COVID-<em>1</em>9, caused by the SARS-CoV-2 virus, is a major source of morbidity and mortality due to its inflammatory effects in the lungs and heart. The p38 MAPK pathway plays a crucial role in the release of pro-inflammatory cytokines such as IL-6 and has been implicated in acute lung injury and myocardial dysfunction. The overwhelming inflammatory response in COVID-<em>1</em>9 infection may be caused by disproportionately upregulated p38 activity, explained by two mechanisms. First, <em>angiotensin</em>-converting enzyme 2 (ACE2) activity is lost during SARS-CoV-2 viral entry. ACE2 is highly expressed in the lungs and heart and converts <em>Angiotensin</em> II into <em>Angiotensin</em> <em>1</em>-<em>7</em>. <em>Angiotensin</em> II signals proinflammatory, pro-vasoconstrictive, pro-thrombotic activity through p38 MAPK activation, which is countered by <em>Angiotensin</em> <em>1</em>-<em>7</em> downregulation of p38 activity. Loss of ACE2 upon viral entry may tip the balance towards destructive p38 signaling through <em>Angiotensin</em> II. Second, SARS-CoV was previously shown to directly upregulate p38 activity via a viral protein, similar to other RNA respiratory viruses that may hijack p38 activity to promote replication. Given the homology between SARS-CoV and SARS-CoV-2, the latter may employ a similar mechanism. Thus, SARS-CoV-2 may induce overwhelming inflammation by directly activating p38 and downregulating a key inhibitory pathway, while simultaneously taking advantage of p38 activity to replicate. Therapeutic inhibition of p38 could therefore attenuate COVID-<em>1</em>9 infection. Interestingly, a prior preclinical study showed protective effects of p38 inhibition in a SARS-CoV mouse model. A number of p38 inhibitors are in the clinical stage and should be considered for clinical trials in serious COVID-<em>1</em>9 infection.

<strong class="sub-title">Keywords:</strong> ACE2; COVID-<em>1</em>9; SARS-CoV-2; p38 MAPK.

In pursuit of the hypothesis that estrogen shifts the vasoconstrictor-vasodilator balance of the renin-<em>angiotensin</em> system, we investigated the cardiovascular responses to administration of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] and <em>angiotensin</em> II (ANG II) in female transgenic (mRen2)2<em>7</em>-positive [Tg(+)] and -negative [Tg(-)] rats in the presence and absence of 3 wk of estrogen replacement therapy. Fifty-three female Tg(-) and Tg(+) rats were oophorectomized and received either <em>1</em><em>7</em> beta-estradiol (<em>1</em>.5 mg/rat s.c. for 3 wk) or vehicle. At the end of 3 wk of estrogen treatment, mean blood pressure was lowered in freely moving chronically cannulated Tg(+) (<em>1</em>59 +/- 4 vs. <em>1</em>45 +/- 5 mmHg, P < 0.05) and Tg(-) (<em>1</em><em>1</em>9 +/- 4 vs. <em>1</em>08 +/- 2 mmHg, P < 0.05) rats. Moreover, the magnitude of the depressor component of the biphasic response to ANG-(<em>1</em>-<em>7</em>) was significantly enhanced in estrogen-treated Tg(+) rats, whereas the pressor component to ANG-(<em>1</em>-<em>7</em>) was attenuated in both Tg(+) and Tg(-) rats. Estrogen replacement significantly attenuated the pressor response to ANG II in both Tg(+) and Tg(-) rats. In addition, estrogen replacement therapy significantly reduced plasma ANG-converting enzyme activity in association with a reduction in circulating levels of ANG II. Tissue levels (kidney and aorta) of ANG-converting enzyme were also reduced with chronic estrogen replacement therapy. On the other hand, estrogen augmented the levels of plasma ANG-(<em>1</em>-<em>7</em>) in Tg(+) animals. Plasma renin activity was unchanged with estrogen treatment. These findings provide the first evidence demonstrating that estrogen is protective against hypertension, possibly by amplifying the vasodilator contributions of ANG-(<em>1</em>-<em>7</em>), while reducing the formation and vasoconstrictor actions of ANG II.

In cultured vascular smooth muscle cells (VSMCs), Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) are expressed constitutively and play a role in <em>angiotensin</em> II (Ang II)-induced intracellular signaling and proliferation. However, little is known regarding the relevance of these proteins to the process of vascular remodeling. The role of JAK and STAT proteins in vascular remodeling and their functional coupling with Ang II were examined in balloon-injured rat carotid artery. Immunoreactive Jak2, Tyk2, Stat<em>1</em>, and Stat3 were not detected in the intact artery. Immunohistostaining showed transient expressions of these JAKs and STATs in medial and neointimal VSMCs at days 2 and 5, respectively, with a peak at day <em>7</em> in both layers. The expressions declined to insignificant levels by day <em>1</em>4. Ang II type <em>1</em> receptors (AT(<em>1</em>)s) were coexpressed in the medial and neointimal VSMCs expressing Jak2 and Stat3. The Jak2 and Stat3 inductions in the injured artery were accompanied by constitutive Jak2 and Stat3 phosphorylations, which were enhanced by ex vivo Ang II stimulation via AT(<em>1</em>). Additionally, a Jak2 inhibitor, AG490, blocked the Ang II-induced Stat3 phosphorylation. Furthermore, local treatment with AG490 inhibited constitutive Stat3 phosphorylation and neointimal VSMC replication and subsequently reduced neointima formation in the injured artery. In conclusion, JAK and STAT proteins were inducible in medial and neointimal VSMCs after vascular injury and were functionally coupled to AT(<em>1</em>). The inductions of JAKs and STATs would be involved in the mechanisms of neointima formation after vascular injury.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is thought to act in an opposing manner to its homologue, <em>angiotensin</em>-converting enzyme (ACE), by inactivating the vasoconstrictor peptide <em>angiotensin</em> II and generating the vasodilatory fragment, <em>angiotensin</em>(<em>1</em>-<em>7</em>). Both ACE and ACE2 are membrane-bound ectoenzymes and may circulate in plasma as a consequence of a proteolytic shedding event. In this study, we show that ACE2 circulates in human plasma, but its activity is suppressed by the presence of an endogenous inhibitor. Partial purification of this inhibitor indicated that the inhibitor is small, hydrophilic and cationic, but not a divalent metal cation. These observations led us to develop a method for removal of the inhibitor, thus allowing detection of plasma ACE2 levels using a sensitive quenched fluorescent substrate-based assay. Using this technique, ACE2 activity measured in plasma from healthy volunteers (n = <em>1</em>8) ranged from <em>1</em>.3<em>1</em> to 8.69 pmol substrate cleaved min-<em>1</em> ml-<em>1</em> (mean +/- s.e.m., 4.44 +/- 0.56 pmol min-<em>1</em> ml-<em>1</em>). Future studies of patients with cardiovascular, renal and liver disease will determine whether plasma ACE2 is elevated in parallel with increased tissue levels observed in these conditions.

