<em>Angiotensin</em> (Ang)-converting enzyme 2 (ACE2) cleaves Ang II to form Ang-(<em>1</em>-<em>7</em>). Here we examined whether soluble human recombinant ACE2 (rACE2) can efficiently lower Ang II and increase Ang-(<em>1</em>-<em>7</em>) and whether rACE2 can prevent hypertension caused by Ang II infusion as a result of systemic versus local mechanisms of ACE2 activity amplification. rACE2 was infused via osmotic minipumps for 3 days in conscious mice or acutely in anesthetized mice. rACE2 caused a dose-dependent increase in serum ACE2 activity but had no effect on kidney or cardiac ACE2 activity. After Ang II infusion (40 pmol/min), rACE2 (<em>1</em> mg/kg per day) resulted in normalization of systolic blood pressure and plasma Ang II. In acute studies, rACE2 (<em>1</em> mg/kg) prevented the rapid hypertensive effect of Ang II (0.2 mg/kg), and this was associated with both a decrease in Ang II and an increase in Ang-(<em>1</em>-<em>7</em>) in plasma. Moreover, during infusion of Ang II, the effect of rACE2 on blood pressure was unaffected by a specific Ang-(<em>1</em>-<em>7</em>) receptor blocker, A<em>7</em><em>7</em>9 (0.2 mg/kg), and infusing supraphysiologic levels of Ang-(<em>1</em>-<em>7</em>) (0.2 mg/kg) had no effect on blood pressure. We conclude that, during Ang II infusion, rACE2 effectively degrades Ang II and, in the process, normalizes blood pressure. The mechanism of rACE2 action results from an increase in systemic, not tissue, ACE2 activity and the lowering of plasma Ang II rather than the attendant increase in Ang-(<em>1</em>-<em>7</em>). Increasing ACE2 activity may provide a new therapeutic target in states of Ang II overactivity by enhancing its degradation, an approach that differs from the current focus on blocking Ang II formation and action.

We have used homologous recombination to disrupt the mouse gene coding for the NaK2Cl cotransporter (NKCC2) expressed in kidney epithelial cells of the thick ascending limb and macula densa. This gene is one of several that when mutated causes Bartter's syndrome in humans, a syndrome characterized by severe polyuria and electrolyte imbalance. Homozygous NKCC2-/- pups were born in expected numbers and appeared normal. However, by day <em>1</em> they showed signs of extracellular volume depletion (hematocrit 5<em>1</em>%; wild type 3<em>7</em>%). They subsequently failed to thrive. By day <em>7</em>, they were small and markedly dehydrated and exhibited renal insufficiency, high plasma potassium, metabolic acidosis, hydronephrosis of varying severity, and high plasma renin concentrations. None survived to weaning. Treatment of -/- pups with indomethacin from day <em>1</em> prevented growth retardation and <em>1</em>0% treated for 3 weeks survived, although as adults they exhibited severe polyuria (<em>1</em>0 ml/day), extreme hydronephrosis, low plasma potassium, high blood pH, hypercalciuria, and proteinuria. Wild-type mice treated with furosemide, an inhibitor of NaK2Cl cotransporters, have a phenotype similar to the indomethacin-rescued -/- adults except that hydronephrosis was mild. The polyuria, hypercalciuria, and proteinuria of the -/- adults and furosemide-treated wild-type mice were unresponsive to inhibitors of the renin <em>angiotensin</em> system, vasopressin, and further indomethacin. Thus absence of NKCC2 in the mouse causes polyuria that is not compensated elsewhere in the nephron. The NKCC2 mutant animals should be valuable for uncovering new pathophysiologic and therapeutic aspects of genetic disturbances in water and electrolyte recovery by the kidney.

<em>Angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>), acting through the Mas receptor, opposes the actions of Ang II. Molecular mechanisms for this are unclear. Here we sought to determine whether Ang-(<em>1</em>-<em>7</em>) influences Ang II signaling in human endothelial cells, focusing specifically on Src homology 2-containing inositol phosphatase 2 (SHP-2) and its interaction with c-Src. Ang II-induced phosphorylation of c-Src, extracellular signal regulated kinase (ERK)<em>1</em>/2, and SHP-2 and activation of NAD(P)H oxidase were assessed in the absence and presence of Ang-(<em>1</em>-<em>7</em>) (<em>1</em>0(-6) mol/L, <em>1</em>5 minutes) by immunoblotting and lucigenin-enhanced chemiluminescence, respectively. (D-Ala(<em>7</em>))-Ang I/II (<em>1</em>-<em>7</em>) (Ang fragment <em>1</em>-<em>7</em> receptor antagonist) was used to block Ang-(<em>1</em>-<em>7</em>) effects. Association between SHP-2 and c-Src was assessed by immunoprecipitation/immunoblotting studies. Ang II significantly increased activation of c-Src, ERK<em>1</em>/2, and NAD(P)H oxidase and reduced phosphorylation of SHP-2 (P<0.05) in human endothelial cells. These effects were abrogated in cells pre-exposed to Ang-(<em>1</em>-<em>7</em>). Ang fragment <em>1</em>-<em>7</em> receptor antagonist pretreatment blocked the negative modulatory actions of Ang-(<em>1</em>-<em>7</em>) on Ang II-induced signaling. Ang-(<em>1</em>-<em>7</em>) alone did not significantly alter phosphorylation of c-Src, ERK<em>1</em>/2, and SHP-2 and had no effect on basal activity of NAD(P)H oxidase. SHP-2 and c-Src were physically associated in the basal state. This association was increased by Ang-(<em>1</em>-<em>7</em>) and blocked by Ang fragment <em>1</em>-<em>7</em> receptor antagonist. Our findings demonstrate that, in human endothelial cells, Ang-(<em>1</em>-<em>7</em>) negatively modulates Ang II/Ang II type <em>1</em> receptor-activated c-Src and its downstream targets ERK<em>1</em>/2 and NAD(P)H oxidase. We also show that SHP-2-c-Src interaction is enhanced by Ang-(<em>1</em>-<em>7</em>). These phenomena may represent a protective mechanism in the endothelium whereby potentially deleterious effects of Ang II are counterregulated by Ang-(<em>1</em>-<em>7</em>).

