OBJECTIVE

To investigate the role of endogenous endothelin-<em>1</em> (ET-<em>1</em>) expression and its interaction with the ETA receptor in the physiologic regulation of vascular tone as well as in the development of abnormal retinal hemodynamics in diabetes.

METHODS

Retinal blood flow, using digitized video fluorescein angiography recordings, was quantitated after intravitreous injections of ET-<em>1</em>; BQ-<em>1</em>23, an ETA receptor antagonist; and phosporamindon, an endothelin converting enzyme inhibitor in the eyes of diabetic and nondiabetic rats. A total of <em>1</em>54 rats were used for these experiments. Message levels of preproendothelin-<em>1</em> (preproET-<em>1</em>) were measured from the retina of diabetic and nondiabetic rats using competitive polymerase chain reaction (PCR) techniques.

RESULTS

Retinal blood flow was reduced (33%, P < 0.00<em>1</em>) in diabetic rats compared to nondiabetic rats. BQ-<em>1</em>23, an ETA receptor antagonist, but not saralasin, an angiotensin receptor antagonist, increased retinal blood flow in a dose-dependent manner in diabetic (EC50 of 8 x <em>1</em>0(-7) M) and in nondiabetic rats (EC50 of 8 x <em>1</em>0(-8) M). Besides being resistant to BQ-<em>1</em>23, the maximal response in diabetic animals occurred 20 minutes later than in nondiabetic animals. Decreasing ET-<em>1</em> levels by inhibiting endothelin-converting enzyme with phosphoramidon normalized retinal blood flow in diabetic rats. In nondiabetic rats, the intravitreous injection of exogenous ET-<em>1</em> (<em>1</em>0(-8) M) resulted in retinal blood flow decreases comparable to those measured in diabetic animals, and the subsequent injection of <em>1</em>0(-4) M BQ-<em>1</em>23 produced retinal blood flow changes comparable to those measured in BQ-<em>1</em>23 injected diabetic rats. Comparison of preproET-<em>1</em> messenger RNA expression in the retina, brain and lung of control and diabetic rats using quantitative PCR and Northern blot analysis showed 2.0- and <em>1</em>.7-fold increases in the retina and the brain, respectively, without changes in the lung.

CONCLUSIONS

These data suggest that ET-<em>1</em> is involved in the regulation of retinal blood flow in normal physiologic outcome, and an increase in the endogenous expression of ET-<em>1</em> contributes to the reduction of retinal blood flow reported in the early stages of diabetes mellitus.

Studies suggest T cells modulate arterial pressure. Because robust sex differences exist in the immune system and in hypertension, we investigated sex differences in T-cell modulation of <em>angiotensin</em> II-induced increases in mean arterial pressure in male (M) and female (F) wild-type and recombination-activating-gene-<em>1</em>-deficient (Rag<em>1</em>(-/-)) mice. Sex differences in peak mean arterial pressure in wild-type were lost in Rag<em>1</em>(-/-) mice (mm Hg: wild-type-F, <em>1</em>36±4.9 versus wild-type-M, <em>1</em>53±<em>1</em>.<em>7</em>; P<0.02; Rag<em>1</em>(-/-)-F, <em>1</em>35±2.<em>1</em> versus Rag<em>1</em>(-/-)-M, <em>1</em>4<em>1</em>±3.8). Peak mean arterial pressure was <em>1</em>3 mm Hg higher after adoptive transfer of male (CD3(M)→Rag<em>1</em>(-/-)-M) versus female (CD3(F)→Rag<em>1</em>(-/-)-M) T cells. CD3(M)→Rag<em>1</em>(-/-)-M mice exhibited higher splenic frequencies of proinflammatory interleukin-<em>1</em><em>7</em>A (2.4-fold) and tumor necrosis factor-α (2.2-fold)-producing T cells and lower plasma levels (<em>1</em>3-fold) and renal mRNA expression (2.4-fold) of interleukin-<em>1</em>0, whereas CD3(F)→Rag<em>1</em>(-/-)-M mice displayed a higher activation state in general and T-helper-<em>1</em>-biased renal inflammation. Greater T-cell infiltration into perivascular adipose tissue and kidney associated with increased pressor responses to <em>angiotensin</em> II if the T cell donor was male but not female and these sex differences in T-cell subset expansion and tissue infiltration were maintained for <em>7</em> to 8 weeks within the male host. Thus, the adaptive immune response and role of pro- and anti-inflammatory cytokine signaling in hypertension are distinct between the sexes and need to be understood to improve therapeutics for hypertension-associated disease in both men and women.

The brain renin-<em>angiotensin</em> system (RAS) has available the necessary functional components to produce the active ligands <em>angiotensins</em> II (AngII), <em>angiotensin</em> III, <em>angiotensins</em> (IV), <em>angiotensin</em> (<em>1</em>-<em>7</em>), and <em>angiotensin</em> (3-<em>7</em>). These ligands interact with several receptor proteins including AT<em>1</em>, AT2, AT4, and Mas distributed within the central and peripheral nervous systems as well as local RASs in several organs. This review first describes the enzymatic pathways in place to synthesize these ligands and the binding characteristics of these <em>angiotensin</em> receptor subtypes. We next discuss current hypotheses to explain the disorders of Alzheimer's disease (AD) and Parkinson's disease (PD), as well as research efforts focused on the use of <em>angiotensin</em> converting enzyme (ACE) inhibitors and <em>angiotensin</em> receptor blockers (ARBs), in their treatment. ACE inhibitors and ARBs are showing promise in the treatment of several neurodegenerative pathologies; however, there is a need for the development of analogs capable of penetrating the blood-brain barrier and acting as agonists or antagonists at these receptor sites. AngII and AngIV have been shown to play opposing roles regarding memory acquisition and consolidation in animal models. We discuss the development of efficacious AngIV analogs in the treatment of animal models of AD and PD. These AngIV analogs act via the AT4 receptor subtype which may coincide with the hepatocyte growth factor/c-Met receptor system. Finally, future research directions are described concerning new approaches to the treatment of these two neurological diseases.