OBJECTIVE

The purpose of this study was to test the hypothesis that ACE2 overexpression may enhance atherosclerotic plaque stability by antagonizing ACE activity and converting <em>angiotensin</em> II to <em>angiotensin</em> <em>1</em>-<em>7</em>.

RESULTS

Atherosclerotic plaques were induced in the abdominal aorta of <em>1</em><em>1</em>4 rabbits by endothelial injury and atherogenic diet. Gene therapy was performed in group A at week 4 and in group B at week <em>1</em>2, respectively. Each group of rabbits were randomly divided into 3 subgroups which received, respectively, a recombinant ACE2 expressing vector (AdACE2), a control vector AdEGFP and AdACE2+A<em>7</em><em>7</em>9, an antagonist of <em>angiotensin</em> <em>1</em>-<em>7</em> receptor. Local ACE2 overexpression attenuated the progression of lesions from week 4 to week 8, but not progression of plaque size from week <em>1</em>2 to week <em>1</em>6. In group B rabbits, local ACE2 overexpression resulted in stable plaque compositions, ie, fewer macrophages, less lipid deposition and more collagen contents, higher plaque stability scores, decreased <em>angiotensin</em> II levels, and increased <em>angiotensin</em> <em>1</em>-<em>7</em> levels in plaque tissues in the AdACE2 subgroup compared with those in the AdEGFP subgroup.

CONCLUSIONS

Overexpression of ACE2 results in stabilized atherosclerotic plaques and the mechanism is probably the conversion of vasoconstrictive <em>angiotensin</em> II to vessel protective <em>angiotensin</em> <em>1</em>-<em>7</em>.

We investigated the endogenous production of apelin and the cardiac and pulmonary effects of its chronic administration in monocrotaline (MCT)-induced pulmonary hypertension (PH). Male Wistar rats were injected with MCT (60 mg/kg sc) or vehicle (day 0). One week later, these animals were randomly treated during <em>1</em><em>7</em> days with pyroglutamylated apelin-<em>1</em>3 (Pyr-AP<em>1</em>3; 200 microg*kg(-<em>1</em>)*day(-<em>1</em>) ip) or a similar volume of saline, resulting in four groups: sham (n = <em>1</em><em>1</em>), sham-AP (n = <em>1</em><em>1</em>), MCT (n = <em>1</em>6), and MCT-AP (n = <em>1</em>3). On day 25, right ventricular (RV) and left ventricular (LV) hemodynamic and morphometric parameters were assessed. Tissue and plasma samples were collected for histological and molecular analysis. When compared with sham, the MCT group presented a significant increase of RV mass (<em>1</em>66 +/- 38%), diameter of cardiomyocyte (40 +/- <em>1</em>0%), myocardial fibrosis (95 +/- 20%), peak systolic pressure (99 +/- 22%), peak rate of ventricular pressure rise (dP/dt(max); <em>7</em>4 +/- 24%), peak rate of ventricular pressure decline (dP/dt(min); <em>7</em>3 +/- <em>1</em>9%), and time constant tau (55 +/- <em>1</em>6%). In these animals, RV expression of apelin (-<em>7</em>3 +/- <em>1</em>0%) and its receptor APJ (-6<em>1</em> +/- 20%) was downregulated, whereas mRNA expression of type B natriuretic peptide (9,606 +/- <em>7</em><em>1</em>3%), <em>angiotensin</em>ogen (<em>1</em>9<em>1</em> +/- <em>1</em>4<em>7</em>%), endothelin-<em>1</em> (RV, 49<em>7</em> +/- <em>1</em>56%; and LV, <em>7</em>99 +/- 309%), plasmatic levels of apelin (<em>1</em>04 +/- 48%), and <em>angiotensin</em> <em>1</em>-<em>7</em> (<em>1</em>6<em>1</em> +/- <em>1</em>5<em>1</em>%) were increased. Chronic treatment with Pyr-AP<em>1</em>3 significantly attenuated or normalized these changes, preventing apelin-APJ mRNA downregulation and PH-induced neurohumoral activation of several vasoconstrictors, which exacerbates apelin-APJ vasodilator effects. Therefore, apelin delayed the progression of RV hypertrophy and diastolic dysfunction. Together, these observations suggest that the apelin-APJ system may play an important role in the pathophysiology of PH, representing a potential therapeutic target since it significantly attenuates RV overload and PH-induced neurohumoral activation.

BACKGROUND

We studied the effects of angiotensin II (Ang II) and diastolic overstretch on the induction of cardiac growth in isometrically contracting muscle preparations from human right atria and left ventricles. We used the gene expression of brain natriuretic peptide (BNP) as a molecular marker of cardiac hypertrophy.