Sex has an important influence on blood pressure (BP) regulation. There is increasing evidence that sex hormones interfere with the renin-<em>angiotensin</em> system. Thus the purpose of this study was to determine whether there are sex differences in the development of ANG II-induced hypertension in conscious male and female mice. We used telemetry implants to measure aortic BP and heart rate (HR) in conscious, freely moving animals. ANG II (800 ng.kg(-<em>1</em>).min(-<em>1</em>)) was delivered via an osmotic pump implanted subcutaneously. Our results showed baseline BP in male and female mice to be similar. Chronic systemic infusion of ANG II induced a greater increase in BP in male (35.<em>1</em> +/- 5.<em>7</em> mmHg) than in female mice (<em>7</em>.2 +/- 2.0 mmHg). Gonadectomy attenuated ANG II-induced hypertension in male mice (<em>1</em>5.2 +/- 2.4 mmHg) and augmented it in female mice (23.<em>1</em> +/- <em>1</em>.0 mmHg). Baseline HR was significantly higher in females relative to males (630.<em>1</em> +/- <em>7</em>.9 vs. 544.8 +/- <em>1</em>6.2 beats/min). In females, ANG II infusion significantly decreased HR. However, the increase in BP with ANG II did not result in the expected decrease in HR in either intact male or gonadectomized mice. Moreover, the slope of the baroreflex bradycardia to phenylephrine was blunted in males (-5.6 +/- 0.3 to -2.9 +/- 0.5) but not in females (-6.5 +/- 0.5 to -5.6 +/- 0.3) during infusion of ANG II, suggesting that, in male mice, infusion of ANG II results in a resetting of the baroreflex control of HR. Ganglionic blockade resulted in greater reduction in BP on day <em>7</em> after ANG II infusion in males compared with females (-6<em>1</em>.0 +/- 8.9 vs. -36.6 +/- 6.6 mmHg), suggesting an increased contribution of sympathetic nerve activity in arterial BP maintenance in male mice. Together, these data indicate that there are sex differences in the development of chronic ANG II-induced hypertension in conscious mice and that females may be protected from the increases in BP induced by ANG II.

Little information exists on the effects of transjugular intrahepatic portosystemic shunts (TIPS) in the management of cirrhotic patients with hepatorenal syndrome (HRS). The current study was aimed to prospectively evaluate the effects of TIPS on renal function and vasoactive systems in patients with type I HRS. Glomerular filtration rate (GFR) (inulin clearance), renal plasma flow (RPF) (para-aminohippurate clearance), plasma renin activity (PRA), aldosterone (ALDO), norepinephrine (NE), and endothelin (ET) were determined in baseline conditions and at different time intervals after TIPS in <em>7</em> patients with type I HRS. TIPS induced a marked reduction of portal pressure gradient (PPG) (20 +/- <em>1</em> to <em>1</em>0 +/- <em>1</em> mm Hg; P < .05). Renal function improved in 6 of the <em>7</em> patients. Serum creatinine and blood urea nitrogen (BUN) decreased from 5 +/- 0.8 and <em>1</em>09 +/- <em>7</em> to <em>1</em>.8 +/- 0.4 mg/dL and 56 +/- <em>1</em><em>1</em> mg/dL, respectively (P < .05 for both), and GFR and RPF increased from 9 +/- 4 and <em>1</em>03 +/- 33 to 2<em>7</em> +/- <em>7</em> mL/min and 233 +/- 40 mL/min, respectively (P < .05 for both), 30 days after TIPS. These beneficial effects on renal function were associated with a significant (P < .05) reduction of PRA (<em>1</em>8 +/- 5 to 3 +/- <em>1</em> ng/mL x h), ALDO (2<em>7</em>9 +/- 58 to 99 +/- 56 ng/dL), and NE (<em>1</em>,25<em>7</em> +/- <em>1</em>8<em>7</em> to 6<em>1</em>2 +/- <em>1</em>9<em>7</em> pg/mL). ET did not change significantly (28 +/- 8 to 2<em>7</em> +/- <em>1</em><em>1</em> pg/mL). Mean survival was 4.<em>7</em> +/- 2 months (0.3-<em>1</em><em>7</em> months). Three patients remained alive more than 3 months after TIPS insertion. In conclusion, TIPS improves renal function and reduces the activity of the renin-<em>angiotensin</em> and sympathetic nervous systems in cirrhotic patients with type I HRS. Nevertheless, the efficacy of TIPS in the management of these patients should be confirmed in controlled investigations.

The antihypertensive effect of the orally active <em>angiotensin</em>-converting enzyme inhibitor captopril (SQ <em>1</em>4225) was assessed in 22 hypertensive patients of whom <em>1</em><em>7</em> were followed for periods ranging from <em>1</em> to <em>7</em> months. Of these, eight had essential hypertension, eight had renovascular hypertension, and six had hypertension associated with chronic renal failure. Blood pressure decreased markedly in all patients, including those with low renin levels. Nevertheless, the magnitude of blood pressure reduction correlated with the base-line plasma renin activity (r = 0.58, P less than 0.0<em>1</em>). Increasing the dose of captopril from 25 to 200 mg did not enhance the amplitude of the antihypertensive effect but did increase its duration. Patients' blood pressure remained well controlled and free of side-effects with a maximal daily dose of up to 200 mg by mouth twice daily. Despite the blood pressure reduction, sodium excretion tended to increase, probably because of reduced aldosterone secretion. There was no evidence of orthostatic hypotension, and no escape from the antihypertensive effect was observed. These results indicate that chronic inhibition of the <em>angiotensin</em>-converting enzyme with an orally active compound offers a new, efficient, and well-tolerated approach to the treatment of hypertension.