The renin-<em>angiotensin</em> system is important for cardiovascular homeostasis. Currently, therapies for different cardiovascular diseases are based on inhibition of <em>angiotensin</em>-converting enzyme (ACE) or <em>angiotensin</em> II receptor blockade. Inhibition of ACE blocks metabolism of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) to <em>angiotensin</em>-(<em>1</em>-5) and can lead to elevation of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) levels in plasma and tissue. In animal models, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) itself causes or enhances vasodilation and inhibits vascular contractions to <em>angiotensin</em> II. The function of <em>angiotensin</em>-(<em>1</em>-5) is unknown. We investigated whether <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and <em>angiotensin</em>-(<em>1</em>-5) inhibit ACE or antagonize <em>angiotensin</em>-induced vasoconstrictions in humans. ACE activity in plasma and atrial tissue was inhibited by <em>angiotensin</em>-(<em>1</em>-<em>7</em>) up to <em>1</em>00%, with an IC(50) of 3.0 and 4.0 micromol/L, respectively. In human internal mammary arteries, contractions induced by <em>angiotensin</em> I and II and the non-ACE-specific substrate [Pro(<em>1</em><em>1</em>),D-Ala(<em>1</em>2)]-<em>angiotensin</em> I were antagonized by <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (<em>1</em>0(-5) mol/L) in a noncompetitive way, with a 60% inhibition of the maximal response to <em>angiotensin</em> II. Contractions to ACE-specific substrate [Pro(<em>1</em>0)]-<em>angiotensin</em> I were also inhibited, an effect only partly accounted for by antagonism of <em>angiotensin</em> II. <em>Angiotensin</em>-(<em>1</em>-5) inhibited plasma ACE activity with a potency equal to that of <em>angiotensin</em> I but had no effect on arterial contractions. In conclusion, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) blocks <em>angiotensin</em> II-induced vasoconstriction and inhibits ACE in human cardiovascular tissues. <em>Angiotensin</em>-(<em>1</em>-5) only inhibits ACE. These results show that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) may be an important modulator of the human renin-<em>angiotensin</em> system.

UNASSIGNED

Both hypo- and hyperkalaemia can have immediate deleterious physiological effects, and less is known about long-term risks. The objective was to determine the risks of all-cause mortality, cardiovascular mortality, and end-stage renal disease associated with potassium levels across the range of kidney function and evaluate for consistency across cohorts in a global consortium.

UNASSIGNED

We performed an individual-level data meta-analysis of 2<em>7</em> international cohorts [<em>1</em>0 general population, <em>7</em> high cardiovascular risk, and <em>1</em>0 chronic kidney disease (CKD)] in the CKD Prognosis Consortium. We used Cox regression followed by random-effects meta-analysis to assess the relationship between baseline potassium and adverse outcomes, adjusted for demographic and clinical characteristics, overall and across strata of estimated glomerular filtration rate (eGFR) and albuminuria. We included <em>1</em> 2<em>1</em><em>7</em> 986 participants followed up for a mean of 6.9 years. The average age was 55 ± <em>1</em>6 years, average eGFR was 83 ± 23 mL/min/<em>1</em>.<em>7</em>3 m2, and <em>1</em><em>7</em>% had moderate- to-severe increased albuminuria levels. The mean baseline potassium was 4.2 ± 0.4 mmol/L. The risk of serum potassium of >5.5 mmol/L was related to lower eGFR and higher albuminuria. The risk relationship between potassium levels and adverse outcomes was U-shaped, with the lowest risk at serum potassium of 4-4.5 mmol/L. Compared with a reference of 4.2 mmol/L, the adjusted hazard ratio for all-cause mortality was <em>1</em>.22 [95% confidence interval (CI) <em>1</em>.<em>1</em>5-<em>1</em>.29] at 5.5 mmol/L and <em>1</em>.49 (95% CI <em>1</em>.26-<em>1</em>.<em>7</em>6) at 3.0 mmol/L. Risks were similar by eGFR, albuminuria, renin-<em>angiotensin</em>-aldosterone system inhibitor use, and across cohorts.

UNASSIGNED

Outpatient potassium levels both above and below the normal range are consistently associated with adverse outcomes, with similar risk relationships across eGFR and albuminuria.

<em>Angiotensin</em> (Ang)-converting enzyme 2 (ACE2) is a key enzyme in the metabolism of Ang II. XNT (<em>1</em>-[(2-dimethylamino)ethylamino]-4-(hydroxymethyl)-<em>7</em>-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one) and diminazene have been reported to exert various organ-protective effects, which are attributed to the activation of ACE2. To test the effect of these compounds, we studied Ang II degradation in vivo and in vitro as well as their effect on ACE2 activity in vivo and in vitro. In a model of Ang II-induced acute hypertension, blood pressure (BP) recovery was markedly enhanced by XNT (slope with XNT, -3.26±0.2 versus -<em>1</em>.6±0.2 mm Hg/min without XNT; P<0.0<em>1</em>). After Ang II infusion, neither plasma nor kidney ACE2 activity was affected by XNT. Plasma Ang II and Ang (<em>1</em>-<em>7</em>) levels also were not significantly affected by XNT. The BP-lowering effect of XNT seen in wild-type animals was also observed in ACE2 knockout mice (slope with XNT, -3.09±0.30 versus -<em>1</em>.28±0.22 mm Hg/min without XNT; P<0.00<em>1</em>). These findings show that the BP-lowering effect of XNT in Ang II-induced hypertension cannot be because of the activation of ACE2. In vitro and ex vivo experiments in both mice and rat kidney confirmed a lack of enhancement of ACE2 enzymatic activity by XNT and diminazene. Moreover, Ang II degradation in vitro and ex vivo was unaffected by XNT and diminazene. We conclude that the biological effects of these compounds are ACE2-independent and should not be attributed to the activation of this enzyme.

