The renin-<em>angiotensin</em> system (RAS) has been traditionally linked to blood pressure and volume regulation mediated through the <em>angiotensin</em> II (ANG II) type <em>1</em> (AT<em>1</em>) receptor. Here we report that ANG II via its ANG II type 2 (AT2) receptor promotes the axonal elongation of postnatal rat retinal explants (postnatal day <em>1</em><em>1</em>) and dorsal root ganglia neurons in vitro, and, moreover, axonal regeneration of retinal ganglion cells after optic nerve crush in vivo. In retinal explants, ANG II (<em>1</em>0(-<em>7</em>)-<em>1</em>0(-5) M) induced neurite elongation via its AT2 receptor, since the effects were mimicked by the AT2 receptor agonist CGP 42<em>1</em><em>1</em>2 (<em>1</em>0(-5) M) and were entirely abolished by costimulation with the AT2 receptor antagonist PD <em>1</em>23<em>1</em><em>7</em><em>7</em> (<em>1</em>0(-5) M), but not by the AT<em>1</em> receptor antagonist losartan (<em>1</em>0(-5) M). To investigate whether ANG II is able to promote axonal regeneration in vivo, we performed optic nerve crush experiments in the adult rats. After ANG II treatment (0.6 nmol), an increased number of growth-associated protein (GAP)-43-positive fibers was detected and the regenerating fibers regularly crossed the lesion site (<em>1</em>.6 mm). Cotreatment with the AT2 receptor antagonist PD <em>1</em>23<em>1</em><em>7</em><em>7</em> (6 nmol), but not with the AT<em>1</em> receptor antagonist losartan (6 nmol), completely abolished the ANG II-induced axonal regeneration, providing for the first time direct evidence for receptor-specific neurotrophic action of ANG II in the central nervous system of adult mammals and revealing a hitherto unknown function of the RAS.

The purpose of this study was to test the hypothesis that overexpression of <em>angiotensin</em>-converting enzyme 2 (ACE2) may favorably affect left ventricular (LV) remodeling and function after myocardial infarction (MI). The left anterior descending coronary artery was ligated to produce anterior MI in <em>1</em>00 Wistar-Kyoto rats that were randomly divided into Ad-ACE2, Ad-ACE2+A<em>7</em><em>7</em>9, Ad-EGFP, model, and sham groups. Two weeks later, rats in the Ad-ACE2 and Ad-EGFP groups received direct intramyocardial injection of Ad-ACE2 and Ad-EGFP, respectively. Rats in the Ad-ACE2+A<em>7</em><em>7</em>9 group received both intramyocardial injection of Ad-ACE2 and a continuous intravenous infusion of A<em>7</em><em>7</em>9 for <em>1</em>5 days. LV volume and systolic function, the extent of myocardial fibrosis, and levels of ACE2, <em>angiotensin</em> II (Ang II), and collagen I protein expression were evaluated. Four weeks after ACE2 gene transfer, the Ad-ACE2 group showed reduced LV volume, extent of myocardial fibrosis, and expression levels of ACE, Ang II, and collagen I in the myocardium, and increased LV ejection fraction and levels of ACE2 activity and expression in comparison with the Ad-EGFP and model groups. These results suggest that ACE2 overexpression attenuated LV fibrosis and improved LV remodeling and systolic function. In conclusion, overexpression of ACE2 favorably affected the pathological process of LV remodeling after MI by inhibiting ACE activity, reducing AngII levels, and up-regulating Ang-(<em>1</em>-<em>7</em>) expression, thus providing a potential therapeutic target in the treatment of heart failure.

Diabetes alters microvascular function in the vascular beds of organs, including the lungs. Cardiovascular complications of pulmonary vascular affectation may be a consequence of the overactivation of the vasoconstrictive and proliferative components of the renin-<em>angiotensin</em> system. We previously reported that pulmonary physiology and surfactant production is improved by the glucagon-like peptide <em>1</em> receptor (GLP-<em>1</em>R) agonist liraglutide (LIR) in a rat model of lung hypoplasia. Because we hypothesized that streptozotocin-induced diabetes rats would show deficiencies in lung function, including surfactant proteins, and develop an imbalance of the renin-<em>angiotensin</em> system in the lungs. This effect would in turn be prevented by long-acting agonists of the GLP-<em>1</em>R, such as LIR. The induction of diabetes reduced the surfactant protein A and B in the lungs and caused the vasoconstrictor component of the renin-<em>angiotensin</em> system to predominate, which in turn increased <em>angiotensin</em> II levels, and ultimately being associated with right ventricle hypertrophy. LIR restored surfactant protein levels and reversed the imbalance in the renin-<em>angiotensin</em> system in this type <em>1</em> diabetes mellitus rat model. Moreover, LIR provoked a strong increase in <em>angiotensin</em>-converting enzyme 2 expression in the lungs of both diabetic and control rats, and in the circulating <em>angiotensin</em>(<em>1</em>-<em>7</em>) in diabetic animals. These effects prompted complete reversion of right ventricle hypertrophy. The consequences of LIR administration were independent of glycemic control and of glucocorticoids, and they involved NK2 homeobox <em>1</em> signaling. This study demonstrates by first time that GLP-<em>1</em>R agonists, such as LIR, might improve the cardiopulmonary complications associated with diabetes.