RESULTS

Northern blot analysis was performed in human atrial muscle preparations, which were either incubated in 10(-6) mol/L Ang II for 45 minutes or diastolically stretched to 120% of optimum muscle length. Similar experiments were performed with human left ventricular muscle preparations. Results were as follows: (1) BNP gene expression increased in human atrial myocardium 4-fold when stimulated by Ang II (n=7, P<0.001). (2) Diastolic overstretch increased BNP expression in a time-dependent manner. The linear regression equations for the BNP/GAPDH ratio as a function of time (hours) were y=1.21+0.62x (P:<0.001) for overstretched preparations and y=1.07-0.01x (P:=NS) for atrial preparations kept at physiological muscle length. (3) In left ventricular human muscle preparations, diastolic overstretch and Ang II increased BNP gene expression as well. (4) In addition, the Ang II subtype 1 receptor blocker losartan was able to block the effects of Ang II and diastolic overstretch.

CONCLUSIONS

Cardiac hypertrophy can be induced in isolated human atrial and left ventricular intact myocardium by Ang II and diastolic overstretch but not by isometric afterload. The fact that the induction of cardiac growth is inhibited by the blockade of Ang II subtype 1 receptors is of scientific and clinical importance.

Cardiac hypertrophy is a process that occurs in response to various mechanical or hormonal stimuli. Stimulation of the renin-<em>angiotensin</em> system is involved in the process of cardiac hypertrophy through mechanisms related to increased peripheral vascular resistance and increased cardiac afterload. In this study we determined whether [Sar<em>1</em>]<em>angiotensin</em> II (ANG II) directly stimulated protein synthesis and cell growth in embryonic chick myocytes in cell culture. Eighteen-day-old embryonic chick myocytes in subconfluent cell culture, incubated in a chemically defined serum-free media, showed a significant increase in total protein content, <em>1</em>8.5, 26.2, and 22.2%, respectively, when exposed to [Sar<em>1</em>]ANG II (<em>1</em> microM/day) for 5, <em>7</em>, and 9 days, respectively. The increase in total protein resulted in part from an increase in the fractional protein synthesis rate of 2<em>1</em>.<em>7</em>, <em>1</em>6.5, and <em>1</em>4.9% at 5, <em>7</em>, and 9 days, respectively. Total DNA and RNA levels did not change significantly following a 4-day exposure to [Sar<em>1</em>]ANG II in subconfluent culture. The relative rate of protein synthesis, determined by pulse labeling for 3 h with [3H]phenylalanine, showed increases of 23.4, 22.9, and <em>1</em><em>7</em>.8% over control after 4, 5, and 6 days of exposure to [Sar<em>1</em>]ANG II. The incorporation of [3H]phenylalanine was blocked by the specific ANG II-receptor antagonist [Sar<em>1</em>,Ile8]ANG II. The data demonstrate a receptor-mediated increase in the rate of protein synthesis in cultured chick myocytes in response to [Sar<em>1</em>]ANG II, with a resultant increase in total cellular protein. This <em>angiotensin</em> peptide appears to directly stimulate protein synthesis in cultured embryonic chick myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac remodelling is a key risk factor for the development of heart failure in the chronic phase following myocardial infarction. Our previous studies have shown an anti-remodelling role of ACE2 (<em>angiotensin</em>-converting enzyme 2) in vivo during hypertension and that these protective effects are mediated through increased circulating levels of Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)]. In the present study, we have demonstrated that cardiac myocytes have modest ACE2 activity, whereas cardiac fibroblasts do not exhibit any endogenous activity. As fibroblasts are the major cell type found in an infarct zone following a myocardial infarction, we examined the effects of ACE2 gene delivery to cultured cardiac fibroblasts after acute hypoxic exposure. Cardiac fibroblasts from 5-day-old Sprague-Dawley rat hearts were grown to confluence and transduced with a lentiviral vector containing murine ACE2 cDNA under transcriptional control by the EF<em>1</em>alpha (elongation factor <em>1</em>alpha) promoter (lenti-ACE2). Transduction of fibroblasts with lenti-ACE2 resulted in a viral dose-dependent increase in ACE2 activity. This was associated with a significant attenuation of both basal and hypoxia/re-oxygenation-induced collagen production by the fibroblasts. Cytokine production, specifically TGFbeta (transforming growth factor beta), by these cells was also significantly attenuated by ACE2 expression. Collectively, these results indicate that: (i) endogenous ACE2 activity is observed in cardiac myocytes, but not in cardiac fibroblasts; (ii) ACE2 overexpression in the cardiac fibroblast attenuates collagen production; and (iii) this prevention is probably mediated by decreased expression of cytokines. We conclude that ACE2 expression, limited to cardiac fibroblasts, may represent a novel paradigm for in vivo therapy following acute ischaemia.

OBJECTIVE

To examine associations among the angiotensin I-converting enzyme (ACE) insertion (I)/deletion (D) polymorphism and the response to a 12-wk (2 d.wk) unilateral, upper-arm resistance training (RT) program in the trained (T, nondominant) and untrained (UT, dominant) arms.

METHODS

Subjects were 631 (mean+/-SEM, 24.2+/-0.2 yr) white (80%) men (42%) and women (58%). The ACE ID genotype was in Hardy-Weinberg equilibrium with frequencies of 23.1, 46.1, and 30.8% for ACE II, ID, and DD, respectively (chi=1.688, P=0.430). Maximum voluntary contraction (MVC) and one-repetition maximum (1RM) assessed peak elbow flexor muscle strength. Magnetic resonance imaging measured biceps muscle cross-sectional area (CSA). Multiple variable and repeated-measures ANCOVA tested whether muscle strength and size differed at baseline and pre- to post-RT among T and UT and ACE ID genotype.