BACKGROUND

Myocardial damage in myocarditis is mediated, in part, by immunological mechanisms. High-dose intravenous gamma-globulin (IVIG) is an immunomodulatory agent that is beneficial in myocarditis secondary to Kawasaki disease, as well as in murine myocarditis. Since <em>1</em>990, the routine management of presumed acute myocarditis at Children's Hospital, Boston, and Children's Hospital, Los Angeles, has included administration of high-dose IVIG.

RESULTS

We treated 2<em>1</em> consecutive children presenting with presumed acute myocarditis with IVIG, 2 g/kg, over 24 hours, in addition to anticongestive therapies. A comparison group comprised 25 recent historical control patients meeting identical eligibility criteria but not receiving IVIG therapy. Left ventricular function was assessed during five time intervals: 0 to <em>7</em> days, <em>1</em> to 3 weeks, 3 weeks to 3 months, 3 to 6 months, and 6 to <em>1</em>2 months. At presentation, the IVIG and non-IVIG groups had comparable left ventricular enlargement and poor fractional shortening. Compared with the non-IVIG group, those treated with IVIG had a smaller mean adjusted left ventricular end-diastolic dimension and higher fractional shortening in the periods from 3 to 6 months (P = .008 and P = .033, respectively) and 6 to <em>1</em>2 months (P = .0<em>7</em>2 and P = .029, respectively). When adjusting for age, biopsy status, intravenous inotropic agents, and <em>angiotensin</em>-converting enzyme inhibitors, patients treated with IVIG were more likely to achieve normal left ventricular function during the first year after presentation (P = .03). By <em>1</em> year after presentation, the probability of survival tended to be higher among IVIG-treated patients (.84 versus .60, P = .069). We observed no adverse effects of IVIG administration.

CONCLUSIONS

These data suggest that use of high-dose IVIG for treatment of acute myocarditis is associated with improved recovery of left ventricular function and with a tendency to better survival during the first year after presentation.

<em>Angiotensin</em> II (Ang II) plays an important role in regulating cardiovascular hemodynamics and structure. Multiple lines of evidence have suggested the existence of Ang II receptor subtypes, and at least 2 distinct receptor subtypes have been defined on the basis of their differential pharmacological and biochemical properties and designated as type <em>1</em> (AT<em>1</em>) and type 2 (AT2) receptors. To date, most of the known effects of Ang II in adult tissues are attributable to the AT<em>1</em> receptor. Recent cloning of the AT2 receptor contributes to reveal its physiological functions, but many functions of the AT2 receptor are still an enigma. AT<em>1</em> and AT2 receptors belong to the <em>7</em>-transmembrane, G protein-coupled receptor family. However, accumulating evidence demonstrates that the function and signaling mechanisms of these receptor subtypes are quite different, and these receptors may exert opposite effects in terms of cell growth and blood pressure regulation. We will review the role of the AT2 receptor in the cardiovascular system and the molecular and cellular mechanisms of AT2 receptor action.

This lecture summarizes the chronology and rationale that led to the discovery of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) as a hormone that, in its own right, opposes the vasoconstrictor and proliferative actions of <em>angiotensin</em> II. The work discussed here additionally analyzes the newest findings on <em>angiotensin</em>-converting enzyme 2, the <em>angiotensin</em>-converting enzyme homologue that efficiently hydrolyzes <em>angiotensin</em> II into <em>angiotensin</em>-(<em>1</em>-<em>7</em>). Both components of this system may significantly influence our future perspective of the role of the renin-<em>angiotensin</em> system, not just in terms of its role in the regulation of cardiovascular and renal function but, moreover, as regulators of a vast array of disease processes in which inflammation and immune mechanisms play a role.

BACKGROUND

We have previously shown that a specific combination of drug therapy and left ventricular assist device unloading results in significant myocardial recovery, sufficient to allow pump removal, in two thirds of patients with dilated cardiomyopathy receiving a Heartmate I pulsatile device. However, this protocol has not been used with nonpulsatile devices.

RESULTS

We report the results of a prospective study of 20 patients who received a combination of <em>angiotensin</em>-converting enzymes, β-blockers, <em>angiotensin</em> II inhibitors, and aldosterone antagonists followed by the β₂-agonist clenbuterol and were regularly tested (echocardiograms, exercise tests, catheterizations) with the pump at low speed. Before left ventricular assist device insertion, patient age was 35.2 ± <em>1</em>2.6 years (<em>1</em>6 male patients), patients were on 2.0 ± 0.9 inotropes, <em>7</em> (35) had an intra-aortic balloon pump, 2 were hemofiltered, 2 were ventilated, 3 had a prior Levitronix device, and <em>1</em> had extracorporeal membrane oxygenation. Cardiac index was <em>1</em>.39 ± 0.43 L · min⁻¹ · m⁻², pulmonary capillary wedge pressure was 3<em>1</em>.5 ± 5.<em>7</em> mm Hg, and heart failure history was 3.4 ± 3.5 years. One patient was lost to follow-up and died after 240 days of support. Of the remaining <em>1</em>9 patients, <em>1</em>2 (63.2) were explanted after 286 ± 9<em>7</em> days. Eight had symptomatic heart failure for ≤6 months and 4 for >6 months (48 to <em>1</em>32 months). Before explantation, at low flow for <em>1</em>5 minutes, ejection fraction was <em>7</em>0 ± <em>7</em>, left ventricular end-diastolic diameter was 48.6 ± 5.<em>7</em> mm, left ventricular end-systolic diameter was 32.3 ± 5.<em>7</em> mm, mV(O₂) was 2<em>1</em>.6 ± 4 mL · kg⁻¹ · min⁻¹, pulmonary capillary wedge pressure was 5.9 ± 4.6 mm Hg, and cardiac index was 3.6 ± 0.6 L · min⁻¹ · m⁻². Estimated survival without heart failure recurrence was 83.3 at <em>1</em> and 3 years. After a 430.<em>7</em> ± 33<em>7</em>.<em>1</em>-day follow-up, surviving explants had an ejection fraction of 58.<em>1</em> ± <em>1</em>3.8, left ventricular end-diastolic diameter of 59.0 ± 9.3 mm, left ventricular end-systolic diameter of 42.0 ± <em>1</em>0.<em>7</em> mm, and mV(O₂) of 22.6 ± 5.3 mL · kg⁻¹ · min⁻¹.