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) [ANG-(<em>1</em>-<em>1</em>2)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(<em>1</em>-<em>1</em>2) being an alternate renin-independent, <em>angiotensin</em>-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac <em>angiotensins</em>. Significant decreases in plasma ANG-(<em>1</em>-<em>1</em>2), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(<em>1</em>-<em>1</em>2), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(<em>1</em>-<em>1</em>2) decreased 34% following nephrectomy, which accompanied <em>7</em>8 and 66% decreases in plasma ANG I and ANG II, respectively (P < 0.05 vs. controls). Contrastingly, cardiac ANG-(<em>1</em>-<em>1</em>2) in anephric rats averaged 2<em>7</em>6 +/- 24 fmol/mg compared with <em>1</em>44 +/- 20 fmol/mg in controls (P < 0.005). Cardiac ANG I and ANG II values were 300 +/- <em>1</em>5 and 62 +/- <em>7</em> fmol/mg, respectively, in anephric rats compared with <em>1</em><em>7</em>2 +/- 8 fmol/mg for ANG I and 42 +/- 4 fmol/mg for ANG II in controls (P < 0.00<em>1</em>). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue <em>angiotensin</em>ogen, ANG I, ANG II, and AT(<em>1</em>) receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in p4<em>7</em>(phox), Rac<em>1</em>, and Nox4 isoform expression. In conclusion, ANG-(<em>1</em>-<em>1</em>2) may be a functional precursor for <em>angiotensin</em> peptide formation in the absence of circulating renin.

Although NAD(P)H oxidase-derived superoxide (O(2)(-)) is increased during the development of <em>angiotensin</em> II (ANG II)-dependent hypertension, vascular regulation at the protein level has not been reported. We have shown that four major components of NAD(P)H oxidase are located primarily in the vascular adventitia as a primary source of vascular O(2)(-). Here we compare vascular levels of O(2)(-) and NAD(P)H oxidase in normotensive and ANG II-infused hypertensive mice and show that, after <em>7</em> days of ANG II infusion (<em>7</em>50 microg. kg(-<em>1</em>). day(-<em>1</em>) ip) in C5<em>7</em>B<em>1</em>/6 mice, systolic blood pressure was increased compared with that after sham infusion, concomitant with increased O(2)(-) in the thoracic aorta as measured using lucigenin (25 microM)-enhanced chemiluminescence. Both p6<em>7</em>(phox) and gp9<em>1</em>(phox) were detectable by Western blotting in aortic homogenates, and we observed increased protein levels of NAD(P)H oxidase subunits. These ANG II-induced increases were normalized by simultaneous treatment with the AT(<em>1</em>) receptor antagonist losartan. Moreover, the primary location of these subunits was the adventitia as detected immunohistochemically. Our results suggest that ANG II-induced increases in O(2)(-) are due to increased adventitial NAD(P)H oxidase activity, brought about by the heightened expression and interaction of its components.

The influence of estrogen on the regulation of cardiovascular function remains a controversial and complex area of investigation. We assessed the effects of estrogen depletion in the congenic mRen(2). Lewis rat, established from the back-cross of the original (mRen2)-2<em>7</em> transgenic onto the Lewis inbred strain. Ovariectomy of heterozygous mRen(2). Lewis at 4 to 5 weeks resulted in a progressive increase in blood pressure compared with the sham surgery congenics at weeks 6 to <em>1</em><em>1</em>. At <em>1</em><em>1</em> weeks, the ovariectomized mRen(2). Lewis (OVX) systolic blood pressure averaged <em>1</em>95+/-3.<em>7</em> mm Hg versus <em>1</em>4<em>1</em>+/-4.0 mm Hg for sham. Plasma <em>Angiotensin</em> (Ang) II, serum ACE activity, plasma renin concentration, as well as urinary excretion of Ang II, 8-isoprostane F2alpha, and endothelin-<em>1</em> were elevated; however, renal mRNA levels of eNOS were suppressed after ovariectomy. Estrogen replacement reduced blood pressure below both the sham and OVX by <em>1</em><em>1</em> weeks (<em>1</em>25+/-2.9 mm Hg, n=<em>7</em>, P<0.0<em>1</em> versus OVX and sham). Moreover, the AT<em>1</em> receptor antagonist olmesartan (CS866; week <em>1</em>2 to <em>1</em>6) essentially normalized blood pressure to <em>1</em><em>1</em>3+/-5.4 mm Hg (n=6, P<0.0<em>1</em> versus OVX and sham). The attenuation of the hypertension was still evident <em>7</em> weeks after complete withdrawal of treatment (<em>1</em>24+/-4.<em>1</em> mm Hg at week 23). In summary, the OVX mRen.2. Lewis exhibited a rapid and sustained increase in blood pressure. Estrogen or olmesartan lowered pressure by a similar extent. We conclude that the ovary exerts considerable influence on the regulation of the blood pressure in the mRen2. Lewis strain, possibly by limiting activation of the renin-<em>angiotensin</em> system.