RESULTS

Baseline muscle strength and size were greater in UT than T (P<0.001) and did not differ among ACE ID genotype in either arm (P>>or= 0.05). In T, MVC increases were greater for ACE II/ID (22%) than DD (17%) (P<0.05), whereas 1RM (51%) and CSA (19%) gains were not different among ACE ID genotype pre- to post-RT (P>>or= 0.05). In UT, MVC increased among ACE II/ID (7%) (P<0.001) but was similar among ACE DD (2%) pre- to post-RT (P>>or= 0.05). In UT, 1RM (11%) and CSA (2%) increases were greater for ACE DD/ID than ACE II (1RM, 7%; CSA, -0.1%) (P<0.05). ACE ID genotype explained approximately 1% of the MVC response to RT in T and approximately 2% of MVC, 2% of 1RM, and 4% of CSA response in UT (P<0.05).

CONCLUSIONS

ACE ID genotype is associated with the contralateral effects of unilateral RT, perhaps more so than with the muscle strength and size adaptations that result from RT.

Maternal separation during early life is an established chronic behavioral model of early life stress in rats. It is known that perinatal adverse environments increase activity of the renin-<em>angiotensin</em> (Ang) system, specifically Ang II, in adulthood. The aim of this study was to investigate whether the effects of early life stress augment the sensitivity of the Ang II pathway. Using Wistar Kyoto rats, the maternal separation (MS) protocol was performed by separating approximately half of the male pups from their mother 3 h/d from days 2 to <em>1</em>4 of life. Pups remaining with the mother at all times were used as controls. Maternal separation did not influence the plasma basal parameters, such as blood glucose, insulin, Ang II, Ang <em>1</em>-<em>7</em> and plasma renin activity. Furthermore, body weight, blood pressure, and heart rate were similar in MS and control rats. The acute pressor response to Ang II was not different in anesthetized MS and control rats. However, the chronic infusion of Ang II (65 ng/min SC) elicited an exaggerated hypertensive response in MS compared with control rats (P<0.05). Surprisingly, HR was dramatically increased during the second week of Ang II infusion in MS compared with control rats (P<0.05). This enhanced Ang II sensitivity was accompanied by a greater vascular inflammatory response in MS versus control rats. Chronic Ang II infusion increased vascular wall structure in both groups similarly. These data indicate that early life stress sensitizes rats to an increased hemodynamic and inflammatory response during Ang II-induced hypertension.

BACKGROUND

Angiotensin II is strongly incriminated in progressive renal injury. There is recent evidence that angiotensin II induces oxidative stress in vitro. We examined the capacity of angiotensin II to induce oxidative stress in vivo and the functional significance of such stress. The capacity of angiotensin II to induce the oxidant-sensitive gene heme oxygenase (HO) in vivo and in vitro was also examined.

METHODS

Angiotensin II was administered via mini-osmotic pumps to rats maintained on standard diets. Indices of oxidative stress, including thiobarbituric acid reactive substance, carbonyl protein content, and HO activity, were determined. Indices of oxidative stress and functional markers were also determined in the DOCA salt model. The effect of angiotensin II was studied in rats maintained on antioxidant-deficient diets so as to examine the functional significance of oxidative stress induced by angiotensin II. We also explored the inductive effect of angiotensin II on HO in vivo and whether such actions occur in vitro.

RESULTS

Angiotensin II administered in vivo increased kidney content of thiobarbituric acid reactive substances protein carbonyl content, and HO activity. These indices were not present in the kidney of rats treated with DOCA salt for three weeks. Such oxidative stress was functionally significant, since the administration of angiotensin II to rats maintained on a prooxidant diet demonstrated increased proteinuria and decreased creatinine clearance. The stimulatory effect on HO activity was due to induction of HO-1 mRNA, with HO-2 mRNA remaining unchanged. Expression of HO-1 was localized to the renal proximal tubules in vivo. We also demonstrate that angiotensin II at concentrations of 10-8 and 10-7 mol/L induces expression of HO-1 mRNA in LLC-PK1 cells.

CONCLUSIONS

Angiotensin II induces oxidative stress in vivo, which contributes to renal injury. This study also demonstrates that angiotensin II induces renal HO activity caused by up-regulation of HO-1 in renal proximal tubules. Finally, angiotensin II directly induces HO-1 in renal proximal tubular epithelial cells in vitro.

BACKGROUND

In proteinuric nephropathies with increasingly severe defects of the glomerular filtering barrier, interstitial fibrogenesis is a major effector of scarring. An early event in this process is the peritubular accumulation of myofibroblasts that express alpha-smooth muscle actin (alpha-SMA) and contribute to abnormal matrix production. Common trigger factors are poorly understood. Enhanced protein trafficking may play a role by up-regulating inflammatory and fibrogenic genes in proximal tubular cells.

METHODS

The remnant kidney model in rats was used to (<em>1</em>) analyze interactions between activated proximal tubular cells, peritubular cells expressing the myofibroblast marker, and inflammatory cells at time intervals (days <em>7</em>, <em>1</em>4, and 30) after surgery, and (2) evaluate the effects of <em>angiotensin</em>-converting enzyme inhibitor (ACEi) on protein trafficking, fibrogenic signaling, and alpha-SMA expression.

RESULTS

Abnormal uptake of ultrafiltered proteins by proximal tubular cells (IgG staining) occurred at an early stage (day <em>7</em>) and was subsequently associated with macrophage and alpha-SMA+ cell accumulation into the peritubular interstitium. alpha-SMA+ cells clustered with macrophages into the interstitium. These changes were associated with appearance of transforming growth factor-beta<em>1</em> (TGF-beta<em>1</em>) mRNA in proximal tubular cells and in the infiltrating cells with time. At day 30, focal alpha-SMA staining also was found in the tubular cells and in peritubular endothelial cells on semithin ultracryosections. ACEi prevented both proteinuria and abnormal protein accumulation in tubular cells, as well as the inflammatory and fibrogenic reaction with peritubular alpha-SMA expression.

CONCLUSIONS

Profibrogenic signaling from both proximal tubular cells on challenge with filtered protein and inflammatory cells is implicated as a key candidate trigger of progressive tubulointerstitial injury.