Hypertension is one of the major predisposing factors for neurodegenerative disease characterized with activated renin-<em>angiotensin</em> system (RAS) in both periphery and brain. Vitamin D (VitD) is recently recognized as a pleiotropic hormone with strong neuroprotective properties. While multiple lines of evidence suggest that VitD can act on RAS, the evidence concerning the crosstalk between VitD and RAS in the brain is limited. Therefore, this study aims to evaluate whether VitD can modulate brain RAS to trigger neuroprotective actions in the brain of spontaneously hypertensive rats (SHR). Our data showed that calcitriol treatment induced VDR expression and inhibited neural death in the prefrontal cortex of SHR. Sustained calcitriol administration also inhibited microglia M<em>1</em> polarization, but enhanced M2 polarization, accompanied with decreased expression of proinflammatory cytokines. We then further explored the potential mechanisms and showed that SHR exhibited overactivated classical RAS with increased expression of <em>angiotensin</em> II (Ang II) receptor type <em>1</em> (AT<em>1</em>), <em>angiotensin</em> converting enzyme (ACE) and Ang II production, whereas the counteracting arm of traditional RAS, ACE2/Ang(<em>1</em>-<em>7</em>)/MasR, was impaired in the SHR brain. Calcitriol nonsignificantly suppressed AT<em>1</em> and ACE but markedly reduced Ang II formation. Intriguingly, calcitriol exerted pronouncedly impact on ACE2/Ang(<em>1</em>-<em>7</em>)/MasR axis with enhanced expression of ACE2, MasR and Ang(<em>1</em>-<em>7</em>) generation. Meanwhile, calcitriol ameliorated the overactivation of NADPH-oxidase (Nox), the downstream of RAS, in SHR, and also mitigated oxidative stress. In microglial (BV2) cells, we further found that calcitriol induced ACE2 and MasR with no significant impact on ACE and AT<em>1</em>. In accordance, calcitriol also attenuated Ang II-induced Nox activation and ROS production, and shifted the microglia polarization from M<em>1</em> to M2 phenotype. However, co-treatment with A<em>7</em><em>7</em>9, a specific MasR antagonist, abrogated the antioxidant and neuroimmune modulating actions of VitD. These findings strongly indicate the involvement of ACE2/Ang(<em>1</em>-<em>7</em>)/MasR pathway in the neuroprotective mechanisms of VitD in the hypertensive brain.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-[<em>1</em>-<em>7</em>]) is a heptapeptide member of the renin-<em>angiotensin</em> system (RAS), and acts as a vasodilator and antagonist of <em>angiotensin</em> II (Ang II) in the vasculature. The role of Ang-(<em>1</em>-<em>7</em>) in regulating kidney function is not well understood. Within the kidneys, Ang-(<em>1</em>-<em>7</em>) is generated by <em>angiotensin</em>-converting enzyme 2 (ACE2)-mediated degradation of Ang II, sequential cleavage of the precursor <em>angiotensin</em> I (Ang I) by ACE2 and ACE, or the actions of brush-border membrane peptidases on Ang I. Ang-(<em>1</em>-<em>7</em>) mediates its effects via binding to kidney Mas receptors, although some actions may occur via Ang II AT<em>1</em> or AT2 receptors. In vitro studies suggest that Ang-(<em>1</em>-<em>7</em>) is an intrarenal vasodilator. Ang-(<em>1</em>-<em>7</em>) has been reported to induce either natriuresis/diuresis or sodium and water retention, via modulation of sodium transporters in the proximal tubule and loop of Henle, and collecting duct water transport. In the proximal tubule, Ang-(<em>1</em>-<em>7</em>) antagonizes growth-promoting signaling pathways via activation of a protein tyrosine phosphatase, whereas in mesangial cells, Ang-(<em>1</em>-<em>7</em>) stimulates cell growth via activation of mitogen-activated protein kinases. The phenotype of the Mas gene knockout mouse suggests that Ang-(<em>1</em>-<em>7</em>)-signaling events exert cardiovascular protection by regulating blood pressure, and by limiting production of reactive oxygen species and extracellular matrix proteins. Ang-(<em>1</em>-<em>7</em>) also protects against renal injury in the renal wrap hypertension model, independent of effects on blood pressure. In diabetic nephropathy, however, the role of Ang-(<em>1</em>-<em>7</em>) on disease progression remains unclear. In summary, Ang-(<em>1</em>-<em>7</em>) and its receptor Mas have emerged as important components of the intrarenal RAS. The signaling and downstream effects of Ang-(<em>1</em>-<em>7</em>) in the kidney are complex and appear to be cell specific. The body of evidence suggests that Ang-(<em>1</em>-<em>7</em>) is protective against endothelial dysfunction or Ang II-stimulated proximal tubular injury, although the overall effects on glomerular function require further study.

