 and was observed on both luminal and basolateral membranes. In accord with these findings, cultured murine UB cells express AT<em>1</em> receptor protein and mRNA. Treatment of UB cells cultured in three-dimensional type I collagen gels with ANG II (<em>1</em>0-<em>7</em> to <em>1</em>0-5 M) elicits a dose-related increase in the number of cells that have primary and secondary branches. These effects of ANG II on UB branching are abrogated by pretreatment with the AT<em>1</em> receptor antagonist candesartan. These data demonstrate a direct and independent role for ANG II acting via AT<em>1</em> receptors on UB cell branching in vitro. The presence of Ao in the stroma and AT<em>1</em> on UB cells supports the notion that cross talk between stroma and epithelial cells is crucial to epithelial branching morphogenesis in the developing kidney.

BACKGROUND

Hypercortisolism as a sign of hypothamamus-pituitary-adrenocortical (HPA) axis overactivity and sleep EEG changes are frequently observed in depression. Closely related to the HPA axis is the renin-<em>angiotensin</em>-aldosterone system (RAAS) as <em>1</em>. adrenocorticotropic hormone (ACTH) is a common stimulus for cortisol and aldosterone, 2. cortisol release is suppressed by mineralocorticoid receptor (MR) agonists 3. <em>angiotensin</em> II (ATII) releases CRH and vasopressin from the hypothalamus. Furthermore renin and aldosterone secretion are synchronized to the rapid eyed movement (REM)-nonREM cycle.

METHODS

Here we focus on the difference of sleep related activity of the RAAS between depressed patients and healthy controls. We studied the nocturnal plasma concentration of ACTH, cortisol, renin and aldosterone, and sleep EEG in 7 medication free patients with depression (<em>1</em> male, 6 females, age: (mean +/-SD) 53.3 +/- <em>1</em>4.4 yr.) and 7 age matched controls (2 males, 5 females, age: 54.7 +/- <em>1</em>9.5 yr.). After one night of accommodation a polysomnography was performed between 23.00 h and 7.00 h. During examination nights blood samples were taken every 20 min between 23.00 h and 7.00 h. Area under the curve (AUC) for the hormones separated for the halves of the night (23.00 h to 3.00 h and 3.00 h to 7.00 h) were used for statistical analysis, with analysis of co variance being performed with age as a covariate.

RESULTS

No differences in ACTH and renin concentrations were found. For cortisol, a trend to an increase was found in the first half of the night in patients compared to controls (p < 0.06). Aldosterone was largely increased in the first (p < 0.05) and second (p < 0.0<em>1</em>) half of the night. Cross correlations between hormone concentrations revealed that in contrast to earlier findings, which included only male subjects, in our primarily female sample, renin and aldosterone secretion were not coupled and no difference between patients and controls could be found, suggesting a gender difference in RAAS regulation. No difference in conventional sleep EEG parameters were found in our sample.

CONCLUSIONS

Hyperaldosteronism could be a sensitive marker for depression. Further our findings point to an altered renal mineralocorticoid sensitivity in patients with depression.

OBJECTIVE

The transactivation of the epidermal growth factor (EGF) receptor appears to be an important central transduction mechanism in mediating diabetes-induced vascular dysfunction. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] via its Mas receptor can prevent the development of hyperglycaemia-induced cardiovascular complications. Here, we investigated whether Ang-(<em>1</em>-<em>7</em>) can inhibit hyperglycaemia-induced EGF receptor transactivation and its classical signalling via ERK<em>1</em>/2 and p38 MAPK in vivo and in vitro.

METHODS

Streptozotocin-induced diabetic rats were chronically treated with Ang-(<em>1</em>-<em>7</em>) or AG<em>1</em>4<em>7</em>8, a selective EGF receptor inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in primary cultures of vascular smooth muscle cells (VSMCs).

RESULTS

Diabetes significantly enhanced phosphorylation of EGF receptor at tyrosine residues Y992, Y<em>1</em>068, Y<em>1</em>086, Y<em>1</em><em>1</em>48, as well as ERK<em>1</em>/2 and p38 MAPK in the mesenteric vasculature bed whereas these changes were significantly attenuated upon Ang-(<em>1</em>-<em>7</em>) or AG<em>1</em>4<em>7</em>8 treatment. In VSMCs grown in conditions of high glucose (25 mM), an Src-dependent elevation in EGF receptor phosphorylation was observed. Ang-(<em>1</em>-<em>7</em>) inhibited both Ang II- and glucose-induced transactivation of EGF receptor. The inhibition of high glucose-mediated Src-dependant transactivation of EGF receptor by Ang-(<em>1</em>-<em>7</em>) could be prevented by a selective Mas receptor antagonist, D-Pro<em>7</em>-Ang-(<em>1</em>-<em>7</em>).

CONCLUSIONS

These results show for the first time that Ang-(<em>1</em>-<em>7</em>) inhibits EGF receptor transactivation via a Mas receptor/Src-dependent pathway and might represent a novel general mechanism by which Ang-(<em>1</em>-<em>7</em>) exerts its beneficial effects in many disease states including diabetes-induced vascular dysfunction.