Macula densa cyclooxygenase 2 (COX-2)-derived prostaglandins serve as important modulators of the renin-<em>angiotensin</em> system, and cross-talk exists between these two systems. Cortical COX-2 induction by <em>angiotensin</em>-converting enzyme (ACE) inhibitors or AT(<em>1</em>) receptor blockers (ARBs) suggests that <em>angiotensin</em> II may inhibit cortical COX-2 by stimulating the AT(<em>1</em>) receptor pathway. In the present studies we determined that chronic infusion of either hypertensive or nonhypertensive concentrations of <em>angiotensin</em> II attenuated cortical COX-2. <em>Angiotensin</em> II infusion reversed cortical COX-2 elevation induced by ACE inhibitors. However, we found that <em>angiotensin</em> II infusion further stimulated cortical COX-2 elevation induced by ARBs, suggesting a potential role for an AT(2) receptor-mediated pathway when the AT(<em>1</em>) receptor was inhibited. Both WT and AT(2) receptor knockout mice were treated for <em>7</em> days with either ACE inhibitors or ARBs. Cortical COX-2 increased to similar levels in response to ACE inhibition in both knockout and WT mice. In WT mice ARBs increased cortical COX-2 more than ACE inhibitors, and this stimulation was attenuated by the AT(2) receptor antagonist PD<em>1</em>233<em>1</em>9. In the knockout mice ARBs led to significantly less cortical COX-2 elevation, which was not attenuated by PD<em>1</em>233<em>1</em>9. PCR confirmed AT(<em>1</em>a) and AT(2) receptor expression in the cultured macula densa cell line MMDD<em>1</em>. <em>Angiotensin</em> II inhibited MMDD<em>1</em> COX-2, and CGP42<em>1</em><em>1</em>2A, an AT(2) receptor agonist, stimulated MMDD<em>1</em> COX-2. In summary, these results demonstrate that macula densa COX-2 expression is oppositely regulated by AT(<em>1</em>) and AT(2) receptors and suggest that AT(2) receptor-mediated cortical COX-2 elevation may mediate physiologic effects that modulate AT(<em>1</em>)-mediated responses.

<em>Angiotensin</em> II AT2 receptors act as a functional antagonist for the AT<em>1</em> receptors in various tissues. We previously reported that activation of the renal AT2 receptors promotes natriuresis and diuresis; however, the mechanism is not known. The present study was designed to investigate whether activation of AT2 receptors affects the activity of Na+-K+-ATPase (NKA), an active tubular sodium transporter, in the proximal tubules isolated from Sprague-Dawley rats. The AT2 receptor agonist CGP-42<em>1</em><em>1</em>2 (<em>1</em>0(-<em>1</em>0)-<em>1</em>0(-<em>7</em>) M) produced a dose-dependent inhibition of NKA activity (9-38%); the inhibition was attenuated by the presence of the AT2 receptor antagonist PD-<em>1</em>233<em>1</em>9 (<em>1</em> microM), suggesting the involvement of the AT2 receptors. The AT<em>1</em> receptor antagonist losartan (<em>1</em> microM) did not affect the CGP-42<em>1</em><em>1</em>2 (<em>1</em>00 nM)-induced inhibition of NKA activity. The presence of guanylyl cyclase inhibitor ODQ (<em>1</em>0 microM) and the nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; <em>1</em>00 microM) abolished the CGP-42<em>1</em><em>1</em>2 (<em>1</em>00 nM)-induced NKA inhibition. ANG II (<em>1</em>00 nM), in the presence of losartan, significantly inhibited NKA activity; the inhibition was attenuated by PD-<em>1</em>233<em>1</em>9. CGP-42<em>1</em><em>1</em>2 also, in a dose-dependent manner, stimulated NO production (approximately 0-230%) and cGMP accumulation (approximately 25-<em>1</em>00%). The CGP-42<em>1</em><em>1</em>2 (<em>1</em>00 nM)-induced NO and cGMP increases were abolished by the AT2 receptor antagonist PD-<em>1</em>233<em>1</em>9, ODQ, and L-NAME. The data suggest that the activation of the AT2 receptor via stimulation of the NO/cGMP pathway causes inhibition of NKA activity in the proximal tubules. This phenomenon provides a plausible mechanism responsible for the AT2 receptor-mediated natriuresis-diuresis in rodents.

OBJECTIVE

We studied the effect of the <em>angiotensin</em> II type <em>1</em> (AT<em>1</em>)-receptor antagonist candesartan on infarct size resulting from regional myocardial ischemia in pigs.

BACKGROUND

The effects of AT<em>1</em>-receptor blockade on infarct size in different species remain controversial and its potential cardioprotective mechanisms are still unclear. In pigs, infarct development closely resembles that observed in humans.

METHODS

A total of 62 enflurane-anesthetized pigs underwent a protocol of 90-min low-flow ischemia and <em>1</em>20-min reperfusion. Systemic hemodynamics (micromanometer), regional myocardial function (sonomicrometry), regional myocardial blood flow (microspheres) and infarct size (TTC [triphenyl tetrazolium chloride]-staining) were determined.

RESULTS

Left ventricular peak pressure decreased with candesartan (<em>1</em> mg/kg i.v.) from 97+/-2 standard error of the mean (SEM) to 86+/-5 mm Hg and was then readjusted by aortic banding. In placebo pigs (n=9), infarct size was 2<em>1</em>.8+/-4.8% of the area at risk. Candesartan (n=7) reduced infarct size to 9.7+/-2.5% (p < 0.05). Pretreatment with the AT2-receptor antagonist PD<em>1</em>233<em>1</em>9 (3 microg/kg/min intracoronarily [i.c.]; n=8), the bradykinin B2-receptor antagonist HOE<em>1</em>40 (0.0<em>1</em> microg/kg/min i.c.; n=8) or the cyclooxygenase inhibitor indomethacin (<em>1</em>0 mg/kg i.v.; n= 8) per se did not affect infarct size but did abolish the reduction of infarct size achieved by candesartan (PD<em>1</em>233<em>1</em>9 + candesartan (n=7): 23.2+/-4.7%; HOE<em>1</em>40 + candesartan (n=7): <em>1</em>8.2+/-4.0%; indomethacin + candesartan (n=8): 2<em>1</em>.<em>1</em>+/-5.2%). Hemodynamics, regional myocardial blood flow during ischemia and the area at risk were comparable among all groups of pigs.