Renin, as a component of the renin-<em>angiotensin</em> system, plays important roles in the regulation of blood pressure, electrolyte homeostasis, and mammalian renal development. Transcription of renin genes is subject to complex developmental and tissue-specific regulation. Progress has been made recently in elucidating the molecular mechanisms involved in renin gene expression. Using mouse As4.<em>1</em> cells, which have many features characteristic of the renin-expressing juxtaglomerular cells of kidney, a proximal promoter region (-<em>1</em>9<em>7</em> to -50 bp) and an enhancer (-2866 to -2625 bp) have been identified in the mouse renin gene, Ren-<em>1</em>(c), that are critical for its expression. The proximal promoter region contains at least <em>7</em> transcription factor-binding sites, including a binding site for the products of Hox, developmental control genes. The enhancer consists of at least <em>1</em><em>1</em> transcription factor-binding sites and is responsive to various signal transduction pathways, including cAMP, retinoic acid, endothelin-<em>1</em>, and cytokines, to alter renin mRNA levels. Sequence highly homologous to the mouse enhancer is also found in the human and rat renin genes. How these regulatory regions function in vivo will be the focus of future study.

To determine the role of <em>angiotensin</em>ogen and <em>angiotensin</em> II type-<em>1</em> (AT<em>1</em>) receptor genes in hypertension, spontaneously hypertensive rats (SHR) were injected with synthetic antisense oligodeoxynucleotides (ODNs), intracerebroventricularly (i.c.v). Antisense ODNs were constructed to bases -5 to +<em>1</em>3 of <em>angiotensin</em>ogen mRNA (<em>1</em>8-mer) and to bases +63 to +<em>7</em><em>7</em> (<em>1</em>5-mer) of <em>angiotensin</em> II type-<em>1</em> receptor mRNA. Hypertension was significantly reduced by the application of 50 micrograms of both antisense ODNs to normotensive levels. The phosphorothioated antisense ODN to the AT<em>1</em> receptor produced long-lasting (<em>7</em> days) decreases in blood pressure. After AT<em>1</em> antisense treatment, AT<em>1</em> receptors were reduced in the paraventricular nucleus (PVN) and in the anterior third ventricle area (AV3V). Following <em>angiotensin</em>ogen antisense treatment, <em>angiotensin</em> II levels were significantly reduced in the brainstem (P < 0.05), indicating arrest of <em>angiotensin</em> II synthesis. The results demonstrate that inhibiting the brain renin-<em>angiotensin</em> system by antisense inhibition of the <em>angiotensin</em>ogen and the AT<em>1</em> receptor genes, lowers high blood pressure in the SHR. The antisense administration to specific genes of the tissue renin-<em>angiotensin</em> system offers the possibility of a new approach to developing antihypertension treatments.

The <em>angiotensin</em> II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-<em>1</em>-{[2'-(5-oxo-4,5-dihydro-<em>1</em>,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-<em>1</em>H-benzimidazole-<em>7</em>-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of ¹²⁵I-Sar¹-Ile⁸-AII to human <em>angiotensin</em> type <em>1</em> receptors with an IC₅₀ of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of <em>7</em>.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC₅₀ values of 6.<em>7</em>, 5.<em>1</em>, 44.9, and <em>1</em>5.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC₅₀ values of 242.5, <em>1</em>9<em>1</em>.6,>><em>1</em>0,000, and>><em>1</em>0,000 nM). AZL also inhibited the accumulation of AII-induced inositol <em>1</em>-phosphate (IP<em>1</em>) in the cell-based assay with an IC₅₀ value of 9.2 nmol; this effect was resistant to washout (IC₅₀ value of 8<em>1</em>.3 nM). Olmesartan and valsartan inhibited IP<em>1</em> accumulation with IC₅₀ values of <em>1</em>2.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC₅₀ value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human <em>angiotensin</em> type <em>1</em> receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'₂ value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.

BACKGROUND

Previous studies suggest that angiotensin II (Ang II) upregulates the expression of tumor necrosis factor (TNF) in nonmyocyte cell types; however, the effect of Ang II on TNF expression in the adult mammalian heart is not known.

RESULTS

To determine whether Ang II was sufficient to provoke TNF biosynthesis in the adult heart, we examined the effects of Ang II in isolated buffer-perfused Langendorff feline hearts. Ang II (10(-7) mol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF mRNA and protein biosynthesis in the heart as well as in cultured adult cardiac myocytes. The effects of Ang II on myocardial TNF mRNA and protein synthesis were mediated through the angiotensin type 1 receptor (AT1R), insofar as an AT1R antagonist (AT1a) blocked the effects of Ang II, whereas an angiotensin type 2 receptor (AT2R) antagonist (AT2a) had no effect. Stimulation with Ang II led to the activation of nuclear factor-kappaB and activator protein-1 (AP-1), two transcription factors that are important for TNF gene expression. Nuclear factor-kappaB activation was accompanied by phosphorylation of IkappaBalpha on serine 32 as well as degradation of IkappaBalpha, suggesting that the effects of Ang II were mediated through an IkappaBalpha-dependent pathway. The important role of protein kinase C (PKC) was suggested by studies in which a phorbol ester triggered TNF biosynthesis, and a PKC inhibitor abrogated Ang II-induced TNF biosynthesis.