Endothelial-to-mesenchymal transition (EndMT) emerges as an important source of fibroblasts. MicroRNA let-<em>7</em> exhibits anti-EndMT effects and fibroblast growth factor (FGF) receptor has been shown to be an important in microRNA let-<em>7</em> expression. The endogenous antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a substrate of <em>angiotensin</em>-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-<em>1</em> 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-<em>7</em> family was suppressed in the diabetic kidney; antifibrotic and anti-EndMT effects of AcSDKP were associated with the restoration of microRNA let-<em>7</em> 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-<em>7</em>.

BACKGROUND

Excess dietary sodium has been linked to the development of hypertension and other cardiovascular diseases. In humans, the effects of sodium consumption on endothelial function have not been separated from the effects on blood pressure. The present study was designed to determine if dietary sodium intake affected endothelium-dependent dilation (EDD) independently of changes in blood pressure.

METHODS

Fourteen healthy salt-resistant adults were studied (9M, 5F; age 33 ± 2.4 years) in a controlled feeding study. After a baseline run-in diet, participants were randomized to a <em>7</em>-day high-sodium (300-350 mmol/day) and <em>7</em>-day low-sodium (20 mmol/day) diet. Salt resistance, defined as a 5 mmHg or less change in a 24-h mean arterial pressure, was individually assessed while on the low-sodium and high-sodium diets and confirmed in the participants undergoing study (low-sodium: 85 ± <em>1</em> mmHg; high-sodium: 85 ± 2 mmHg). EDD was determined in each participant via brachial artery flow-mediated dilation on the last day of each diet.

RESULTS

Sodium excretion increased during the high-sodium diet (P < 0.0<em>1</em>). EDD was reduced on the high-sodium diet (low: <em>1</em>0.3 ± 0.9%, high: <em>7</em>.3 ± 0.<em>7</em>%; P < 0.05). The high-sodium diet significantly suppressed plasma renin activity (PRA), plasma angiotensin II, and aldosterone (P < 0.05).

CONCLUSIONS

These data demonstrate that excess salt intake in humans impairs endothelium-dependent dilation independently of changes in blood pressure.

Ischemia-reperfusion (I/R) is a model of acute kidney injury (AKI) that is characterized by vasoconstriction, oxidative stress, apoptosis and inflammation. Previous studies have shown that activation of the renin-<em>angiotensin</em> system (RAS) may contribute to these processes. <em>Angiotensin</em> converting enzyme 2 (ACE2) metabolizes <em>angiotensin</em> II (Ang II) to <em>angiotensin</em>-(<em>1</em>-<em>7</em>), and recent studies support a beneficial role for ACE2 in models of chronic kidney disease. However, the role of ACE2 in models of AKI has not been fully elucidated. In order to test the hypothesis that ACE2 plays a protective role in AKI we assessed I/R injury in wild-type (WT) mice and ACE2 knock-out (ACE2 KO) mice. ACE2 KO and WT mice exhibited similar histologic injury scores and measures of kidney function at 48 hours after reperfusion. Loss of ACE2 was associated with increased neutrophil, macrophage, and T cell infiltration in the kidney. mRNA levels for pro-inflammatory cytokines, interleukin-<em>1</em>β, interleukin-6 and tumour necrosis factor-α, as well as chemokines macrophage inflammatory protein 2 and monocyte chemoattractant protein-<em>1</em>, were increased in ACE2 KO mice compared to WT mice. Changes in inflammatory cell infiltrates and cytokine expression were also associated with greater apoptosis and oxidative stress in ACE2 KO mice compared to WT mice. These data demonstrate a protective effect of ACE2 in I/R AKI.

Elevated circulating fatty acid-binding protein 4 (FABP4/A-FABP/aP2), an adipokine, is associated with obesity, insulin resistance, hypertension and cardiovascular events. However, how circulating FABP4 level is modified by pharmacological agents remains unclear. We here examined the effects of <em>angiotensin</em> II receptor blockers (ARBs) on serum FABP4 level. First, essential hypertensives were treated with ARBs: candesartan (8 mg day(-<em>1</em>); n=<em>7</em>) for 2 weeks, olmesartan (20 mg day(-<em>1</em>); n=9) for <em>1</em>2 weeks, and valsartan (80 mg day(-<em>1</em>); n=94) or telmisartan (40 mg day(-<em>1</em>); n=9<em>1</em>) for 8 weeks added to amlodipine (5 mg day(-<em>1</em>)). Treatment with ARBs significantly decreased blood pressure and serum FABP4 concentrations by 8-20% without significant changes in adiposity or lipid variables, though the M value determined by hyperinsulinemic-euglycemic glucose clamp, a sensitive index of insulin sensitivity, was significantly increased by candesartan. Next, alterations in FABP4 secretion from 3T3-L<em>1</em> adipocytes were examined under several agents. Lipolytic stimulation of the β-adrenoceptor in 3T3-L<em>1</em> adipocytes by isoproterenol increased FABP4 secretion, and conversely, insulin suppressed FABP4 secretion. However, treatment of 3T3-L<em>1</em> adipocytes with <em>angiotensin</em> II or ARBs for 2 h had no effect on gene expression or secretion of FABP4 regardless of β-adrenoceptor stimulation. In conclusion, treatment with structurally different ARBs similarly decreases circulating FABP4 concentrations in hypertensive patients as a class effect of ARBs, which is not attributable to blockade of the <em>angiotensin</em> II receptor in adipocytes. Reduction of FABP4 levels by ARBs might be involved in suppression of cardiovascular events.