CONCLUSIONS

Reduction of infarct size by the AT<em>1</em>-receptor antagonist candesartan in pigs involves <em>angiotensin</em> II type 2 receptor (AT2) activation, bradykinin and prostaglandins.

Recent studies have shown that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] interacts with kinins and augments bradykinin (BK)-induced vasodilator responses by an unknown mechanism. In this study, we evaluated whether the potentiation of the BK-induced vasodilation by Ang-(<em>1</em>-<em>7</em>) may be attributable to inhibition of BK metabolism, release of nitric oxide, or both. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers. <em>1</em>25I-[Tyr0]-BK metabolism was determined in vascular rings by assessing the degradation of the peptide by high-performance liquid chromatography. Ang-(<em>1</em>-<em>7</em>) augmented the vasodilation induced by BK in a concentration-dependent manner in rings preconstricted with the thromboxane analog U466<em>1</em>9. The EC50 of BK (2.45 +/- 0.5<em>1</em> nmol/L versus 0.3<em>7</em> +/- 0.08 nmol/L) was shifted leftward by 6.6-fold in the presence of 2 mumol/L concentration of Ang-(<em>1</em>-<em>7</em>). The response was specific for BK. since Ang-(<em>1</em>-<em>7</em>) did not augment the vasodilation induced by either acetylcholine (0.05 mumol/L) or sodium nitroprusside (0.<em>1</em> mumol/L). Moreover, neither <em>angiotensin</em> I nor <em>angiotensin</em> II (Ang II) duplicated the augmented BK response of Ang-(<em>1</em>-<em>7</em>). Pretreatment of vascular rings with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NA; <em>1</em>00 mumol/L) completely abolished the effects of Ang-(<em>1</em>-<em>7</em>) on BK-induced vasodilation whereas pretreatment with indomethacin (<em>1</em>0 mumol/L) was without effect. The potent specific BK B2 receptor antagonist, Hoe <em>1</em>40. nearly abolished the BK and the Ang-(<em>1</em>-<em>7</em>) potentiated responses at 2 mumol/L, whereas at a lower concentration (20 nmol/L) Hoe <em>1</em>40 shifted the response curve to the right for both Ang-(<em>1</em>-<em>7</em>) and vehicle; however, the augmented response to Ang-(<em>1</em>-<em>7</em>) persisted. Preincubation of vascular rings with 20 mumol/L of the AT<em>1</em> (CV<em>1</em><em>1</em>9<em>7</em>4), AT2 (PD<em>1</em>233<em>1</em>9), or nonselective (Sar<em>1</em> Thr8-Ang II) receptor antagonists had no significant effect on the Ang-(<em>1</em>-<em>7</em>)-enhanced vasodilator response to BK. Lisinopril (2 mumol/L) significantly enhanced the BK-induced vasodilator response while at the same time it abolished the synergistic action of Ang-(<em>1</em>-<em>7</em>) on BK. In addition, pretreatment with 2 mumol/L Ang-(<em>1</em>-<em>7</em>) significantly inhibited the degradation of <em>1</em>25I-[Tyr0]-BK and the appearance of the BK-(<em>1</em>-<em>7</em>) and BK-(<em>1</em>-5) metabolites in coronary vascular rings. Ang-(<em>1</em>-<em>7</em>) inhibited purified canine <em>angiotensin</em> converting enzyme activity with an IC50 of 0.65 mumol/L. In conclusion. Ang-(<em>1</em>-<em>7</em>) acts as a local synergistic modulator of kinin-induced vasodilation by inhibiting <em>angiotensin</em> converting enzyme and releasing nitric oxide.

The present study was performed to investigate whether transient receptor potential (TRPC)6 participated in Ca(2+) signaling of glomerular mesangial cells (MCs) and expression of this protein was altered in diabetes. Western blots and real-time PCR were used to evaluate the expression level of TRPC6 protein and mRNA, respectively. Cell-attached patch-clamp and fura-2 fluorescence measurements were utilized to assess <em>angiotensin</em> II (ANG II)-stimulated membrane currents and Ca(2+) responses in MCs. In cultured human MCs, high glucose significantly reduced expression of TRPC6 protein, but there was no effect on either TRPC<em>1</em> or TRPC3. The high glucose-induced effect on TRPC6 was time and dose dependent with the maximum effect observed on day <em>7</em> and at 30 mM glucose, respectively. In glomeruli isolated from streptozotocin-induced diabetic rats, TRPC6, but not TRPC<em>1</em>, was markedly reduced compared with the glomeruli of control rats. Furthermore, TRPC6 mRNA in MCs was also significantly decreased by high glucose as early as <em>1</em> day after treatment with maximal reduction on day 4. Patch-clamp experiments showed that ANG II-stimulated membrane currents in MCs were significantly attenuated or enhanced by knockdown or overexpression of TRPC6, respectively. Fura-2 fluorescence measurements revealed that the ANG II-induced Ca(2+) influxes were markedly inhibited in MCs with TRPC6 knockdown, reminiscent of the impaired Ca(2+) entry in response to ANG II in high glucose-treated MCs. These results suggest that the TRPC6 protein expression in MCs was downregulated by high glucose and the deficiency of TRPC6 protein might contribute to the impaired Ca(2+) signaling of MCs seen in diabetes.