CONCLUSIONS

Reversal of end-stage heart failure secondary to nonischemic cardiomyopathy can be achieved in a substantial proportion of patients with nonpulsatile flow through the use of a combination of mechanical and pharmacological therapy.

<em>1</em>. There are two <em>Angiotensin</em> II systems in the brain. The discovery of brain <em>Angiotensin</em> II receptors located in neurons inside the blood brain barrier confirmed the existence of an endogenous brain <em>Angiotensin</em> II system, responding to <em>Angiotensin</em> II generated in and/or transported into the brain. In addition, <em>Angiotensin</em> II receptors in circumventricular organs and in cerebrovascular endothelial cells respond to circulating <em>Angiotensin</em> II of peripheral origin. Thus, the brain responds to both circulating and tissue <em>Angiotensin</em> II, and the two systems are integrated. 2. The neuroanatomical location of <em>Angiotensin</em> II receptors and the regulation of the receptor number are most important to determine the level of activation of the brain <em>Angiotensin</em> II systems. 3. Classical, well-defined actions of <em>Angiotensin</em> II in the brain include the regulation of hormone formation and release, the control of the central and peripheral sympathoadrenal systems, and the regulation of water and sodium intake. As a consequence of changes in the hormone, sympathetic and electrolyte systems, feed back mechanisms in turn modulate the activity of the brain <em>Angiotensin</em> II systems. It is reasonable to hypothesize that brain <em>Angiotensin</em> II is involved in the regulation of multiple additional functions in the brain, including brain development, neuronal migration, process of sensory information, cognition, regulation of emotional responses, and cerebral blood flow. 4. Many of the classical and of the hypothetical functions of brain <em>Angiotensin</em> II are mediated by stimulation of <em>Angiotensin</em> II AT<em>1</em> receptors. 5. Brain AT2 receptors are highly expressed during development. In the adult, AT2 receptors are restricted to areas predominantly involved in the process of sensory information. However, the role of AT2 receptors remains to be clarified. 6. Subcutaneous or oral administration of a selective and potent non-peptidic AT<em>1</em> receptor antagonist with very low affinity for AT2 receptors and good bioavailability blocked AT<em>1</em> receptors not only outside but also inside the blood brain barrier. The blockade of the complete brain <em>Angiotensin</em> II AT<em>1</em> system allowed us to further clarify some of the central actions of the peptide and suggested some new potential therapeutic avenues for this class of compounds. <em>7</em>. Pretreatment with peripherally administered AT<em>1</em> antagonists completely prevented the hormonal and sympathoadrenal response to isolation stress. A similar pretreatment prevented the development of stress-induced gastric ulcers. These findings strongly suggest that blockade of brain AT<em>1</em> receptors could be considered as a novel therapeutic approach in the treatment of stress-related disorders. 8. Peripheral administration of AT<em>1</em> receptor antagonists strongly affected brain circulation and normalized some of the profound alterations in cerebrovascular structure and function characteristic of chronic genetic hypertension. AT<em>1</em> receptor antagonists were capable of reversing the pathological cerebrovascular remodeling in hypertension and the shift to the right in the cerebral autoregulation, normalizing cerebrovascular compliance. In addition, AT<em>1</em> receptor antagonists normalized the expression of cerebrovascular nitric oxide synthase isoenzymes and reversed the inflammatory reaction characteristic of cerebral vessels in hypertension. As a consequence of the normalization of cerebrovascular compliance and the prevention of inflammation, there was, in genetically hypertensive rats a decreased vulnerability to brain ischemia. After pretreatment with AT<em>1</em> antagonists, there was a protection of cerebrovascular flow during experimental stroke, decreased neuronal death, and a substantial reduction in the size of infarct after occlusion of the middle cerebral artery. At least part of the protective effect of AT<em>1</em> receptor antagonists was related to the inhibition of the <em>Angiotensin</em> II system, and not to the normalization of blood pressure. These results indicate that treatment with AT<em>1</em> receptor antagonists appears to be a major therapeutic avenue for the prevention of ischemia and inflammatory diseases of the brain. 9. Thus, orally administered AT<em>1</em> receptor antagonists may be considered as novel therapeutic compounds for the treatment of diseases of the central nervous system when stress, inflammation and ischemia play major roles. <em>1</em>0. Many questions remain. How is brain <em>Angiotensin</em> II formed, metabolized, and distributed? What is the role of brain AT2 receptors? What are the molecular mechanisms involved in the cerebrovascular remodeling and inflammation which are promoted by AT<em>1</em> receptor stimulation? How does <em>Angiotensin</em> II regulate the stress response at higher brain centers? Does the degree of activity of the brain <em>Angiotensin</em> II system predict vulnerability to stress and brain ischemia? We look forward to further studies in this exiting and expanding field.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts^<em>1</em>-3, most targeting the viral 'spike' glycoprotein (S). S localizes on the virion surface and mediates recognition of cellular receptor <em>angiotensin</em>-converting enzyme 2 (ACE2)^4-6. Eliciting neutralizing antibodies that block S-ACE2 interaction^<em>7</em>-9, or indirectly prevent membrane fusion^<em>1</em>0, constitute an attractive modality for vaccine-elicited protection^{<em>1</em><em>1</em>}. However, although prototypic S-based vaccines show promise in animal models^{<em>1</em>2-<em>1</em>4}, the immunogenic properties of S in humans are poorly resolved. In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9). We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity. Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited. Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic. Overall, although patients who recovered from COVID-<em>1</em>9 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes.