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

<em>Angiotensin</em> converting enzyme 2 (ACE2), a newly recognized homolog of ACE that converts <em>angiotensin</em> II (Ang II) to <em>angiotensin</em>-<em>1</em>-<em>7</em> (Ang-(<em>1</em>-<em>7</em>)), is found in vascular smooth muscle cells. Expression of ACE2 may be a local determinant of vascular Ang-(<em>1</em>-<em>7</em>) production and, when increased, may augment the increasingly recognized protective effects of this peptide within injured tissues. We previously showed that treatment with the <em>angiotensin</em> II type <em>1</em> (AT<em>1</em>) receptor blocker (ARB) olmesartan increased aortic ACE2 and Ang-(<em>1</em>-<em>7</em>) in conjunction with improved vascular remodeling in spontaneously hypertensive rats (SHR). In the present study, we investigated balloon injury-related ACE2 in the vasculature by determining the effect of sustained AT<em>1</em> blockade on ACE2 protein expression in the carotid arteries of <em>1</em>2-week-old male SHR treated with either vehicle (n=5) or <em>1</em>0 mg/kg olmesartan (n=5) in drinking water for <em>1</em>4 days. Olmesartan treatment caused a 6<em>1</em>% reduction in the cross-sectional area of the neointima, from 0.2<em>7</em>+/-0.0<em>1</em> mm2 in vehicle-treated rats to 0.<em>1</em><em>1</em>+/-0.0<em>1</em> mm2 in olmesartan-treated rats. In contrast, olmesartan treatment had no effect on the medial area of injured or uninjured carotid arteries compared to that in vehicle-treated rats. Quantitative analysis of ACE2 immunostaining intensity in the carotid artery of SHR was significantly greater (p<0.05) in the neointima of olmesartan-treated SHR compared to that in vehicle-treated animals. In contrast, ACE2 immunostaining intensity was not quantitatively different in uninjured carotid arteries of olmesartan and vehicle-treated animals. These studies suggest that changes in ACE2 within the vascular system of SHR are regulated by a factor other than arterial pressure.

<em>1</em>. The responses of the smooth muscle of the capsule and blood vessels of the isolated, perfused human spleen to sympathetic nerve stimulation, adrenaline, noradrenaline, <em>angiotensin</em>, oxytocin, vasopressin, isoprenaline and acetylcholine have been investigated and compared with those of dog spleen.2. Stimulation of the postganglionic sympathetic nerves to the human spleen at frequencies of 3-<em>1</em>0 Hz evoked graded vasoconstriction but very small changes in spleen volume.3. The injection of adrenaline and noradrenaline in doses of 0.25-25 mug to the human spleen produced graded increases in splenic vascular resistance with very small decreases in spleen volume.4. Administration of the alpha-adrenoceptor blocking drug phenoxybenzamine completely abolished or considerably reduced the vascular responses of the human spleen to sympathetic nerve stimulation or the injection of noradrenaline.5. The vascular action of adrenaline was often reversed to elicit a vasodilatation after phenoxybenzamine suggesting the presence of beta-adrenoceptors in the vascular bed. This was confirmed by the administration of isoprenaline which induced a marked reduction in vascular resistance of the human spleen.6. The polypeptides <em>angiotensin</em> and vasopressin induced a marked vasoconstriction in the human spleen without changes in the spleen volume. These effects were uninfluenced by the administration of phenoxybenzamine.<em>7</em>. The polypeptide oxytocin caused a slight vasodilatation in the human spleen, an effect almost exactly mimicked by the preservative chlorobutanol.8. Preliminary experiments suggest that noradrenaline is the transmitter released by the postganglionic nerves to the human spleen.9. These results provide direct evidence that the normal human spleen, unlike that of the dog, does not have a reservoir function. It is suggested that contractions of the enlarged human spleen may occur in various pathological conditions.

BACKGROUND

<em>Angiotensin</em>-converting enzyme (ACE) 2, a homolog of ACE, converts <em>angiotensin</em> (Ang) II into Ang(<em>1</em>-<em>7</em>), and the vasoprotective effects of Ang(<em>1</em>-<em>7</em>) have been documented. We explored the hypothesis that serum autoantibodies to ACE2 predispose patients with connective tissue diseases to constrictive vasculopathy, pulmonary arterial hypertension (PAH), or persistent digital ischemia.

METHODS

Serum was examined from 42 patients with systemic lupus erythematosus (SLE), scleroderma, or mixed connective tissue disease. Eighteen vasculopathy patients with PAH (five cases) and/or persistent digital ischemia (<em>1</em>6 cases) were compared with 24 patients without these vasculopathies (control patients) for serum reactivity to purified recombinant human ACE2, using an ELISA.

RESULTS

The sera from <em>1</em><em>7</em> of the <em>1</em>8 (94%) vasculopathy patients had ELISA scores above the baseline level determined using control sera from 28 healthy subjects, and the mean ELISA score in the vasculopathy patients was significantly higher than that in the control patients (P<0.0005). The relative activity of serum ACE2, which was defined using a reference serum, correlated inversely with the ELISA scores for serum anti-ACE2 antibodies in the <em>1</em>8 vasculopathy patients (R2=0.68<em>7</em>2). The IgG fraction from vasculopathy patients, but not from healthy subjects, inhibited ACE2 activities in vitro. Consistent with this, immunosuppressive therapy given to one SLE patient with digital necrosis markedly decreased the anti-ACE2 antibody titer and restored serum ACE2 activity.