CONCLUSIONS

These studies suggest that Ang II provokes TNF biosynthesis in the adult mammalian heart through a PKC-dependent pathway.

The type-2 (AT(2)) <em>angiotensin</em> (Ang) II receptor has been characterized as potentially counterregulatory to the actions of Ang II at its type-<em>1</em> (AT(<em>1</em>)) receptor. We investigated the effects of Ang II and CGP-42<em>1</em><em>1</em>2A (CGP), a selective peptide AT(2) receptor agonist, on blood pressure (BP) in rats with or without pharmacological blockade of the AT(<em>1</em>) receptor with losartan (LOS) or valsartan (VAL). In anesthetized rats (n=5 per group) receiving normal sodium intake, Ang II (200 pmol/kg per minute IV) alone increased BP from a control of <em>1</em><em>1</em>2+/-3 to <em>1</em>68+/-<em>7</em> mm Hg (P<0.00<em>1</em>) and LOS (30 mg/kg) alone decreased BP to 89+/-<em>7</em> mm Hg (P<0.000<em>1</em> from control). Ang II administered together with LOS decreased BP further to <em>7</em><em>1</em>+/-4 mm Hg (P<0.0000<em>1</em> from control and LOS alone). AT(2) receptor antagonist PD <em>1</em>23,3<em>1</em>9 (PD) completely blocked the hypotensive response to LOS combined with Ang II (P=NS from control). In conscious rats (n=5 per group) receiving normal sodium intake, VAL (<em>1</em>0 mg/kg) alone decreased BP from a control of 98+/-5 to 86+/-3 mm Hg (P<0.0000<em>1</em>). Ang II combined with VAL induced a consistent, highly significant decline in BP for 6 days to a nadir of 69+/-3 mm Hg (P<0.0<em>1</em> versus daily VAL alone). PD completely blocked the chronic hypotensive response to the combination of Ang II and VAL to control levels before VAL administration. In another study in conscious rats (n=5 per group), CGP (<em>7</em>0 microg/kg per minute) also decreased BP in VAL-treated conscious rats. BP was <em>1</em><em>1</em>9+/-3 mm Hg during the control period, decreased to 86+/-6 mm Hg during 3 days of VAL alone, (P<0.0000<em>1</em>) and decreased further to 65+/-<em>7</em> mm Hg (P<0.00<em>1</em> from daily VAL alone) with <em>7</em> days of CGP in the presence of VAL. In the absence of VAL, CGP decreased BP for 4 consecutive days, and this response was blocked by PD. Also, the CGP-induced decrease in BP over a <em>7</em>-day period was blocked by N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. The results strongly suggest that the AT(2) receptor induces a systemic vasodilator response mediated by NO that counterbalances the vasoconstrictor action of Ang II at the AT(<em>1</em>) receptor.

We determined the mechanism accounting for the removal and metabolism of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] in 2<em>1</em> anesthetized spontaneously hypertensive (SHR), <em>1</em>8 age-matched normotensive Sprague-Dawley (SD), and 36 mRen-2 transgenic (TG+) rats. Animals of all 3 strains were provided with tap water or tap water containing losartan, lisinopril, or a combination of lisinopril and losartan for 2 weeks. On the day of the experiment, Ang-(<em>1</em>-<em>7</em>) was infused for a period of <em>1</em>5 minutes at a rate of 2<em>7</em>8 nmol . kg-<em>1</em> . min-<em>1</em>. After this time, samples of arterial blood were collected rapidly at regular intervals for the assay of plasma Ang-(<em>1</em>-<em>7</em>) levels by radioimmunoassay. Infusion of Ang-(<em>1</em>-<em>7</em>) had a minimal effect on vehicle-treated SD rats but elicited a biphasic pressor/depressor response in vehicle-treated SHR and TG+ rats. In lisinopril-treated rats, Ang-(<em>1</em>-<em>7</em>) infusion increased blood pressure, whereas losartan treatment abolished the pressor component of the response without altering the secondary fall in arterial pressure. Combined treatment with lisinopril and losartan abolished the cardiovascular response to Ang-(<em>1</em>-<em>7</em>) in all 3 strains. In vehicle-treated SD, SHR and TG+ the half-life (t<em>1</em>/2) of Ang-(<em>1</em>-<em>7</em>) averaged <em>1</em>0+/-<em>1</em>, <em>1</em>0+/-<em>1</em>, and 9+/-<em>1</em> seconds, respectively. Lisinopril alone or in combination with losartan produced a statistically significant rise in the half-life of Ang-(<em>1</em>-<em>7</em>) in all 3 strains of rats. Plasma clearance of Ang-(<em>1</em>-<em>7</em>) was significantly greater in the untreated SD rats compared with either the SHR or TG+ rat. Lisinopril treatment was associated with reduced clearance of Ang-(<em>1</em>-<em>7</em>) in all 3 strains. Concurrent experiments in pulmonary membranes from SD and SHR showed a statistically significant inhibition of <em>1</em>25I-Ang-(<em>1</em>-<em>7</em>) metabolism in the presence of lisinopril. These studies showed for the first time that the very short half-life of Ang-(<em>1</em>-<em>7</em>) in the circulation is primarily accounted for peptide metabolism by ACE. These findings suggest a novel role of ACE in the regulation of the production and metabolism of the two primary active hormones of the renin <em>angiotensin</em> system.