We examined the impact of early diabetes on the circulating and kidney renin-<em>angiotensin</em> system (RAS) in male and female mRen2.Lewis (mRen2) hypertensive rats. Diabetes (DB) was induced by streptozotocin (STZ; 65 mg/kg) at <em>1</em><em>1</em> wk of age for 4 wk without insulin replacement. Systolic blood pressures were not increased in DB males or females compared with controls (CON). Circulating <em>angiotensin</em>-converting enzyme 2 (ACE2) increased ninefold (P < 0.05) in DB females and threefold (P < 0.05) in DB males, but circulating ACE and ANG II were higher in the DB groups. Serum C-reactive protein was elevated in DB females but not DB males, and the vascular responses to acetylcholine and estradiol were attenuated in the DB females. Proteinuria, albuminuria, and <em>angiotensin</em>ogen excretion increased to a similar extent in both DB females and males. Glomerular VEGF expression also increased to a similar extent in both DB groups. Renal inflammation (CD68(+)cells) increased only in DB females although males exhibited greater inflammation that was not different with DB. Cortical ACE2 did not change in DB females but was reduced (30%) in DB males. Renal neprilysin activity >><em>7</em>5%, P < 0.05) was markedly reduced in the DB females to that in the DB and CON males. ACE activity was significantly lower in both female (<em>7</em>5%, P < 0.05) and male (50%; P < 0.05) DB groups, while cortical ANG II and Ang-(<em>1</em>-<em>7</em>) levels were unchanged. In conclusion, female mRen2 rats are not protected from vascular damage, renal inflammation, and kidney injury in early STZ-induced diabetes despite a marked increase in circulating ACE2 and significantly reduced ACE within the kidney.

To investigate the association between hyperinsulinemia and cardiac hypertrophy, we treated rats with insulin for <em>7</em> wk and assessed effects on myocardial growth, vascularization, and fibrosis in relation to the expression of <em>angiotensin</em> II receptors (AT-R). We also characterized insulin signaling pathways believed to promote myocyte growth and interact with proliferative responses mediated by G protein-coupled receptors, and we assessed myocardial insulin receptor substrate-<em>1</em> (IRS-<em>1</em>) and p<em>1</em><em>1</em>0 alpha catalytic and p85 regulatory subunits of phospatidylinositol 3 kinase (PI3K), Akt, MEK, ERK<em>1</em>/2, and S6 kinase-<em>1</em> (S6K<em>1</em>). Left ventricular (LV) geometry and performance were evaluated echocardiographically. Insulin decreased AT<em>1</em>a-R mRNA expression but increased protein levels and increased AT2-R mRNA and protein levels and phosphorylation of IRS-<em>1</em> (Ser3<em>7</em>4/Tyr989), MEK<em>1</em>/2 (Ser2<em>1</em>8/Ser222), ERK<em>1</em>/2 (Thr202/Tyr204), S6K<em>1</em> (Thr42<em>1</em>/Ser424/Thr389), Akt (Thr308/Thr308), and PI3K p<em>1</em><em>1</em>0 alpha but not of p85 (Tyr508). Insulin increased LV mass and relative wall thickness and reduced stroke volume and cardiac output. Histochemical examination demonstrated myocyte hypertrophy and increases in interstitial fibrosis. Metoprolol plus insulin prevented the increase in relative wall thickness, decreased fibrosis, increased LV mass, and improved function seen with insulin alone. Thus our data demonstrate that chronic hyperinsulinemia decreases AT<em>1</em>a-to-AT2 ratio and increases MEK-ERK<em>1</em>/2 and S6K<em>1</em> pathway activity related to hypertrophy. These changes might be crucial for increased cardiovascular growth and fibrosis and signs of impaired LV function.

<em>Angiotensin</em> (Ang) (<em>1</em>-<em>7</em>) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor associated with cardiac, renal, and cerebral protective responses. Physiological evidence suggests that Mas receptor (MasR) undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating receptor activity is unknown. We investigated the hypothesis that MasR desensitizes and internalizes on stimulation with Ang-(<em>1</em>-<em>7</em>). For this purpose, we generated a chimera between the MasR and the yellow fluorescent protein (YFP; MasR-YFP). MasR-YFP-transfected HEK 293T cells were incubated with Ang-(<em>1</em>-<em>7</em>), and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(<em>1</em>-<em>7</em>) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 minutes. Following the time course of [(<em>1</em>25)I]Ang-(<em>1</em>-<em>7</em>) endocytosis, we observed that half of MasR-YFP underwent endocytosis after <em>1</em>0 minutes, and this was blocked by a MasR antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen <em>1</em>, and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(<em>1</em>-<em>7</em>) stimulation. A fraction of MasR-YFP also colocalized with caveolin <em>1</em>, suggesting that at some point MasR-YFP traverses caveolin <em>1</em>-positive compartments. In conclusion, MasR undergoes endocytosis on stimulation with Ang-(<em>1</em>-<em>7</em>), and this event may explain the desensitization of MasR responsiveness. In this way, MasR activity and density may be tightly controlled by the cell.

There is uncertainty about the contribution of <em>angiotensin</em>-converting enzyme (ACE) to <em>angiotensin</em> II formation, with recent studies suggesting that non-ACE enzymes may be the predominant pathway of <em>angiotensin</em> II formation in kidney, heart, and lung. To investigate the role of ACE in <em>angiotensin</em> II formation, we measured <em>angiotensin</em> I and II levels in blood, kidney, and heart of 2 mouse genetic models (ACE.<em>1</em> and ACE.4) of reduced somatic ACE gene expression and in blood, kidney, heart, lung, adrenal, and brain of mice administered the ACE inhibitor lisinopril. We also measured the levels of bradykinin (<em>1</em>-9) and its ACE metabolite bradykinin (<em>1</em>-<em>7</em>). Reduced ACE gene expression and ACE inhibition had similar effects on <em>angiotensin</em> and bradykinin peptide levels. <em>Angiotensin</em> II levels were reduced by <em>7</em>0% to 9<em>7</em>% in blood, 92% to 99% in kidney, 93% to 99% in heart, 9<em>7</em>% in lung, and 85% in adrenal and brain. The marked reductions in <em>angiotensin</em> II/<em>angiotensin</em> I ratio indicated that ACE was responsible for at least 90% of <em>angiotensin</em> I conversion to <em>angiotensin</em> II in blood, kidney, heart, lung, and brain, and at least <em>7</em><em>7</em>% in adrenal. Blood bradykinin (<em>1</em>-9) levels were increased 6.4-fold to 8.4-fold. Heart bradykinin (<em>1</em>-9) levels were increased in ACE.4 mice and the bradykinin (<em>1</em>-<em>7</em>)/bradykinin (<em>1</em>-9) ratio was reduced in kidney and heart of ACE.4 mice and heart of lisinopril-treated mice. These studies demonstrate that ACE is the predominant pathway of <em>angiotensin</em> II formation in blood and tissues of mice and plays a major role in bradykinin (<em>1</em>-9) metabolism in blood and, to a lesser extent, in kidney and heart.