CONCLUSIONS

Autoantibodies to ACE2 may be associated with constrictive vasculopathies.

The RAS (renin-<em>angiotensin</em> system) is now recognized as an important regulator of liver fibrosis and portal pressure. Liver injury stimulates the hepatic expression of components of the RAS, such as ACE (<em>angiotensin</em>-converting enzyme) and the AT(<em>1</em>) receptor [AngII (<em>angiotensin</em> II) type <em>1</em> receptor], which play an active role in promoting inflammation and deposition of extracellular matrix. In addition, the more recently recognized structural homologue of ACE, ACE2, is also up-regulated. ACE2 catalyses the conversion of AngII into Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)], and there is accumulating evidence that this 'alternative axis' of the RAS has anti-fibrotic, vasodilatory and anti-proliferative effects, thus counterbalancing the effects of AngII in the liver. The RAS is also emerging as an important contributor to the pathophysiology of portal hypertension in cirrhosis. Although the intrahepatic circulation in cirrhosis is hypercontractile in response to AngII, resulting in increased hepatic resistance, the splanchnic vasculature is hyporesponsive, promoting the development of the hyperdynamic circulation that characterizes portal hypertension. Both liver fibrosis and portal hypertension represent important therapeutic challenges for the clinician, and there is accumulating evidence that RAS blockade may be beneficial in these circumstances. The present review outlines new aspects of the RAS and explores its role in the pathogenesis and treatment of liver fibrosis and portal hypertension.

BACKGROUND

Blockade of the renin angiotensin system (RAS) via angiotensin I converting enzyme (ACE) inhibition reduces growth of colorectal cancer (CRC) liver metastases in a mouse model. In this work we defined the expression of the various components of the RAS in both tumor and liver during the progression of this disease.

METHODS

Immunohistochemistry and quantitative RT-PCR was used to examine RAS expression in a mouse CRC liver metastases model. CRC metastases and liver tissue was assessed separately at key stages of CRC liver metastases development in untreated (control) mice and in mice treated with the ACE inhibitor captopril (750 mg/kg/day). Non-tumor induced (sham) mice indicated the effect of tumors on normal liver RAS. The statistical significance of multiple comparisons was determined using one-way analysis of variance followed by Bonferroni adjustment with SAS/STAT software.

RESULTS

Reduced volume of CRC liver metastases with captopril treatment was evident. Local RAS of CRC metastases differed from the surrounding liver, with lower angiotensin II type 1 receptor (AT1R) expression but increased ANG-(1-7) receptor (MasR) compared to the liver. The AT1R localised to cancer and stromal infiltrating cells, while other RAS receptors were detected in cancer cells only. Tumor induction led to an initial increase in AT1R and ACE expression while captopril treatment significantly increased ACE expression in the final stages of tumor growth. Conversely, captopril treatment decreased expression of AT1R and angiotensinogen.

CONCLUSIONS

These results demonstrate significant changes in RAS expression in the tumor-bearing captopril treated liver and in CRC metastases. The data suggests the existence of a tumor-specific RAS that can be independently targeted by RAS blockade.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) plays an important role as a member of the renin-<em>angiotensin</em>-aldosterone system (RAAS) in regulating the conversion of <em>angiotensin</em> II (Ang II) into <em>angiotensin</em> (<em>1</em>-<em>7</em>) (Ang [<em>1</em>-<em>7</em>]). But at the same time, while expressed on the surface of human cells, ACE2 is the entry receptor for SARS-CoV-2. Expression of this receptor has been described in several types of cells, including hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), which raises a concern that the virus may infect and damage the stem cell compartment. We demonstrate for the first time that ACE2 and the entry-facilitating transmembrane protease TMPRSS2 are expressed on very small CD<em>1</em>33⁺CD34⁺Lin^-CD45^- cells in human umbilical cord blood (UCB), which can be specified into functional HSCs and EPCs. The existence of these cells known as very small embryonic-like stem cells (VSELs) has been confirmed by several laboratories, and some of them may correspond to putative postnatal hemangioblasts. Moreover, we demonstrate for the first time that, in human VSELs and HSCs, the interaction of the ACE2 receptor with the SARS-CoV-2 spike protein activates the Nlrp3 inflammasome, which if hyperactivated may lead to cell death by pyroptosis. Based on this finding, there is a possibility that human VSELs residing in adult tissues could be damaged by SARS-CoV-2, with remote effects on tissue/organ regeneration. We also report that ACE2 is expressed on the surface of murine bone marrow-derived VSELs and HSCs, although it is known that murine cells are not infected by SARS-CoV-2. Finally, human and murine VSELs express several RAAS genes, which sheds new light on the role of these genes in the specification of early-development stem cells. Graphical Abstract •Human VSELs and HSCs express ACE2 receptor for SARS-CoV2 entry. •Interaction of viral spike protein with ACE2 receptor may hyperactivate Nlrp3 inflammasome which induces cell death by pyroptosis. •SARS-CoV2 may also enter cells and eliminate them by cell lysis. •What is not shown since these cells express also Ang II receptor they may hyperactivate Nlrp3 inflammasome in response to Ang II which may induce pyroptosis. Our data indicates that Ang <em>1</em>-<em>7</em> may have a protective effect.