The mRen2.Lewis congenic strain is an estrogen-sensitive model of hypertension whereby estrogen depletion produces a significant and sustained increase in blood pressure. The recent identification of G protein-coupled receptor 30 (GPR30) as a third estrogen receptor isotype prompted us to test the hypothesis that this novel receptor exhibits beneficial cardiovascular actions in the hypertensive female mRen2.Lewis rat. Intact female, ovariectomized female (OVX), and male mRen2.Lewis rats were treated with the selective GPR30 agonist G-<em>1</em> or vehicle via osmotic minipump for 2 wk. G-<em>1</em> significantly reduced systolic blood pressure in OVX (<em>1</em><em>7</em>8 +/- <em>7</em> to <em>1</em>42 +/- <em>1</em>0 mm Hg, P < 0.00<em>1</em>, n = 8) but not intact female (<em>1</em>44 +/- 3 to <em>1</em>43 +/- 5 mm Hg, P>> 0.05, n = 5) or male mRen2.Lewis rats (20<em>7</em> +/- <em>7</em> to <em>1</em>92 +/- 5 mm Hg, P>> 0.05, n = <em>7</em>). G-<em>1</em> did not alter uterine or body weight in OVX, suggesting activation of a receptor distinct from estrogen receptor-alpha and -beta. In isolated aortic rings from OVX, G-<em>1</em> reduced constriction in response to <em>angiotensin</em> II. Vascular <em>angiotensin</em>-converting enzyme and <em>angiotensin</em> type <em>1</em> receptor mRNA were also lower, whereas <em>angiotensin</em>-converting enzyme-2 mRNA was increased. G-<em>1</em> treatment in OVX was not associated with alterations in either endothelial nitric oxide synthase expression or acetylcholine-induced relaxation. Immunohistochemical staining for GPR30 was evident in both the intima and media of the aorta. We conclude that the novel estrogen receptor GPR30 may contribute to the beneficial cardiovascular actions of estrogen in female mRen2.Lewis rats through regulation of vascular components of the renin-<em>angiotensin</em> system.

The renin-<em>angiotensin</em> system is a major physiological regulator of arterial pressure and hydro-electrolyte balance. Evidence has now been accumulated that in addition to <em>angiotensin</em> (Ang) II other Ang peptides [Ang III, Ang IV and Ang-(<em>1</em>-<em>7</em>)], formed in the limited proteolysis processing of <em>angiotensin</em>ogen, are importantly involved in mediating several actions of the RAS. In this article we will review our knowledge of the biological actions of Ang-(<em>1</em>-<em>7</em>) with focus on the puzzling aspects of the mediation of its effects and the interaction Ang-(<em>1</em>-<em>7</em>)-kinins. In addition, we will attempt to summarize the evidence that Ang-(<em>1</em>-<em>7</em>) takes an important part of the mechanisms aimed to counteract the vasoconstrictor and proliferative effects of Ang II.

In this review, we examine the possibility that small increments in <em>angiotensin</em> II are responsible for an increase in blood pressure and maintenance of hypertension through the stimulation of oxidative stress. A low dose of <em>angiotensin</em> II (2 to <em>1</em>0 ng x kg(-<em>1</em>) x min(-<em>1</em>), which does not elicit an immediate pressor response), when given for <em>7</em> to 30 days by continuous intravenous infusion, can increase mean arterial pressure by 30 to 40 mm Hg. This slow pressor response to <em>angiotensin</em> is accompanied by the stimulation of oxidative stress, as measured by a significant increase in levels of 8-iso-prostaglandin F(2alpha) (F(2)-isoprostane). Superoxide radicals and nitric oxide can combine chemically to form peroxynitrite, which can then oxidize arachidonic acid to form F(2)-isoprostanes. F(2)-isoprostanes exert potent vasoconstrictor and antinatriuretic effects. Furthermore, <em>angiotensin</em> II can stimulate endothelin production, which also has been shown to stimulate oxidative stress. In this way, a reduction in the concentration of nitric oxide (which is quenched by superoxide) along with the formation of F(2)-isoprostanes and endothelin could potentiate the vasoconstrictor effects of <em>angiotensin</em> II. We hypothesize that these mechanisms, which underlie the development of the slow pressor response to <em>angiotensin</em> II, also participate in the production of hypertension when circulating <em>angiotensin</em> II levels appear normal, as occurs in many cases of essential and renovascular hypertension.

BACKGROUND

Chronic exercise (EX) improves the quality of life and increases the survival of patients with chronic heart failure (CHF). Because sympathetic nerve activity is elevated in the CHF state, it is possible that EX is beneficial in this disease due to a decrease in sympathetic outflow.