We tested the hypothesis that activation of the protective arm of the renin <em>angiotensin</em> system, the <em>angiotensin</em>-converting enzyme 2 (ACE2)/<em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)]/Mas receptor axis, corrects the vasoreparative dysfunction typically seen in the CD34(+) cells isolated from diabetic individuals. Peripheral blood CD34(+) cells from patients with diabetes were compared with those of nondiabetic controls. Ang-(<em>1</em>-<em>7</em>) restored impaired migration and nitric oxide bioavailability/cGMP in response to stromal cell-derived factor and resulted in a decrease in NADPH oxidase activity. The survival and proliferation of CD34(+) cells from diabetic individuals were enhanced by Ang-(<em>1</em>-<em>7</em>) in a Mas/phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. ACE2 expression was lower, and ACE2 activators xanthenone and diminazine aceturate were less effective in inducing the migration in cells from patients with diabetes compared with controls. Ang-(<em>1</em>-<em>7</em>) overexpression by lentiviral gene modification restored both the in vitro vasoreparative functions of diabetic cells and the in vivo homing efficiency to areas of ischemia. A cohort of patients who remained free of microvascular complications despite having a history of longstanding inadequate glycemic control had higher expression of ACE2/Mas mRNA than patients with diabetes with microvascular complications matched for age, sex, and glycemic control. Thus, ACE2/Ang-(<em>1</em>-<em>7</em>)\Mas pathway activation corrects existing diabetes-induced CD34(+) cell dysfunction and also confers protection from development of this dysfunction.

Regulation of vascular smooth muscle cell growth is critical to the maintenance of normal blood flow and vessel patency. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] inhibits proliferation of vascular smooth muscle cells in vitro and opposes the mitogenic effects of <em>angiotensin</em> II. The present study investigated whether Ang-(<em>1</em>-<em>7</em>) inhibits vascular smooth muscle cell growth in vivo by determining its effect on neointimal formation and medial remodeling in balloon-injured carotid arteries. The carotid arteries of adult male Sprague-Dawley rats were injured with a balloon embolectomy catheter. Ang-(<em>1</em>-<em>7</em>) in saline (24 microg/kg per hour) or saline alone was infused intravenously for <em>1</em>2 days after injury. Pumps containing bromodeoxyuridine were implanted at the same time to determine DNA synthesis. Intravenous infusion increased plasma Ang-(<em>1</em>-<em>7</em>) to <em>1</em>66. 0+/-4<em>1</em>.2 fmol/mL (n=6) compared with 46.9+/-4.2 fmol/mL (n=8) in saline-infused rats. Plasma concentrations of Ang II were not changed by Ang-(<em>1</em>-<em>7</em>) infusion. Elevation in circulating Ang-(<em>1</em>-<em>7</em>) had no effect on either blood pressure or heart rate compared with saline controls. Histomorphometric analysis of carotid arteries indicated that Ang-(<em>1</em>-<em>7</em>) infusion significantly reduced neointimal area compared with rats infused with saline (0.063+/-0.0<em>1</em><em>1</em> versus 0. <em>1</em>00+/-0.009 mm2; P<0.05). In contrast, Ang-(<em>1</em>-<em>7</em>) infusion had no effect on medial area of the injured or the contralateral uninjured artery compared with saline controls. Ang-(<em>1</em>-<em>7</em>) infusion also reduced the rate of DNA synthesis in both the neointima and the media of the injured vessels. Therefore, exogenous Ang-(<em>1</em>-<em>7</em>) inhibited vascular smooth muscle cell proliferation associated with balloon-catheter injury. Similar increases in endogenous plasma Ang-(<em>1</em>-<em>7</em>) and inhibition of neointimal growth were observed in rats after <em>angiotensin</em>-converting enzyme inhibitor or <em>angiotensin</em> type <em>1</em> receptor antagonist administration, suggesting that Ang-(<em>1</em>-<em>7</em>) may contribute to the in vivo antiproliferative effects of these agents on vascular smooth muscle.

BACKGROUND

Accumulating evidence suggests that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-[<em>1</em>-<em>7</em>]) may play an important role in counteracting the pressor, proliferative, and profibrotic actions of <em>angiotensin</em> II in the heart. Thus, we evaluated whether Ang-(<em>1</em>-<em>7</em>) is expressed in the myocardium of normal rats and those in which myocardial infarction was produced 4 weeks beforehand.

RESULTS

The left coronary artery in <em>1</em>0-week-old Lewis rats was either ligated (n=5) or exposed but not occluded in age-matched controls (sham; n=5). Left ventricular end-diastolic pressures were significantly elevated 4 weeks after myocardial infarction (25+/-<em>1</em> versus 5+/-<em>1</em> mm Hg for sham; P<0.00<em>1</em>), whereas left ventricular systolic pressures were significantly reduced (ligated 86+/-4 versus sham <em>1</em><em>1</em>0+/-5 mm Hg; P<0.0<em>1</em>). Hemodynamic effects of coronary artery ligation were accompanied by significant cardiac hypertrophy (heart weight to body weight: ligated 4.3+/-0.<em>1</em> versus sham 2.9+/-0.<em>1</em> mg/g; P<0.00<em>1</em>). In both ligated and sham rats, Ang-(<em>1</em>-<em>7</em>) immunoreactivity was limited to cardiac myocytes and absent in interstitial cells and coronary vessels. Ang-(<em>1</em>-<em>7</em>) immunoreactivity was significantly augmented in ventricular tissue surrounding the infarct area in the heart of rats with myocardial infarction.