<strong class="sub-title"> Keywords: </strong> ACE2; COVID<em>1</em>9; Cytokine storm; Hematopoietic stem cells; Nlrp3 inflammasome; Pyroptosis; SARS-CoV-2; Spike protein; VSELs.

Over activation of the renin-<em>angiotensin</em>-aldosterone system is known to be cardiotoxic but the potential injurious effects on the skeletal musculature have not been investigated. Male Wistar rats were given subcutaneous injections of aldosterone (<em>1</em> microg-<em>1</em>0 mg kg-<em>1</em>) and killed <em>7</em> h later, or continuous infusion (<em>1</em> mg kg-<em>1</em> d-<em>1</em>) and killed 48 h later. The role of the mineralocorticoid receptor in mediating aldosterone-induced apoptosis in vivo was investigated using spironolactone (200 mg kg-<em>1</em>). The number of apoptotic (caspase 3 positive) myocytes was counted on cryosections of the heart, soleus and Tibialis Anterior muscles. Injections of aldosterone induced significant (P<0.05) cardiomyocyte apoptosis (peak=2.46+/-0.6 per <em>1</em>0(4) viable myocytes) over the range of <em>1</em>00 microg-<em>1</em>0 mg kg-<em>1</em>, whereas only administration of <em>1</em> mg kg-<em>1</em> induced significant (P<0.05) apoptosis (2.4<em>7</em>+/-0.8 per <em>1</em>0(4) viable myocytes) in the soleus muscle. In contrast, no apoptosis was detected in the striated muscles after administration of only the vehicle. Infusion of aldosterone induced less apoptosis than the same dose (<em>1</em> mg kg-<em>1</em>) given as a single injection. Prior administration of spironolactone significantly (P<0.05) protected the heart (90%) and soleus muscle (<em>7</em>9%) against the apoptosis induced by a single injection of <em>1</em> mg kg-<em>1</em> aldosterone. These data confirm a myotoxic effect of aldosterone on the heart and provide the first description of aldosterone-induced myocyte apoptosis in skeletal muscle. High circulating levels of aldosterone are clearly capable of damaging all types of striated muscle and this may lend support to the concept that heart failure is a generalised, rather than cardiac-specific, myopathy.

The hypothesis that TNF receptor <em>1</em>-deficient (TNFR<em>1</em>(-/-)) mice display blood pressure (BP) and renal functional responses that differ from wild-type (WT) mice was tested in an <em>angiotensin</em> II (ANG II)-dependent model of hypertension. Basal systolic BP (SBP), mean arterial pressure, diastolic BP, heart rate (HR), and pulse pressure were similar in WT and TNFR<em>1</em>(-/-) mice. Infusion of ANG II for <em>7</em> days elevated SBP to a greater extent in TNFR<em>1</em>(-/-) compared with WT mice; pulse pressure was also elevated in TNFR<em>1</em>(-/-). HR decreased in TNFR<em>1</em>(-/-) mice infused with ANG II, an effect prominent on day <em>1</em>. Basal urinary albumin excretion was similar in WT and TNFR<em>1</em>(-/-) mice but was higher in TNFR<em>1</em>(-/-) in response to ANG II infusion. Water intake and urine volume were increased by ANG II infusion; this increase was higher in TNFR<em>1</em>(-/-) vs. WT mice, whereas body weight and food intake were unaffected. Baseline creatinine clearance (Ccr), urinary sodium excretion, and fractional excretion of sodium (FE(Na)%) were similar in vehicle-treated WT and TNFR<em>1</em>(-/-) mice. ANG II infusion for <em>7</em> days increased Ccr and filtered load of sodium in TNFR<em>1</em>(-/-) but not WT mice, whereas it elicited an increase in FE(Na)% and urinary sodium excretion in WT but not TNFR<em>1</em>(-/-) mice. ANG II also inhibited renal TNFR<em>1</em> mRNA accumulation while increasing that of TNFR2. These findings indicate deletion of TNFR<em>1</em> is associated with an exacerbated SBP response, decrease in HR, and altered renal function in ANG II-dependent hypertension.

BACKGROUND

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is an endogenous, heptapeptide hormone with anti-proliferative and anti-angiogenic properties. The primary objective of this study was to determine whether Ang-(<em>1</em>-<em>7</em>) effectively reduces prostate cancer metastasis in mice.

METHODS

Human PC3 prostate cancer cells were injected into the aortic arch via the carotid artery of SCID mice pre-treated with Ang-(<em>1</em>-<em>7</em>) or injected into the tibia of athymic mice, administered Ang-(<em>1</em>-<em>7</em>) for 5 weeks beginning 2 weeks post-injection. Tumor growth and volume were determined by bioluminescent and magnetic resonance imaging. The presence of tumors was confirmed by hematoxylin and eosin staining; TRAP histochemistry was used to identify osteolytic lesions. The effect of Ang-(<em>1</em>-<em>7</em>) on osteoclastogenesis was assessed in differentiated bone marrow cells.