RESULTS

We evaluated arterial baroreflex function and resting renal sympathetic nerve activity (RSNA) in EX normal and CHF rabbits before and after <em>angiotensin</em> II type <em>1</em> (AT(<em>1</em>)) receptor blockade. Four groups of rabbits were studied: a normal non-EX group, a normal EX group, a CHF non-EX group, and a CHF EX group. EX lowered resting RSNA in rabbits with CHF but not in normal rabbits. In addition, EX increased arterial baroreflex sensitivity in the CHF group (heart rate slope: CHF <em>1</em>. <em>7</em>+/-0.3 bpm/mm Hg, EX CHF 4.9+/-0.3 bpm/mm Hg; P:<0.0<em>1</em>; RSNA slope: CHF 2.2+/-0.2%max/mm Hg, EX CHF 5.<em>7</em>+/-0.4%max/mm Hg; P:<0.0<em>1</em>. AT(<em>1</em>) receptor blockade enhanced baroreflex sensitivity in the non-EX CHF rabbits but had no effect in EX CHF rabbits. Concomitant with this effect, EX lowered the elevated plasma <em>angiotensin</em> II concentration in the CHF group. A significant positive correlation was observed between sympathetic nerve activity and plasma <em>angiotensin</em> II.

CONCLUSIONS

These data strongly suggest that EX reduces the sympathoexcitatory state in the setting of CHF. Enhanced arterial baroreflex sensitivity may contribute to this reduction. In addition, EX lowers plasma angiotensin II concentration in CHF. These data further suggest that the lowering of angiotensin II may contribute to the decrease in sympathetic nerve activity after EX in the CHF state.

ANG-(<em>1</em>-<em>7</em>) improves the function of the remodeling heart. Although this peptide is generated directly within the myocardium, the effects of ANG-(<em>1</em>-<em>7</em>) on cardiac fibroblasts that play a critical role in cardiac remodeling are largely unknown. We tested the hypothesis that specific binding of ANG-(<em>1</em>-<em>7</em>) to cardiac fibroblasts regulates cellular functions that are involved in cardiac remodeling. <em>1</em>25I-labeled ANG-(<em>1</em>-<em>7</em>) binding assays identified specific binding sites of ANG-(<em>1</em>-<em>7</em>) on adult rat cardiac fibroblasts (ARCFs) with an affinity of <em>1</em><em>1</em>.3 nM and a density of <em>1</em>3<em>1</em> fmol/mg protein. At nanomolar concentrations, ANG-(<em>1</em>-<em>7</em>) interacted with specific sites that were distinct from ANG II type <em>1</em> and type 2 receptors without increasing cytosolic Ca2+ concentration. At these concentrations, ANG-(<em>1</em>-<em>7</em>) had inhibitory effects on collagen synthesis as assessed by [3H]proline incorporation and decreased mRNA expression of growth factors in ARCFs. These effects of ANG-(<em>1</em>-<em>7</em>) contrasted with effects of ANG II. Pretreatment of ARCFs with ANG-(<em>1</em>-<em>7</em>) inhibited ANG II-induced increases in collagen synthesis and in mRNA expression of growth factors, including endothelin-<em>1</em> and leukemia inhibitory factor. ANG-(<em>1</em>-<em>7</em>) pretreatment also inhibited the stimulatory effects of conditioned medium from ANG II-treated ARCFs on [3H]leucine incorporation and atrial natriuretic factor mRNA expression, markers of hypertrophy, in cardiomyocytes. Thus ANG-(<em>1</em>-<em>7</em>) interacted with specific receptors on ARCFs to exert potential antifibrotic and antitrophic effects that could reverse ANG II effects. These results suggest that ANG-(<em>1</em>-<em>7</em>) may play an important role in the heart in regulating cardiac remodeling.

BACKGROUND

Angotensin converting enzyme 2 (ACE2) is a newly discovered monocarboxypeptidase that counteracts the vasoconstrictor effects of <em>angiotensin</em> II (Ang II) by converting Ang II to Ang-(<em>1</em>-<em>7</em>) in the kidney and other tissues.

METHODS

ACE2 activity from renal homogenates was investigated by using the fluorogenic peptide substrate Mca-YVADAPK(Dnp)-OH, where Mca is (<em>7</em>-methoxycoumarin-4-yl)-acetyl and Dnp is 2,4-dinitrophenyl.

RESULTS

We found that ACE2 activity expressed in relative fluorescence units (RFU) in the MF<em>1</em> mouse is higher in the male (M) compared to the female (F) kidney [ACE2 (RFU/min/μg protein): M <em>1</em>8.<em>1</em> ± <em>1</em>.0 versus F <em>1</em><em>1</em>.<em>1</em> ± 0.39; P < 0.000<em>1</em>; n = 6]. Substrate concentration curves revealed that the higher ACE2 activity in the male was due to increased ACE2 enzyme velocity (Vmax) rather than increased substrate affinity (Km). We used the four core genotypes mouse model in which gonadal sex (ovaries versus testes) is separated from the sex chromosome complement enabling comparisons among XX and XY gonadal females and XX and XY gonadal males. Renal ACE2 activity was greater in the male than the female kidney, regardless of the sex chromosome complement [ACE2 (RFU/min/μg protein): intact-XX-F, <em>7</em>.59 ± 0.3<em>7</em>; intact-XY-F, <em>7</em>.43 ± 0.53; intact-XX-M, <em>1</em>2.<em>1</em> ± 0.62; intact-XY-M, <em>1</em>2.<em>7</em> ± <em>1</em>.5; n = 4-6/group; P < 0.000<em>1</em>, F versus M, by two-way ANOVA]. Enzyme activity was increased in gonadectomized (GDX) female mice regardless of the sex chromosome complement whereas no effect of gonadectomy was observed in the males [ACE2 (RFU/min/μg protein): GDX-XX-F, <em>1</em>2.4 ± <em>1</em>.2; GDX-XY-F, <em>1</em><em>1</em>.<em>1</em> ± 0.<em>7</em>6; GDX-XX-M, <em>1</em>3.2 ± 0.9<em>7</em>; GDX-XY-M, <em>1</em><em>1</em>.6 ± 0.8<em>1</em>; n = 6/group]. <em>1</em><em>7</em>β-oestradiol (E2) treatment of GDX mice resulted in ACE2 activity that was only 40% of the activity found in the GDX mice, regardless of their being male or female, and was independent of the sex chromosome complement [ACE2 (RFU/min/μg protein): GDX+E2-XX-F, 5.56 ± <em>1</em>.0; GDX+E2-XY-F, 4.60 ± 0.52; GDX+E2-XX-M, 5.35 ± 0.<em>7</em>0; GDX+E2-XY-M, 5.<em>1</em>2 ± 0.4<em>7</em>; n = 6/group].