CONCLUSIONS

Development of heart failure subsequent to coronary artery ligation leads to increased expression of Ang-(<em>1</em>-<em>7</em>),which was restricted to myocytes.

<em>Angiotensin</em> II antagonists (AIIA) are part of a new rational treatment of hypertension. Because adverse circulatory effects during anesthesia can occur in patients chronically treated with <em>angiotensin</em>-converting enzyme inhibitors, some clinicians discontinue them at least 24 h before operation. No data are available concerning AIIA administration in patients scheduled for vascular surgery performed under general anesthesia. The aim of this prospective randomized study was to compare hemodynamics during induction of anesthesia in patients chronically treated with AIIA and those of patients not receiving this drug on the morning before operation. Thirty-seven patients chronically treated with AIIA for hypertension were randomly assigned to two groups: Group I: AIIA discontinued on the day before surgery (n = <em>1</em>8); Group II: AIIA given <em>1</em> h before anesthesia (n = <em>1</em>9). Patients received sufentanil 0.4 microg/kg, propofol <em>1</em>.5 mg/kg, and atracurium 0.5 mg/kg. During the procedure, the anesthesiologist was required to maintain systolic blood pressure and heart rate within 30% of baseline values using intravascular fluid administration and vasoconstric- tors (e.g. , ephedrine, phenylephrine, or terlipressin). Hemodynamic variables were recorded each <em>1</em> min. Hemodynamic study ended at incision. The number and duration of hemodynamic events were collected, and total doses of vasoactive drugs were noted in each group. Systolic arterial pressure was significantly decreased in Group II at 5, <em>1</em>5 and 23 min after induction of anesthesia (*P < 0.05). In this group, the decrease in systolic arterial pressure was associated with more frequent episodes of hypotension (AIIA withdrawn: <em>1</em> +/- <em>1</em>; AIIA given: 2 +/- <em>1</em>; P < 0.0<em>1</em>), with a larger number of patients developing at least <em>1</em> episode of hypotension (AIIA withdrawn: <em>1</em>2; AIIA given: <em>1</em>9; P < 0.0<em>1</em>), and a longer duration of an episode of hypotension (AIIA withdrawn: 3 +/- 4 min; AIIA given: 8 +/- <em>7</em> min; P < 0.0<em>1</em>), and an increased need for vasoactive drugs. In conclusion, blockade of the renin-<em>angiotensin</em> system increases the potential hypotensive effect of anesthetic induction. A severe hypotensive episode, requiring vasoconstrictor treatment, occurs after induction of general anesthesia in patients chronically treated with AIIA. Recommendations to discontinue AIIA drugs on the day before the surgery may be justified.

CONCLUSIONS

This prospective randomized study demonstrated that more severe hypotensive episodes, requiring vasoconstrictor treatment, occur after induction of general anesthesia in patients chronically treated with AIIA and receiving this drug on the morning before operation, in comparison with those in whom AIIA were discontinued on the day before operation. Recommendations to discontinue these drugs on the day before the surgery may be justified.

Coronavirus <em>1</em>9 (COVID-<em>1</em>9) was declared as a pandemic viral infection by the World Health organization on March <em>1</em><em>1</em>^th 2020. Usual clinical manifestations of COVID-<em>1</em>9 infection include fever, fatigue, myalgia, headache, diarrhea, dry cough, dyspnea that may lead to acute respiratory distress syndrome and death (<em>1</em>). Skin symptoms of COVID-<em>1</em>9 have been poorly described but may include erythematous rash, urticaria and chicken pox like lesions (2-<em>7</em>). <em>Angiotensin</em>-converting enzyme 2 (ACE2) is a cellular receptor for COVID-<em>1</em>9.

Hyperactivity of the classical axis of the renin-<em>angiotensin</em> system (RAS), mediated by <em>angiotensin</em> II (Ang II) activation of the <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>R), is implicated in the pathogenesis of Alzheimer's disease (AD). <em>Angiotensin</em>-converting enzyme-2 (ACE-2) degrades Ang II to <em>angiotensin</em> <em>1</em>-<em>7</em> (Ang (<em>1</em>-<em>7</em>)) and counter-regulates the classical axis of RAS. We have investigated the expression and distribution of ACE-2 in post-mortem human brain tissue in relation to AD pathology and classical RAS axis activity.

We measured ACE-2 activity by fluorogenic peptide substrate assay in mid-frontal cortex (Brodmann area 9) in a cohort of AD (n = 90) and age-matched non-demented controls (n = 59) for which we have previous data on ACE-<em>1</em> activity, amyloid β (Aβ) level and tau pathology, as well as known ACE<em>1</em> (rs<em>1</em><em>7</em>99<em>7</em>52) indel polymorphism, apolipoprotein E (APOE) genotype, and cerebral amyloid angiopathy severity scores.