The in vivo efficacy of many therapeutic peptides is hampered by their rapid proteolytic degradation. Cyclization of these therapeutic peptides is an excellent way to render them more resistant against breakdown. Here, we describe the enzymatic introduction of a thioether ring in <em>angiotensin</em> [Ang-(<em>1</em>-<em>7</em>)], a heptapeptide that plays a pivotal role in the renin-<em>angiotensin</em> system and possesses important therapeutic activities. The lactic acid bacterium Lactococcus lactis, equipped with the plasmid-based nisin modification machinery, was used to produce thioether-bridged Ang-(<em>1</em>-<em>7</em>). The resulting cyclized Ang-(<em>1</em>-<em>7</em>) is fully resistant against purified <em>angiotensin</em>-converting enzyme, has significantly increased stability in homogenates of different organs and in plasma derived from pig, and displays a strongly (34-fold) enhanced survival in Sprague-Dawley (SD) rats in vivo. With respect to functional activity, cyclized Ang-(<em>1</em>-<em>7</em>) induces relaxation of precontracted SD rat aorta rings in vitro. The magnitude of this effect is 2-fold larger than that obtained for natural Ang-(<em>1</em>-<em>7</em>). The Ang-(<em>1</em>-<em>7</em>) receptor antagonist D-Pro(<em>7</em>)-Ang-(<em>1</em>-<em>7</em>), which completely inhibits the activity of natural Ang-(<em>1</em>-<em>7</em>), also abolishes the vasodilation by cyclized Ang-(<em>1</em>-<em>7</em>), providing evidence that cyclized Ang-(<em>1</em>-<em>7</em>) also interacts with the Ang-(<em>1</em>-<em>7</em>) receptor. Taken together, applying a highly innovative enzymatic peptide stabilization method, we generated a stable Ang-(<em>1</em>-<em>7</em>) analog with strongly enhanced therapeutic potential.

Renal fibrosis is the hallmark of virtually all progressive kidney diseases and strongly correlates with the deterioration of kidney function. The renin-<em>angiotensin</em>-aldosterone system blockade is central to the current treatment of patients with chronic kidney disease (CKD) for the renoprotective effects aimed to prevent or slow progression to end-stage renal disease (ESRD). However, the incidence of CKD is still increasing, and there is a critical need for new therapeutics. Here, we review novel strategies targeting various components implicated in the fibrogenic pathway to inhibit or retard the loss of kidney function. We focus, in particular, on antifibrotic approaches that target transforming growth factor (TGF)-β<em>1</em>, a key mediator of kidney fibrosis, and exciting new data on the role of autophagy. Bone morphogenetic protein (BMP)-<em>7</em> and connective tissue growth factor (CTGF) are highlighted as modulators of profibrotic TGF-β activity. BMP-<em>7</em> has a protective role against TGF-β<em>1</em> in kidney fibrosis, whereas CTGF enhances TGF-β-mediated fibrosis. We also discuss recent advances in the development of additional strategies for antifibrotic therapy. These include strategies targeting chemokine pathways via CC chemokine receptors <em>1</em> and 2 to modulate the inflammatory response, inhibition of phosphodiesterase to restore nitric oxide-cyclic 3',5'-guanosine monophosphate function, inhibition of nicotinamide adenine dinucleotide phosphate oxidase <em>1</em> and 4 to suppress reactive oxygen species production, and inhibition of endothelin <em>1</em> or tumor necrosis factor α to ameliorate progressive renal fibrosis. Furthermore, a brief overview of some of the biomarkers of kidney fibrosis is currently being explored that may improve the ability to monitor antifibrotic therapies. It is hoped that evidence based on the preclinical and clinical data discussed in this review leads to novel antifibrotic therapies effective in patients with CKD to prevent or delay progression to ESRD.

BACKGROUND

Osteopontin (OP) has been identified in cultured rat cardiac fibroblasts, where it contributes to <em>angiotensin</em> II (AII)-induced remodeling processes; in cultured cardiomyocytes; and in macrophages in cardiac tissues with inflammation. However, the presence of OP has not been reported in histological sections of myocardial tissue. In the present study, we investigated (<em>1</em>) the regulation of OP mRNA expression in cultured rat cardiomyocytes; (2) the localization of OP mRNA in neonatal and adult normal and hypertrophied rat hearts; and (3) the histology of OP expression in myocardial specimens from humans either with myocyte hypertrophy or with no pathological changes.