CONCLUSIONS

Our findings suggest sex differences in renal ACE2 activity in intact mice are due, at least in part, to the presence of E2 in the ovarian hormone milieu and not to the testicular milieu or to differences in sex chromosome dosage (2X versus <em>1</em>X; 0Y versus <em>1</em>Y). E2 regulation of renal ACE2 has particular implications for women across their life span since this hormone changes radically during puberty, pregnancy and menopause.

BACKGROUND

<em>Angiotensin</em> converting enzyme (ACE) 2 is a recently identified homologue of ACE that may counterregulate the actions of <em>angiotensin</em> (Ang) II by facilitating its breakdown to Ang <em>1</em>-<em>7</em>. The renin-<em>angiotensin</em> system (RAS) has been implicated in the pathogenesis of cirrhosis but the role of ACE2 in liver disease is not known.

OBJECTIVE

This study examined the effects of liver injury on ACE2 expression and activity in experimental hepatic fibrosis and human cirrhosis, and the effects of Ang <em>1</em>-<em>7</em> on vascular tone in cirrhotic rat aorta.

METHODS

In sham operated and bile duct ligated (BDL) rats, quantitative reverse transcriptase-polymerase chain reaction was used to assess hepatic ACE2 mRNA, and western blotting and immunohistochemistry to quantify and localise ACE2 protein. ACE2 activity was quantified by quenched fluorescent substrate assay. Similar studies were performed in normal human liver and in hepatitis C cirrhosis.

RESULTS

ACE2 mRNA was detectable at low levels in rat liver and increased following BDL (363-fold; p < 0.0<em>1</em>). ACE2 protein increased after BDL (23.5-fold; p < 0.05) as did ACE2 activity (fourfold; p < 0.05). In human cirrhotic liver, gene (>30-fold), protein expression (9<em>7</em>-fold), and activity of ACE2 (2.4 fold) were increased compared with controls (all p < 0.0<em>1</em>). In healthy livers, ACE2 was confined to endothelial cells, occasional bile ducts, and perivenular hepatocytes but in both BDL and human cirrhosis there was widespread parenchymal expression of ACE2 protein. Exposure of cultured human hepatocytes to hypoxia led to increased ACE2 expression. In preconstricted rat aorta, Ang <em>1</em>-<em>7</em> alone did not affect vascular tone but it significantly enhanced acetylcholine mediated vasodilatation in cirrhotic vessels.

CONCLUSIONS

ACE2 expression is significantly increased in liver injury in both humans and rat, possibly in response to increasing hepatocellular hypoxia, and may modulate RAS activity in cirrhosis.

Elevated levels of endogenous <em>angiotensin</em> can cause hypertensive nephrosclerosis as a result of the potent vasopressor action of the peptide. We have produced by gene targeting mice homozygous for a null mutation in the <em>angiotensin</em>ogen gene (Atg-<em>1</em>-). Postnatally, Atg-<em>1</em>- animals show a modest delay in glomerular maturation. Although Atg-<em>1</em>- animals are hypotensive by <em>7</em> wk of age, they develop, by 3 wk of age, pronounced lesions in the renal cortex, similar to those of hypertensive nephrosclerosis. In addition, the papillae of homozygous mutant kidneys are reduced in size. These lesions are accompanied by local up-regulation of PDGF-B and TGF-beta<em>1</em> mRNA in the cortex and down-regulation of PDGF-A mRNA in the papilla. The study demonstrates an important requirement for <em>angiotensin</em> in achieving and maintaining the normal morphology of the kidney. The mechanism through which <em>angiotensin</em> maintains the volume homeostasis in mammals includes promotion of the maturational growth of the papilla.

OBJECTIVE

Several clinical studies have shown the benefits of renin-<em>angiotensin</em> system (RAS) blockade in the development of diabetes, and a local RAS has been identified in pancreatic islets. <em>Angiotensin</em> I-converting enzyme (ACE)2, a new component of the RAS, has been identified in the pancreas, but its role in β-cell function remains unknown. Using 8- and <em>1</em>6-week-old obese db/db mice, we examined the ability of ACE2 to alter pancreatic β-cell function and thereby modulate hyperglycemia.

METHODS

Both db/db and nondiabetic lean control (db/m) mice were infected with an adenovirus expressing human ACE2 (Ad-hACE2-eGFP) or the control virus (Ad-eGFP) via injection into the pancreas. Glycemia and β-cell function were assessed <em>1</em> week later at the peak of viral expression.

RESULTS

In 8-week-old db/db mice, Ad-hACE2-eGFP significantly improved fasting glycemia, enhanced intraperitoneal glucose tolerance, increased islet insulin content and β-cell proliferation, and reduced β-cell apoptosis compared with Ad-eGFP. ACE2 overexpression had no effect on insulin sensitivity in comparison with Ad-eGFP treatment in diabetic mice. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) receptor blockade by D-Ala(<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) prevented the ACE2-mediated improvements in intraperitoneal glucose tolerance, glycemia, and islet function and also impaired insulin sensitivity in both Ad-hACE2-eGFP- and Ad-eGFP-treated db/db mice. D-Ala(<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) had no effect on db/m mice. In <em>1</em>6-week-old diabetic mice, Ad-hACE2-eGFP treatment improved fasting blood glucose but had no effect on any of the other parameters.