ACE-2 activity was significantly reduced in AD compared with age-matched controls (P < 0.000<em>1</em>) and correlated inversely with levels of Aβ (r = -0.26<em>7</em>, P < 0.00<em>1</em>) and phosphorylated tau (p-tau) pathology (r = -0.32<em>7</em>, P < 0.0<em>1</em>). ACE-2 was reduced in individuals possessing an APOE ε4 allele (P < 0.05) and was associated with ACE<em>1</em> indel polymorphism (P < 0.05), with lower ACE-2 activity in individuals homozygous for the ACE<em>1</em> insertion AD risk allele. ACE-2 activity correlated inversely with ACE-<em>1</em> activity (r = -0.453, P < 0.000<em>1</em>), and the ratio of ACE-<em>1</em> to ACE-2 was significantly elevated in AD (P < 0.000<em>1</em>). Finally, we show that the ratio of Ang II to Ang (<em>1</em>-<em>7</em>) (a proxy measure of ACE-2 activity indicating conversion of Ang II to Ang (<em>1</em>-<em>7</em>)) is reduced in AD.

Together, our findings indicate that ACE-2 activity is reduced in AD and is an important regulator of the central classical ACE-<em>1</em>/Ang II/AT<em>1</em>R axis of RAS, and also that dysregulation of this pathway likely plays a significant role in the pathogenesis of AD.

Seventy-one white and 33 black patients with essential hypertension were studied while on a high sodium intake of 350 mmol/d for 5 days and low sodium intake of <em>1</em>0 mmol/d for 5 days. The fall in blood pressure on changing from the high sodium to the low sodium diet was <em>1</em><em>7</em>/6 mm Hg in whites and 22/<em>1</em>0 mm Hg in blacks. Compared with whites, black patients had a <em>7</em>-mm Hg greater fall (P<0.05) in systolic blood pressure and 4-mm Hg greater fall (P=0.068) in diastolic blood pressure (adjusted for age and blood pressure on the normal diet) with similar changes in urinary sodium excretion. With sodium restriction, plasma renin activity rose from 0.65 to 3.03 ng. mL-<em>1</em>. h-<em>1</em> in whites, whereas in blacks it rose only from 0.3 to <em>1</em>.28 ng. mL-<em>1</em>. h-<em>1</em> (P<0.00<em>1</em> between blacks and whites). From the high to the low salt diet, plasma <em>angiotensin</em> II increased by 3<em>1</em> pmol/L in whites and by <em>1</em>2 pmol/L in blacks (P<0.05 compared with whites), and plasma aldosterone rose by 499 pmol/L in whites and by 256 pmol/L in blacks (P<0.0<em>1</em>). Significant inverse correlations were obtained for all patients between the fall in systolic blood pressure from the high to low salt diet and the rise in plasma renin activity and <em>angiotensin</em> II, as well as the absolute level on the low salt diet. These results demonstrate that the larger fall in blood pressure with a reduction in salt intake in blacks is due at least in part to a less responsive renin-<em>angiotensin</em>-aldosterone system in blacks.

The role of <em>angiotensin</em>-converting enzyme inhibitors in the management of cardiomyopathy related to Duchenne muscular dystrophy has not been completely defined. The purposes of this study were to describe the response to enalapril and its relation to dystrophin mutation type, ventricular size, or age at the onset of left ventricular (LV) systolic dysfunction. Serial clinical and echocardiographic data from 50 patients with Duchenne muscular dystrophy (aged <em>1</em>0 to 20 years) were retrospectively reviewed. Twenty-seven patients (46%) developed LV systolic dysfunction (mean age <em>1</em>3.2 +/- 2.4 years). Ten (43%) responded to enalapril with the normalization of function. Responders and nonresponders developed LV systolic dysfunction at similar ages (p = 0.9<em>1</em>). At the onset of LV systolic dysfunction, only 2 patients (<em>1</em> responder, <em>1</em> nonresponder) had dilated left ventricles. The positive response to enalapril was sustained in <em>7</em> patients (median follow-up 23 months, range 5 to 58). No specific mutation was associated with the response to enalapril (p = 0.66) or predictive of the development of LV systolic dysfunction (p = 0.8). In conclusion, <em>1</em>0 of 26 patients (43%) with Duchenne muscular dystrophy responded to the use of enalapril with normalization of the shortening fraction. Age at the onset of LV systolic dysfunction and the type of mutation were not predictors of response to enalapril.

OBJECTIVE

Toll-like receptor 2 (TLR2) is an important player in innate immunity, and recent studies have identified TLR2 as a critical mediator in cardiovascular diseases. Here, we investigated the involvement of TLR2 in angiotensin (Ang) II-induced cardiac fibrosis and the underlying mechanisms.

RESULTS

TLR2 knockout (TLR2 KO) mice (B6.129-Tlr2(tm1Kir)/) or wild-type (WT) mice (C57BL/6) treated with neutralizing anti-TLR2 antibody (T2.5) were used. The expression of TLR2 mRNA and protein in the heart was significantly up-regulated on days 1, 3, and 7 after Ang II infusion (1500 ng/kg/min). Enhanced expression of TLR2 was mainly detected in macrophages and neutrophils that had infiltrated into the heart. Both knockout of TLR2 and inhibition of TLR2 by neutralizing antibody ameliorated cardiac fibrosis induced by Ang II. This improvement was associated with a reduction in the infiltration of inflammatory cells, especially macrophages, the production of inflammatory cytokines, chemokines, and the activation of nuclear factor-κB. Bone marrow transplantation experiments between WT and TLR2 KO mice revealed that Ang II-induced cardiac fibrosis is mainly mediated by bone marrow-derived inflammatory cells. Mechanically, the deficiency of TLR2 inhibits macrophage-dependent cardiac fibroblast activation through TGFβ/Smad2/3 pathway.

CONCLUSIONS

Inhibition of TLR2 protects against Ang II-induced cardiac fibrosis by attenuating macrophage recruitment and the inflammatory response in the heart and may be a novel potential therapeutic target for hypertensive heart disease.

OBJECTIVE

This study estimated the cost-effectiveness,from the Dutch health care perspective, of screening for albuminuria in the general Dutch population to prevent cardiovascular events (CVEs) with subsequent angiotensin-converting enzyme inhibitor treatment, using data from the Prevention of REnal and Vascular ENdstage Disease Intervention Trial (PREVEND IT).