RESULTS

Cultured neonatal cardiomyocytes expressed OP mRNA and were immunoreactive for OP. Endothelin-<em>1</em> (ET-<em>1</em>) and norepinephrine (NE) increased both OP and atrial natriuretic peptide (ANP) mRNA levels twofold to threefold (P<.0<em>1</em>). OP mRNA was prominent in ventricular tissue from neonatal and adult rats with renovascular hypertension and aortic banding, whereas barely detectable levels were observed in normal adult cardiac tissue. ANP and OP mRNA levels in normal and hypertrophied ventricles correlated (r2=.87, P<.00<em>1</em>). OP immunoreactivity and mRNA transcripts were predominantly found in cardiomyocytes not associated with inflammatory cells in sections from neonatal and adult hypertrophied hearts. No staining was detectable in normal adult hearts. Human myocardium with extensive fibrosis and cardiomyocyte hypertrophy obtained from explanted hearts with either idiopathic (n=5) or ischemic cardiomyopathy (n=7) demonstrated substantial myocyte immunoreactivity for both OP and ANP in right and left ventricles that was not associated with leukocyte infiltration. In situ hybridization identified cardiomyocytes as the major source of OP mRNA transcripts in these hearts. In contrast, OP immunoreactivity was not detectable in four of five endomyocardial biopsies with normal histology.

CONCLUSIONS

The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.

BACKGROUND

Hypovolemic shock of marked severity and duration may progress to cardiovascular collapse unresponsive to volume replacement and drug intervention. On the basis of clinical observations, we investigated the action of vasopressin in an animal model of this condition.

RESULTS

In <em>7</em> dogs, prolonged hemorrhagic shock (mean arterial pressure [MAP] of approximately 40 mm Hg) was induced by exsanguination into a reservoir. After approximately 30 minutes, progressive reinfusion was needed to maintain MAP at approximately 40 mm Hg, and by approximately <em>1</em> hour, despite complete restoration of blood volume, the administration of norepinephrine approximately 3 micrograms . kg(-<em>1</em>). min(-<em>1</em>) was required to maintain this pressure. At this moment, administration of vasopressin <em>1</em> to 4 mU. kg(-<em>1</em>). min(-<em>1</em>) increased MAP from 39+/-6 to <em>1</em>28+/-9 mm Hg (P<0.00<em>1</em>), primarily because of peripheral vasoconstriction. In 3 dogs subjected to similar prolonged hemorrhagic shock, <em>angiotensin</em> II <em>1</em>80 ng. kg(-<em>1</em>). min(-<em>1</em>) had only a marginal effect on MAP (45+/-<em>1</em>2 to 49+/-<em>1</em>5 mm Hg). Plasma vasopressin was markedly elevated during acute hemorrhage but fell from 3<em>1</em>9+/-66 to 29+/-9 pg/mL before administration of vasopressin (P<0.0<em>1</em>).

CONCLUSIONS

Vasopressin is a uniquely effective pressor in the irreversible phase of hemorrhagic shock unresponsive to volume replacement and catecholamine vasopressors. Vasopressin deficiency may contribute to the pathogenesis of this condition.

BACKGROUND

<em>Angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) attenuates the development of heart failure. In addition to its local effects on cardiovascular tissue, Ang-(<em>1</em>-<em>7</em>) also stimulates bone marrow, which harbors cells that might complement the therapeutic effect of Ang-(<em>1</em>-<em>7</em>). We studied the effects of Ang-(<em>1</em>-<em>7</em>) either produced locally in the heart or subcutaneously injected during the development of heart failure induced by myocardial infarction (MI) and explored the role of cardiovascular progenitor cells in promoting the effects of this heptapeptide.

RESULTS

Effects of Ang-(<em>1</em>-<em>7</em>) on bone marrow-derived mononuclear cells in rodents, particularly endothelial progenitor cells, were investigated in vitro and in vivo in rats, in mice deficient for the putative Ang-(<em>1</em>-<em>7</em>) receptor Mas, and in mice overexpressing Ang-(<em>1</em>-<em>7</em>) exclusively in the heart. Three weeks after MI induction through permanent coronary artery occlusion, effects of Ang-(<em>1</em>-<em>7</em>) either produced locally in the heart or injected into the subcutaneous space were investigated. Ang-(<em>1</em>-<em>7</em>) stimulated proliferation of endothelial progenitor cells isolated from sham or infarcted rodents. The stimulation was blunted by A<em>7</em><em>7</em>9, a Mas receptor blocker, or by Mas deficiency. Infusion of Ang-(<em>1</em>-<em>7</em>) after MI increased the number of c-kit- and vascular endothelial growth factor-positive cells in infarcted hearts, inhibited cardiac hypertrophy, and improved cardiac function 3 weeks after MI, whereas cardiomyocyte-derived Ang-(<em>1</em>-<em>7</em>) had no effect.

CONCLUSIONS

Our data suggest circulating rather than cardiac Ang-(<em>1</em>-<em>7</em>) to be beneficial after MI. This beneficial effect correlates with a stimulation of cardiac progenitor cells in vitro and in vivo. This characterizes the heptapeptide as a promising new tool in stimulating cardiovascular regeneration under pathophysiological conditions.