The kidney is an important target for the actions of the renin-<em>angiotensin</em> system (RAS) and this tissue contains a complete local RAS that expresses the bioactive peptides <em>angiotensin</em> II (ANG II) and Ang-(<em>1</em>-<em>7</em>). We find both <em>angiotensin</em> type <em>1</em> (AT(<em>1</em>)R) and type 2 (AT(2)R) receptors expressed on renal nuclei that stimulate reactive oxygen species and nitric oxide (NO), respectively. Since Ang-(<em>1</em>-<em>7</em>) also exhibits actions within the kidney and the Ang-(<em>1</em>-<em>7</em>)/Mas receptor protein contains a nuclear localization sequence, we determined the expression of Ang-(<em>1</em>-<em>7</em>) receptors in nuclei isolated from the kidneys of young adult sheep. Binding studies with (<em>1</em>25)I-[Sar(<em>1</em>)Thr(8)]-ANG II revealed sites sensitive to the Ang-(<em>1</em>-<em>7</em>) antagonist [d-Ala(<em>7</em>)]-Ang-(<em>1</em>-<em>7</em>) (DALA, A<em>7</em><em>7</em>9), as well as to AT(2) and AT(<em>1</em>) antagonists. Incubation of Ang-(<em>1</em>-<em>7</em>) [<em>1</em>0(-<em>1</em>5) to <em>1</em>0(-9) M] with isolated cortical nuclei elicited a dose-dependent increase in the fluorescence of the NO indicator [4-amino-5-methylamino-2',<em>7</em>']-difluorofluorescein diacetate. The NO response to Ang-(<em>1</em>-<em>7</em>) was abolished by the NO inhibitor N-nitro-l-arginine methyl ester and DALA, but not the AT(<em>1</em>) antagonist losartan or the AT(2) blocker PD<em>1</em>233<em>1</em>9. Immunofluorescent studies utilizing the Ang-(<em>1</em>-<em>7</em>)/Mas receptor antibody revealed immunolabeling of the proximal tubules but not staining within the glomerulus in cortical sections of the sheep kidney. In the nuclear fraction of isolated proximal tubules, immunoblots revealed the precursor <em>angiotensin</em>ogen and renin, as well as functional activity for ACE, ACE2, and neprilysin. We conclude that renal nuclei express Ang-(<em>1</em>-<em>7</em>)/Mas receptors that are functionally linked to NO formation. The marked sensitivity of the intracellular NO response to Ang-(<em>1</em>-<em>7</em>) implicates a functional role of the Ang-(<em>1</em>-<em>7</em>) axis within the nucleus. Moreover, evidence for the precursor and enzymatic components of the RAS within the nuclear compartment of the proximal tubules provides a potential pathway for the intracellular generation of Ang-(<em>1</em>-<em>7</em>).

The antithrombotic effect of <em>angiotensin</em>(Ang)-(<em>1</em>-<em>7</em>) has been reported, but the mechanism of this effect is not known. We investigated the participation of platelets and receptor Mas-related mechanisms in this action. We used Western blotting to test for the presence of Mas protein in rat platelets and used fluorescent-labeled FAM-Ang-(<em>1</em>-<em>7</em>) to determine the specific binding for Ang-(<em>1</em>-<em>7</em>) and its displacement by the receptor Mas antagonist A-<em>7</em><em>7</em>9 in rat platelets and in Mas(-/ -) and Mas(+/+) mice platelets. To test whether Ang-(<em>1</em>-<em>7</em>) induces NO release from platelets, we used the NO indicator DAF-FM. In addition we examined the role of Mas in the Ang-(<em>1</em>-<em>7</em>) antithrombotic effect on induced thrombi in the vena cava of male Mas(-/ -) and Mas(+/+) mice. The functional relevance of Mas in hemostasis was evaluated by determining bleeding time in Mas(+/+) and Mas(-/ -) mice. We observed the presence of Mas protein in platelets, as indicated by Western Blot, and displacement of the binding of fluorescent Ang-(<em>1</em>-<em>7</em>) to rat platelets by A-<em>7</em><em>7</em>9. Furthermore, in Mas(+/+) mouse platelets we found specific binding for Ang-(<em>1</em>-<em>7</em>), which was absent in Mas(-/ -) mouse platelets. Ang-(<em>1</em>-<em>7</em>) released NO from rat and Mas(+/+) mouse platelets, and A-<em>7</em><em>7</em>9 blocked this effect. The NO release stimulated by Ang-(<em>1</em>-<em>7</em>) was abolished in Mas(-/ -) mouse platelets. Ang-(<em>1</em>-<em>7</em>) inhibited thrombus formation in Mas(+/+) mice. Strikingly, this effect was abolished in Mas(-) (/) (-)mice. Moreover, Mas deficiency resulted in a significant decrease in bleeding time (8.50 +/- <em>1</em>.4<em>7</em> vs. 4.28 +/- 0.66 min). This study is the first to show the presence of Mas protein and specific binding for Ang-(<em>1</em>-<em>7</em>) in rat and mouse platelets. Our data also suggest that the Ang-(<em>1</em>-<em>7</em>) antithrombotic effect involves Mas-mediated NO release from platelets. More importantly, we showed that the antithrombotic effect of Ang-(<em>1</em>-<em>7</em>) in vivo is Mas dependent and that Mas is functionally important in hemostasis.