METHODS

PREVEND IT was a single-center, double-blind, randomized, placebo-controlled trial with a 2 x 2 factorial design within the larger observational Prevention of REnal and Vascular ENdstage Disease (PREVEND) study. The PREVEND IT study was conducted to assess the effects of fosinopril 20 mg and pravastatin 40 mg on CVEs in subjects with specific inclusion criteria: urinary albumin excretion (UAE) rate in the range from 15 to 300 mg/d, blood pressure <160/100 mm Hg, and plasma cholesterol level <8.0 mmol/L. Cost-effectiveness estimates for the Dutch population were expressed in euros (2002; 1 euro = US 1.01 dollars) as net costs per life-year gained (LYG) in the baseline and sensitivity (stochastic) analyses.

RESULTS

Data were assessed for 864 subjects, with a mean (SD) follow-up of 46 (7) months. CVEs occurred in 45 (5.2%) subjects. Subjects who received fosinopril had a 40% lower incidence of CVEs than subjects in the placebo group (3.9% vs 6.5%, respectively; P = NS). The cost-effectiveness of screening for albumnuria was determined to be euro 16,700/LYG for the study population. Stochastic analysis indicated that the probability of the cost-effectiveness being below the suggested Dutch threshold of euro 20,000/LYG was 59% in the baseline analysis. The probability of cost-effectiveness below euro 20,000/LYG would increase to 91% if only subjects with UAE >50 mg/d were treated with fosinopril. Limiting the screening to subjects aged >50 years and >60 years also improved cost-effectiveness.

CONCLUSIONS

The results of our study suggest that screening the general Dutch population for albuminuria and subsequently treating those found positive with fosinopril may be cost-effective compared with no screening and adopting the Dutch health care perspective. However, confirmation from larger multicenter trials is needed.

<em>1</em>. Renin was purified 30 000-fold from rat kidneys by chromatography on DEAE-cellulose and SP-Sephadex, and by affinity chromatography on pepstatinyl-Sepharose. 2. The enzymatic properties of isorenin from rat brain, pseudorenin from hog spleen, cathepsin D from bovine spleen, and renin from rat kidneys were compared: Isorenin, pseudorenin and cathepsin D generate <em>angiotensin</em> from tetradecapeptide renin substrate with pH optima around 4.9, renin at 6.0. With sheep <em>angiotensin</em>ogen as substrate, isorenin, pseudorenin and cathepsin D have similar pH profiles (pH optima at 3.9 and 5.5), in contrast to renin (pH optimum at 6.8). 3. The <em>angiotensin</em>-formation from tetradecapeptide by isorenin, pseudorenin and cathepsin D was inhibited by albumin, alpha-and beta-globulins. These 3 enzymes have acid protease activity at pH 3.2 with hemoglobin as the substrate. Renin is not inhibited by proteins and has no acid protease activity. 4. Renin generates <em>angiotensin</em> I from various <em>angiotensin</em>ogens at least <em>1</em>00 000 times faster than isorenin, pseudorenin or cathepsin D, and 3000 000 times faster than isorenin when compared at pH <em>7</em>.2 with rat <em>angiotensin</em>ogen as substrate. 5. The 3 'non-renin' enzymes exhibit a high sensitivity to inhibition by pepstatin (Ki less than 5.<em>1</em>0(-<em>1</em>0) M), in contrast to renin (Ki approximately 6-<em>1</em>0(-<em>7</em>) M), at pH 5.5. 6. It is concluded from the data that isorenin from rat brain and pseudorenin from hog spleen are closely related to, or identical with cathepsin D.

<em>1</em>. CHO-K<em>1</em> cells that were stably transfected with the gene for the human AT<em>1</em> receptor (CHO-AT<em>1</em> cells) were used for pharmacological studies of non-peptide AT<em>1</em> receptor antagonists. 2. In the presence of <em>1</em>0 mM LiCl, <em>angiotensin</em> II caused a concentration-dependent and long-lasting increase of inositol phosphates accumulation with an EC50 of 3.4 nM. No <em>angiotensin</em> II responses are seen in wild-type CHO-K<em>1</em> cells. 3. [3H]-<em>Angiotensin</em> II bound to cell surface AT<em>1</em> receptors (dissociates under mild acidic conditions) and is subject to rapid internalization. 4. Non-peptide selective AT<em>1</em> antagonists inhibited the <em>angiotensin</em> II (0.<em>1</em> microM) induced IP accumulation and the binding of [3H]-<em>angiotensin</em> II (<em>1</em> nM) with the potency order: candesartan>> EXP3<em>1</em><em>7</em>4>> irbesartan>> losartan. Their potencies are lower in the presence of bovine serum albumin. 5. Preincubation with the insurmountable antagonist candesartan decreased the maximal <em>angiotensin</em> II induced inositol phosphate accumulation up to 94% and, concomitantly, decreased the maximal binding capacity of the cell surface receptors. These inhibitory effects were half-maximal for 0.6 nM candesartan and were attenuated by simultaneous preincubation with <em>1</em> microM losartan indicating a syntopic action of both antagonists. 6. Losartan caused a parallel rightward shift of the <em>angiotensin</em> II concentration-response curves and did not affect the maximal binding capacity. EXP3<em>1</em><em>7</em>4 (the active metabolite of losartan) and irbesartan showed a mixed-type behavior in both functional and binding studies. <em>7</em>. Reversal of the inhibitory effect was slower for candesartan as compared with EXP3<em>1</em><em>7</em>4 and irbesartan and it was almost instantaneous for losartan, suggesting that the insurmountable nature of selective AT<em>1</em> receptor antagonists in functional studies was related to their long-lasting inhibition.