RESULTS

Pre-treatment with Ang-(<em>1</em>-<em>7</em>) prevented metastatic tumor formation following intra-aortic injection of PC3 cells, while 83% of untreated mice developed tumors in metastatic sites. Circulating VEGF was significantly higher in control mice compared to mice administered Ang-(<em>1</em>-<em>7</em>). A 5-week regimen of the heptapeptide hormone attenuated intra-tibial tumor growth; Ang-(<em>1</em>-<em>7</em>) was significantly higher in the tibia of treated mice than in control animals. Osteoclastogenesis was reduced by 50% in bone marrow cells differentiated in the presence of Ang-(<em>1</em>-<em>7</em>), suggesting that the heptapeptide hormone prevents the formation of osteolytic lesions to reduce tumor survival in the bone microenvironment.

CONCLUSIONS

These findings suggest that Ang-(<em>1</em>-<em>7</em>) may serve as an anti-angiogenic and anti-metastatic agent for advanced prostate cancer. By extension, the heptapeptide hormone may provide effective therapy for bone metastasis produced from primary tumors of the lung and breast.

The tubuloglomerular feedback (TGF) responses of the spontaneously hypertensive rat (SHR) are under exaggerated regulation by <em>angiotensin</em> II (Ang II) type <em>1</em> receptors (AT<em>1</em>-R). Since AT<em>1</em>-Rs enhance oxygen radical (O2-) generation, we tested the hypothesis that the exaggerated TGF was due to a diminished blunting by macula densa (MD)-derived nitric oxide (NO) because of excessive AT<em>1</em>-R-dependent generation of O2-. Groups of SHR and control Wistar-Kyoto (WKY) rats received vehicle (Veh), the AT<em>1</em>-R antagonist candesartan (Cand; 3 mg. kg-<em>1</em>. day-<em>1</em>), or nonspecific therapy with hydralazine + hydrochlorothiazide + reserpine (HHR) for two weeks. Compared with WKY rats, the elevated mean arterial pressure of SHR (WKY <em>1</em>25 +/- 2 vs. SHR <em>1</em>63 to <em>7</em><em>7</em>9 mm Hg, P < 0.00<em>1</em>) was reduced (P < 0.00<em>1</em>) similarly in SHR by Cand and HHR (<em>1</em>2<em>1</em> +/- 5 and <em>1</em><em>1</em>6 +/- 5 mm Hg, P = NS). The SHR had an increased maximal TGF response (change in stop flow pressure during luminal perfusion of fluid: SHR <em>1</em><em>1</em>.2 +/- 0.5 vs. WKY 8.3 +/- 0.4 mm Hg, P < 0.0<em>1</em>) and a reduced TGF response to blockade of neuroneal NO synthase (nNOS) in the MD with luminal <em>7</em>-nitroindazole (<em>7</em>-NI: DeltaTGF in WKY 2.8 +/- 0.4 vs. SHR <em>1</em>.<em>1</em> +/- 0.6 mm Hg, P < 0.05). Although the elevated TGF responses of SHR were normalized by both HHR and Cand, only Cand restored a normal TGF response to luminal perfusion of the MD with <em>7</em>-NI (DeltaTGF with <em>7</em>-NI in SHR: Veh + <em>1</em>.8 +/- 0.4 vs. Cand + 3.4 +/- 0.5 mm Hg, P < 0.05). To abrogate the local effects of O2-, tempol (a membrane-permeable superoxide dismutase mimetic) was perfused into the efferent arteriole. During tempol, SHR given vehicle or HHR had a much increased response to blockade of nNOS with <em>7</em>-NI (DeltaTGF in SHR with <em>7</em>-NI during tempol: Veh 6.3 +/- <em>1</em>.0 and HHR 4.5 +/- 0.8 mm Hg, P < 0.0<em>1</em> vs. no tempol for both), implying that the effects of NO had been prevented because of excessive O2-. In contrast, the TGF response to <em>7</em>-NI in SHR given Cand was unaffected by tempol (DeltaTGF with <em>7</em>-NI during tempol: 2.9 +/- 0.9, P = NS, compared with no tempol). In conclusion, TGF responses of SHR are exaggerated because of the effects of hypertension and AT<em>1</em>-R. AT<em>1</em>-R blockade specifically diminishes oxidative stress and restores NO signaling in the juxtaglomerular apparatus of the SHR.

BACKGROUND

Agents inhibiting the renin-<em>angiotensin</em>-aldosterone (RAAS) system have an important role in slowing the progression of chronic kidney disease. We evaluated the hypothesis that the addition of an aldosterone receptor antagonist to an <em>angiotensin</em>-converting enzyme (ACE) inhibitor and <em>angiotensin</em> II type <em>1</em> (AT-<em>1</em>) receptor blocker (ARB) (triple RAAS blockade) may provide an additional benefit compared with an ACE inhibitor and ARB (double RAAS blockade).

METHODS

Randomized open controlled crossover study.

METHODS

<em>1</em>8 whites (7 women, <em>1</em><em>1</em> men) from the Outpatient Department of Nephrology with chronic nondiabetic proteinuric kidney diseases, mean age 42.4 +/- <em>1</em>.9 years (SEM).