<em>Angiotensin</em> II exerts a mitogenic effect in several in vitro models, but a direct effect on erythroid progenitors has not been documented. <em>Angiotensin</em>-converting enzyme inhibitors and losartan, an <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>) antagonist, ameliorate posttransplant erythrocytosis, without altering serum erythropoietin levels. We studied erythroid differentiation and the effect of <em>angiotensin</em> II on proliferation of erythroid progenitors by culturing CD34+ hematopoietic progenitor cells in liquid serum-free medium favoring growth of erythroid precursors. Aliquots of cells were collected every third day, and were used for RNA preparation. AT<em>1</em> mRNA was detected after 6 d. In these same samples, erythroid-specific mRNA (erythropoietin receptor) was also detected. AT<em>1</em> protein was detected in <em>7</em>-d-old burst-forming units-erythroid colonies by Western blotting. The CD34+ cell liquid cultures were used to incubate erythroid precursors with <em>angiotensin</em> II from days 6-9. After incubation, cells were transferred to semisolid medium and cultured with erythropoietin. <em>Angiotensin</em> II increased proliferation of early erythroid progenitors, defined as increased numbers of burst-forming units-erythroid colonies. Losartan completely abolished this stimulatory effect of <em>angiotensin</em> II. Moreover, we observed increased numbers of erythroid progenitors in the peripheral blood of posttransplant erythrocytosis patients. Thus, activation of AT<em>1</em> with <em>angiotensin</em> II enhances erythropoietin-stimulated erythroid proliferation in vitro. A putative defect in the <em>angiotensin</em> II/AT<em>1</em> pathway may contribute to the pathogenesis of posttransplant erythrocytosis.

The renin–<em>angiotensin</em> system (RAS) has recently been extended by the addition of a novel axis consisting of the <em>angiotensin</em>-converting enzyme 2 (ACE2), the heptapeptide <em>angiotensin</em> (<em>1</em>–<em>7</em>) (Ang-(<em>1</em>–<em>7</em>)), and the G protein-coupled receptor Mas. ACE2 converts the vasoconstrictive and pro-oxidative peptide <em>angiotensin</em> II (Ang II) into Ang-(<em>1</em>–<em>7</em>) which exerts vasodilatory and antioxidative effects via its receptor Mas. Thereby, ACE2 regulates the local actions of the RAS in cardiovascular tissues and the ACE2/Ang-(<em>1</em>–<em>7</em>)/Mas axis exerts protective actions in hypertension, diabetes, and other cardiovascular disorders. Consequently, this novel RAS axis represents a promising therapeutic target for cardiovascular and metabolic diseases.

<em>Angiotensin</em> II (AngII) type <em>1</em> receptor blockers (ARBs) have been effectively used in hypertension and cardiac remodeling. However, the differences among them are still unclear. We designed this study to examine and compare the effects of several ARBs widely used in clinics, including Olmesartan, Candesartan, Telmisartan, Losartan, Valsartan and Irbesartan, on the ACE-AngII-AT<em>1</em> axis and the ACE2-Ang(<em>1</em>-<em>7</em>)-Mas axis during the development of cardiac remodeling after pressure overload. Although all of the six ARBs, attenuated the development of cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC) for 2 or 4weeks in the wild-type mice evaluated by echocardiography and hemodynamic measurements, the degree of attenuation by Olmesartan, Candesartan and Losartan tended to be larger than that of the other three drugs tested. Additionally, the degree of downregulation of the ACE-AngII-AT<em>1</em> axis and upregulation of the ACE2-Ang(<em>1</em>-<em>7</em>)-Mas axis was higher in response to Olmesartan, Candesartan and Losartan administration in vivo and in vitro. Moreover, in angiotensinogen-knockdown mice, TAC-induced cardiac hypertrophy and heart failure were inhibited by Olmesartan, Candesartan and Losartan but not by Telmisartan, Valsartan and Irbesartan administration. Furthermore, only Olmesartan and Candesartan could downregulate the ACE-AngII-AT<em>1</em> axis and upregulate the ACE2-Ang(<em>1</em>-<em>7</em>)-Mas axis in vitro. Our data suggest that Olmesartan, Candesartan and Losartan could effectively inhibit pressure overload-induced cardiac remodeling even when with knockdown of Ang II, possibly through upregulation of the expression of the ACE2-Ang(<em>1</em>-<em>7</em>)-Mas axis and downregulation of the expression of the ACE-AngII-AT<em>1</em> axis. In contrast, Telmisartan, Valsartan and Irbesartan only played a role in the presence of AngII, and Losartan had no effect in the presence of AngII in vitro.

It has been shown that endothelial NO synthase (eNOS) uncoupling occurs in hypertension and atherosclerosis. However, its causal role in vascular pathogenesis has not been characterized previously. Here, we challenged eNOS preuncoupled hyperphenylalaninemia (hph)-<em>1</em> mice (deficient in eNOS cofactor tetrahydrobiopterin biosynthetic enzyme GTPCHI) with <em>angiotensin</em> II (Ang II; 0.<em>7</em> mg/kg per day, <em>1</em>4 days). Both wild-type and hph-<em>1</em> groups developed hypertension similarly up to day 6 to <em>7</em>. Thereafter, ≈<em>1</em>4% of Ang II-infused (0.<em>7</em> mg/kg per day) hph-<em>1</em> mice (n=<em>7</em>2) started to die suddenly of ruptured abdominal aortic aneurysm (AAA). Among the survivors, 65% developed AAA, resulting in a total morbidity rate of <em>7</em>9%. In contrast, none of the Ang II-infused wild-type mice died or developed AAA. Ang II progressively deteriorated eNOS uncoupling in hph-<em>1</em> mice while augmenting tetrahydrobiopterin and nitric oxide (NO(·)) deficiencies. The abundance of the tetrahydrobiopterin salvage enzyme dihydrofolate reductase in the endothelium was decreased in hph-<em>1</em> mice and further diminished by Ang II infusion. Intriguingly, restoration of dihydrofolate reductase expression by oral administration of folic acid or overexpression of dihydrofolate reductase completely prevented AAA formation in Ang II-infused hph-<em>1</em> mice while attenuating progressive uncoupling of eNOS. Folic acid also attenuated vascular remodeling and inflammation characterized by medial elastin breakdown and augmented matrix metalloproteinase 2 activity and activation of matrix metalloproteinase 9, as well as macrophage infiltration. In conclusion, these data innovatively suggest a causal role of eNOS uncoupling/tetrahydrobiopterin deficiency in AAA formation. Therefore, oral folic acid administration, endothelium-targeted dihydrofolate reductase gene therapy, and perhaps other countermeasures directed against eNOS uncoupling could be used as new therapeutics for AAA.