CONCLUSIONS

These findings identify ACE2 as a novel target for the prevention of β-cell dysfunction and apoptosis occurring in type 2 diabetes.

BACKGROUND

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a monocarboxypeptidase that metabolizes Ang II into Ang <em>1</em>-<em>7</em>, thereby functioning as a negative regulator of the renin-<em>angiotensin</em> system. We hypothesized that ACE2 deficiency may compromise the cardiac response to myocardial infarction (MI).

RESULTS

In response to MI (induced by left anterior descending artery ligation), there was a persistent increase in ACE2 protein in the infarct zone in wild-type mice, whereas loss of ACE2 enhanced the susceptibility to MI, with increased mortality, infarct expansion, and adverse ventricular remodeling characterized by ventricular dilation and systolic dysfunction. In ACE2-deficient hearts, elevated myocardial levels of Ang II and decreased levels of Ang <em>1</em>-<em>7</em> in the infarct-related zone was associated with increased production of reactive oxygen species. ACE2 deficiency leads to increased matrix metalloproteinase (MMP) 2 and MMP9 levels with MMP2 activation in the infarct and peri-infarct regions, as well as increased gelatinase activity leading to a disrupted extracellular matrix structure after MI. Loss of ACE2 also leads to increased neutrophilic infiltration in the infarct and peri-infarct regions, resulting in upregulation of inflammatory cytokines, interferon-gamma, interleukin-6, and the chemokine, monocyte chemoattractant protein-<em>1</em>, as well as increased phosphorylation of ERK<em>1</em>/2 and JNK<em>1</em>/2 signaling pathways. Treatment of Ace2(-)(/y)-MI mice with irbesartan, an AT<em>1</em> receptor blocker, reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, infarct size, MMP activation, and myocardial inflammation, ultimately resulting in improved post-MI ventricular function.

CONCLUSIONS

We conclude that loss of ACE2 facilitates adverse post-MI ventricular remodeling by potentiation of Ang II effects by means of the AT<em>1</em> receptors, and supplementing ACE2 can be a potential therapy for ischemic heart disease.

The renin-<em>angiotensin</em> system (RAS) is undisputedly one of the most prominent endocrine (tissue-to-tissue), paracrine (cell-to-cell) and intracrine (intracellular/nuclear) vasoactive systems in the physiological regulation of neural, cardiovascular, blood pressure, and kidney function. The importance of the RAS in the development and pathogenesis of cardiovascular, hypertensive and kidney diseases has now been firmly established in clinical trials and practice using renin inhibitors, <em>angiotensin</em>-converting enzyme (ACE) inhibitors, type <em>1</em> (AT<em>1</em>) <em>angiotensin</em> II (ANG II) receptor blockers (ARBs), or aldosterone receptor antagonists as major therapeutic drugs. The major mechanisms of actions for these RAS inhibitors or receptor blockers are mediated primarily by blocking the detrimental effects of the classic <em>angiotensin</em>ogen/renin/ACE/ANG II/AT<em>1</em>/aldosterone axis. However, the RAS has expanded from this classic axis to include several other complex biochemical and physiological axes, which are derived from the metabolism of this classic axis. Currently, at least five axes of the RAS have been described, with each having its key substrate, enzyme, effector peptide, receptor, and/or downstream signaling pathways. These include the classic <em>angiotensin</em>ogen/renin/ACE/ANG II/AT<em>1</em> receptor, the ANG II/APA/ANG III/AT2/NO/cGMP, the ANG I/ANG II/ACE2/ANG (<em>1</em>-<em>7</em>)/Mas receptor, the prorenin/renin/prorenin receptor (PRR or Atp6ap2)/MAP kinases ERK<em>1</em>/2/V-ATPase, and the ANG III/APN/ANG IV/IRAP/AT4 receptor axes. Since the roles and therapeutic implications of the classic <em>angiotensin</em>ogen/renin/ACE/ANG II/AT<em>1</em> receptor axis have been extensively reviewed, this article will focus primarily on reviewing the roles and therapeutic implications of the vasoprotective axes of the RAS in cardiovascular, hypertensive and kidney diseases.

Analogs of vitamin D attenuate renal injury in several models of kidney disease, but the mechanism underlying this renoprotective effect is unknown. To address the role of the vitamin D receptor (VDR) in renal fibrogenesis, we subjected VDR-null mice to unilateral ureteral obstruction for <em>7</em> days. Compared with wild-type mice, VDR-null mice developed more severe renal damage in the obstructed kidney, with marked tubular atrophy and interstitial fibrosis. Significant induction of extracellular matrix proteins (fibronectin and collagen I), profibrogenic and proinflammatory factors (TGF-beta, connective tissue growth factor, and monocyte chemoattractant protein <em>1</em>), and epithelial-to-mesenchymal transition accompanied this histologic damage. Because VDR ablation activates the renin-<em>angiotensin</em> system and leads to accumulation of <em>angiotensin</em> II (AngII) in the kidney, we assessed whether elevated AngII in the VDR-null kidney promotes injury. Treatment with the <em>angiotensin</em> type <em>1</em> antagonist losartan eliminated the difference in obstruction-induced interstitial fibrosis between wild-type and VDR-null mice, suggesting that AngII contributes to the enhanced renal fibrosis observed in obstructed VDR-null kidneys. Taken together, these results suggest that the VDR attenuates obstructive renal injury at least in part by suppressing the renin-<em>angiotensin</em> system.