METHODS

In the 8-week run-in period, all participants received the ACE inhibitor cilazapril (5 mg), the ARB telmisartan (80 mg), and the diuretic hydrochlorothiazide (<em>1</em>2.5 mg) as double RAAS blockade to achieve the target blood pressure of less than <em>1</em>30/80 mm Hg. Participants were then randomly assigned to 2 treatment sequences, either the addition of spironolactone (25 mg) (triple RAAS blockade) through 8 weeks followed by double RAAS blockade through 8 weeks (sequence <em>1</em>) or double RAAS blockade followed by triple RAAS blockade (sequence 2).

METHODS

24-hour urine protein excretion (primary end point) and markers of tubular injury and fibrosis (secondary end points). Analysis was performed using analysis of variance for repeated measurements.

RESULTS

At baseline, mean serum creatinine level was <em>1</em>.<em>1</em>6 +/- 0.09 mg/dL (<em>1</em>03 +/- 8 micromol/L), estimated glomerular filtration rate was <em>1</em>07.8 mL/min (95% confidence interval, 93 to <em>1</em>40.9 [<em>1</em>.8 mL/s; 95% confidence interval, <em>1</em>.55 to 2.35; Cockcroft-Gault formula), and 24-hour mean proteinuria was 0.97 +/- 0.<em>1</em>8 g. Mean urine protein excretion was 0.7 g/24 h (95% confidence interval, 0.48 to 0.92) less after triple RAAS blockade than after double RAAS blockade (P = 0.0<em>1</em>), without change in blood pressure. Urine excretion of N-acetyl-beta-d-glucosaminidase (P = 0.02) and amino-terminal propeptide of type III procollagen (P = 0.05) also significantly decreased. Potassium levels increased significantly after triple therapy (P = 0.02). However, no patient was withdrawn because of adverse effects.

CONCLUSIONS

Absence of blinding, small sample size, short treatment period, absence of histological assessment.

CONCLUSIONS

Administration of an aldosterone receptor antagonist in addition to double RAAS blockade with an ACE inhibitor and ARB may slow the progression of chronic kidney disease. Additional studies are necessary to confirm this result.

The bactericidal effectiveness of liposomal polymyxin B against Pseudomonas aeruginosa was investigated in an animal model of pulmonary infection. Polymyxin B was incorporated into liposomes composed of <em>1</em>,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) (2:<em>1</em>). Lung infection was induced in rats following intratracheal instillation of <em>1</em>0(<em>7</em>) colony-forming units (CFU) of P. aeruginosa (ATCC 2<em>7</em>853) embedded in agar beads. Starting on day 3 post-infection, animals were treated daily, for 3 consecutive days, with saline, empty liposomes, free polymyxin B, or liposomal polymyxin B (2mg polymyxin B/kg body weight) by intratracheal instillation; animals were killed 24hr after the third drug instillation. Treatment of infected animals with liposomal polymyxin B significantly reduced the pulmonary bacterial counts (3.<em>7</em>+/-0.4log CFU/paired lungs) as compared with that of free polymyxin B (5.<em>1</em>+/-0.2log CFU/paired lungs). Treatment of infected animals with empty liposomes gave pulmonary bacterial counts similar to those obtained from the saline-treated group. Pulmonary infection with P. aeruginosa also resulted in lung injury as evidenced by increases in wet lung weight and decreases in <em>angiotensin</em> converting enzyme activity as well as increases in myeloperoxidase activity, an index of the inflammatory response. Treatment with free polymyxin B ameliorated the lung injuries induced by the microorganism, a protective effect that was more pronounced in the liposomal polymyxin B-treated group. The levels of polymyxin B in the lungs of the infected animals treated with the liposomal suspension were significantly higher (42.8+/-6.2 microg/paired lungs) compared with those treated with the free drug (8.2+/-0.4 microg/paired lungs). These data suggest that direct delivery of liposomal polymyxin B to the lung can be effective in the treatment of pulmonary infection with P. aeruginosa by enhancing retention of the antibiotic in the lung.

To understand the regulation of the vasoactive peptides bradykinin, <em>angiotensin</em> II, calcitonin gene-related peptide (CGRP), and neuropeptide Y (NPY), their proteolytic catabolism by cultured rat aortic vascular smooth muscle cells and A<em>7</em>r5 cells was investigated. Endopeptidase-24.<em>1</em><em>1</em> (EC 3.4.24.<em>1</em><em>1</em>, CD <em>1</em>0) was responsible for the final inactivation of bradykinin, <em>angiotensin</em> II, and CGRP, but not of NPY, which was degraded by a different metallo-endopeptidase. Exopeptidases, namely the aminopeptidases A (EC 3.4.<em>1</em><em>1</em>.<em>7</em>), N (EC 3.4.<em>1</em><em>1</em>.2, CD <em>1</em>3), and P (EC 3.4.<em>1</em><em>1</em>.9) and the carboxypeptidases M (EC 3.4.<em>1</em><em>7</em>.<em>1</em>2) and P (EC 3.4.<em>1</em><em>7</em>.<em>1</em>6), were important for their differential, receptor subtype-specific activation or inactivation. Aminopeptidase A and N generated <em>angiotensins</em> III and IV from <em>angiotensin</em> II. Aminopeptidase P liberated the terminal amino acids from bradykinin and NPY, yielding the Y2 receptor specific-agonist NPY(2-36). Carboxypeptidase P produced AT II(<em>1</em>-<em>7</em>) and carboxypeptidase M produced the BK<em>1</em> receptor agonist [des-Arg9]bradykinin. Thus, peptidases at the surface of vascular smooth muscle cells exert a complex influence on the level of biologically active vasoactive peptides.