Skeletal muscle is a tissue that shows the most plasticity in the body; it can change in response to physiological and pathological stimuli. Among the diseases that affect skeletal muscle are myopathy-associated fibrosis, insulin resistance, and muscle atrophy. A common factor in these pathologies is the participation of the renin-<em>angiotensin</em> system (RAS). This system can be functionally separated into the classical and nonclassical RAS axis. The main components of the classical RAS pathway are <em>angiotensin</em>-converting enzyme (ACE), <em>angiotensin</em> II (Ang-II), and Ang-II receptors (AT receptors), whereas the nonclassical axis is composed of ACE2, <em>angiotensin</em> <em>1</em>-<em>7</em> [Ang (<em>1</em>-<em>7</em>)], and the Mas receptor. Hyperactivity of the classical axis in skeletal muscle has been associated with insulin resistance, atrophy, and fibrosis. In contrast, current evidence supports the action of the nonclassical RAS as a counter-regulator axis of the classical RAS pathway in skeletal muscle. In this review, we describe the mechanisms involved in the pathological effects of the classical RAS, advances in the use of pharmacological molecules to inhibit this axis, and the beneficial effects of stimulation of the nonclassical RAS pathway on insulin resistance, atrophy, and fibrosis in skeletal muscle.

OBJECTIVE

To study the effects of addition of spironolactone to angiotensin-converting enzyme (ACE) inhibition or angiotensin II (AngII) receptor antagonism on proteinuria, blood pressure (BP) and renal function in overt type 2 diabetic nephropathy.

METHODS

A placebo-controlled, double-blind, parallel-group trial in patients from two outpatient clinics with a follow-up of 1 year.

METHODS

Type 2 diabetic patients with macroalbuminuria, despite long-term use of an ACE inhibitor or AngII receptor blocker were allocated to spironolactone, 25-50 mg once daily (n = 29) or placebo (n = 30). Urinary albumin to creatinine ratio, BP and biochemical parameters were measured at regular intervals.

RESULTS

Five patients of the spironolactone and one of the placebo group developed hyperkalemia and had to be excluded. Compared to other patients their baseline serum creatinine [161 (123-248) versus 88 (72-170) micromol/l] and potassium concentrations (4.7 +/- 0.3 versus 4.2 +/- 0.2 mmol/l) were elevated (P < 0.001). Albuminuria decreased by 40.6% [95% confidence interval (CI) 23.4-57.8%] and BP by 7 mmHg (2-12 mmHg)/3 mmHg (1-6 mmHg) with spironolactone, but did not change with placebo. Estimated glomerular filtration rate (eGFR) during the 1-year follow-up declined on average by 12.9 ml/min per 1.73 m (9.5-16.5 ml/min per 1.73 m) in the spironolactone and by 4.9 ml/min per 1.73 m (0.8-8.9 ml/min per 1.73 m) in the placebo group (P = 0.004). This decline was progressive in the placebo but leveled off in the spironolactone group. In the spironolactone group changes in albuminuria and GFR were correlated (r = 0.48, P = 0.007).

CONCLUSIONS

Addition of spironolactone to an ACE inhibitor or AngII receptor blocker is associated with a marked and sustained antiproteinuric effect, which in part relates to the more pronounced reduction in GFR.

<em>Angiotensin</em>-converting enzyme-2 (ACE-2) is a homolog of ACE that preferentially forms <em>angiotensin</em>-(ANG)-<em>1</em>-<em>7</em> from <em>angiotensin</em> II (ANG II). We investigated the cardioprotective effects of telmisartan, a well-known <em>angiotensin</em> receptor blockers (ARBs) against experimental autoimmune myocarditis (EAM). EAM was induced in Lewis rats by immunization with porcine cardiac myosin. The rats were divided into two groups and treated with telmisartan (<em>1</em>0 mg/kg/day) or vehicle for 2<em>1</em> days. Myocardial functional parameters were significantly improved by treatment with telmisartan compared with vehicle-treated rats. Telmisartan lowered myocardial protein expressions of NADPH oxidase subunits 3-nitrotyrosine, p4<em>7</em>phox, p6<em>7</em> phox, Nox-4 and superoxide production significantly than vehicle-treated rats. In contrast myocardial protein levels of ACE-2, ANG <em>1</em>-<em>7</em> mas receptor were upregulated in the telmisartan treated group compared with those of vehicle-treated rats. The myocardial protein expression levels of tumor necrosis factor receptor (TNFR)-associated factor (TRAF)-2, C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP) <em>7</em>8 were decreased in the telmisartan treated rats compared with those of vehicle-treated rats. In addition, telmisartan treatment significantly decreased the protein expression levels of phospho-p38 mitogen-activated protein kinase (MAPK), phospho-JNK, phospho-ERK and phospho (MAPK) activated protein kinase-2 than with those of vehicle-treated rats. Moreover, telmisartan significantly decreased the production of proinflammatory cytokines, myocardial apoptotic markers and caspase-3 positive cells compared with those of vehicle-treated rats. Therefore, we suggest that telmisartan was beneficial protection against heart failure in rats, at least in part by suppressing inflammation, oxidative stress, ER stress as well as signaling pathways through the modulation of ACE2/ANG<em>1</em>-<em>7</em>/Mas receptor axis.