Osteopontin (OPN), also called cytokine Eta-<em>1</em>, expressed in the myocardium co-incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. <em>Angiotensin</em> II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen-activated protein kinases (ERK<em>1</em>/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II- and cytokine-induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 +/- 0.3-folds; P < 0.05; n = <em>7</em>), while interleukin-<em>1</em>beta (IL-<em>1</em>beta), tumor necrosis factor (TNF-alpha), and interferon-gamma (IFN-gamma) had no effect. A combination of Ang II with IL-<em>1</em>beta or TNF-alpha, not IFN-gamma, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II- and Ang II+ IL-<em>1</em>beta-stimulated increases in OPN mRNA. Ang II activated ERK<em>1</em>/2 within 5 min of treatment, not JNKs. IL-<em>1</em>beta activated ERK<em>1</em>/2 and JNKs within <em>1</em>5 min of treatment. A combination of Ang II and IL-<em>1</em>beta activated ERK<em>1</em>/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-<em>1</em>beta-stimulated activation of ERK<em>1</em>/2, while partially inhibiting JNKs. PD98059, ERK<em>1</em>/2 pathway inhibitor, and SP600<em>1</em>25, JNKs inhibitor, partially inhibited Ang II + IL-<em>1</em>beta-stimulated increases in OPN mRNA. A combination of PD98059 and SP600<em>1</em>25 almost completely inhibited Ang II + IL-<em>1</em>beta-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-<em>1</em>beta and TNF-alpha act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS-mediated activation of ERK<em>1</em>/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts.

OBJECTIVE

The detailed role of angiotensin II in salt-exacerbated stroke is unclear. We examined the role of angiotensin II in salt-accelerated stroke of stroke-prone spontaneously hypertensive rats (SHRSP).

METHODS

Salt-loaded SHRSP were orally given the angiotensin II type 1 (AT1) receptor blocker candesartan (0.3 to 3 mg/kg per day) and calcium channel blocker amlodipine (1 mg/kg per day), and the effects on stroke (n=61) and brain superoxide were compared between them. We also examined the effect of angiotensin II infusion (200 ng/kg per min) on brain superoxide production and blood-brain barrier.

RESULTS

Despite the comparable hypotensive effect between candesartan and amlodipine, candesartan prolonged survival of salt-loaded SHRSP much more than amlodipine (P<0.01), being associated with more improvement of cerebral arteriolar thickening, cerebral arteriolar cell proliferation, and hippocampal CA1 neuronal cell reduction (1024.9+/-20.6 versus 724.9+/-22.8 cells/mm2; P<0.01; n=7 to 10 in each group) in SHRSP by candesartan (P<0.05) than amlodipine. Salt loading increased superoxide and NADPH oxidase activity in brain cortex and hippocampus of SHRSP, and this increase was prevented by candesartan (P<0.01) but not amlodipine. Angiotensin II infusion, via AT1 receptor, directly increased brain superoxide by 1.8-fold (P<0.05; n=6 to 7 in each group) and impaired blood-brain barrier in salt-loaded SHRSP by 1.7-fold (P<0.05), and this increase in brain superoxide and blood-brain barrier impairment was prevented by tempol as well as candesartan.

CONCLUSIONS

Excess salt, via oxidative stress, accelerates stroke, and angiotensin II, via AT1 receptor, plays a pivotal role in brain superoxide production of SHRSP by excess salt.

The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-<em>angiotensin</em> system (RAS) is involved in the development and progression of cardiac hypertrophy, remodeling, and heart failure. Estrogen attenuates the effects of an activated circulating RAS; however, its role in regulating the cardiac RAS is unclear. Bilateral oophorectomy (OVX; n = <em>1</em><em>7</em>) or sham-operation (Sham; n = <em>1</em>3) was performed in 4-week-old, female mRen2.Lewis rats. At <em>1</em><em>1</em> weeks of age, the rats were randomized and received either <em>1</em><em>7</em> β-estradiol (E2, 36 µg/pellet, 60-day release, n = 8) or vehicle (OVX-V, n = 9) for 4 weeks. The rats were sacrificed, and blood and hearts were used to determine protein and/or gene expression of circulating and tissue RAS components. E2 treatment minimized the rise in circulating <em>angiotensin</em> (Ang) II and aldosterone produced by loss of ovarian estrogens. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(<em>1</em>-<em>7</em>) content, chymase gene expression, and mast cell number. Neither OVX nor OVX+E2 altered cardiac expression or activity of renin, <em>angiotensin</em>ogen, <em>angiotensin</em>-converting enzyme (ACE), and Ang II type <em>1</em> receptor (AT<em>1</em>R). E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(<em>1</em>-<em>7</em>) mas receptor, in comparison to sham-operated and OVX littermates. E2 treatment appears to inhibit upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly by reducing chymase-dependent Ang II formation. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.