Hypertension is the most common modifiable risk factor for cardiovascular disease and death, and lowering blood pressure with antihypertensive drugs reduces target organ damage and prevents cardiovascular disease outcomes. Despite a plethora of available treatment options, a substantial portion of the hypertensive population has uncontrolled blood pressure. The unmet need of controlling blood pressure in this population may be addressed, in part, by developing new drugs and devices/procedures to treat hypertension and its comorbidities. In this Compendium Review, we discuss new drugs and interventional treatments that are undergoing preclinical or clinical testing for hypertension treatment. New drug classes, eg, inhibitors of vasopeptidases, aldosterone synthase and soluble epoxide hydrolase, agonists of natriuretic peptide A and vasoactive intestinal peptide receptor 2, and a novel mineralocorticoid receptor antagonist are in phase II/III of development, while inhibitors of aminopeptidase A, dopamine β-hydroxylase, and the intestinal Na(+)/H(+) exchanger 3, agonists of components of the <em>angiotensin</em>-converting enzyme 2/<em>angiotensin</em>(<em>1</em>-<em>7</em>)/Mas receptor axis and vaccines directed toward <em>angiotensin</em> II and its type <em>1</em> receptor are in phase I or preclinical development. The two main interventional approaches, transcatheter renal denervation and baroreflex activation therapy, are used in clinical practice for severe treatment resistant hypertension in some countries. Renal denervation is also being evaluated for treatment of various comorbidities, eg, chronic heart failure, cardiac arrhythmias and chronic renal failure. Novel interventional approaches in early development include carotid body ablation and arteriovenous fistula placement. Importantly, none of these novel drug or device treatments has been shown to prevent cardiovascular disease outcomes or death in hypertensive patients.

Importance: Biological data are lacking with respect to risk of vertical transmission and mechanisms of fetoplacental protection in maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Objective: To quantify SARS-CoV-2 viral load in maternal and neonatal biofluids, transplacental passage of anti-SARS-CoV-2 antibody, and incidence of fetoplacental infection.

<strong class="sub-title"> Design, setting, and participants: </strong> This cohort study was conducted among pregnant women presenting for care at 3 tertiary care centers in Boston, Massachusetts. Women with reverse transcription-polymerase chain reaction (RT-PCR) results positive for SARS-CoV-2 were recruited from April 2 to June <em>1</em>3, 2020, and follow-up occurred through July <em>1</em>0, 2020. Contemporaneous participants without SARS-CoV-2 infection were enrolled as a convenience sample from pregnant women with RT-PCR results negative for SARS-CoV-2.

Exposures: SARS-CoV-2 infection in pregnancy, defined by nasopharyngeal swab RT-PCR.

Main outcomes and measures: The main outcomes were SARS-CoV-2 viral load in maternal plasma or respiratory fluids and umbilical cord plasma, quantification of anti-SARS-CoV-2 antibodies in maternal and cord plasma, and presence of SARS-CoV-2 RNA in the placenta.

<strong class="sub-title"> Results: </strong> Among <em>1</em>2<em>7</em> pregnant women enrolled, 64 with RT-PCR results positive for SARS-CoV-2 (mean [SD] age, 3<em>1</em>.6 [5.6] years) and 63 with RT-PCR results negative for SARS-CoV-2 (mean [SD] age, 33.9 [5.4] years) provided samples for analysis. Of women with SARS-CoV-2 infection, 23 (36%) were asymptomatic, 22 (34%) had mild disease, <em>7</em> (<em>1</em><em>1</em>%) had moderate disease, <em>1</em>0 (<em>1</em>6%) had severe disease, and 2 (3%) had critical disease. In viral load analyses among <em>1</em>0<em>7</em> women, there was no detectable viremia in maternal or cord blood and no evidence of vertical transmission. Among <em>7</em><em>7</em> neonates tested in whom SARS-CoV-2 antibodies were quantified in cord blood, <em>1</em> had detectable immunoglobuilin M to nucleocapsid. Among 88 placentas tested, SARS-CoV-2 RNA was not detected in any. In antibody analyses among 3<em>7</em> women with SARS-CoV-2 infection, anti-receptor binding domain immunoglobin G was detected in 24 women (65%) and anti-nucleocapsid was detected in 26 women (<em>7</em>0%). Mother-to-neonate transfer of anti-SARS-CoV-2 antibodies was significantly lower than transfer of anti-influenza hemagglutinin A antibodies (mean [SD] cord-to-maternal ratio: anti-receptor binding domain immunoglobin G, 0.<em>7</em>2 [0.5<em>7</em>]; anti-nucleocapsid, 0.<em>7</em>4 [0.44]; anti-influenza, <em>1</em>.44 [0.80]; P < .00<em>1</em>). Nonoverlapping placental expression of SARS-CoV-2 receptors <em>angiotensin</em>-converting enzyme 2 and transmembrane serine protease 2 was noted.

Conclusions and relevance: In this cohort study, there was no evidence of placental infection or definitive vertical transmission of SARS-CoV-2. Transplacental transfer of anti-SARS-CoV-2 antibodies was inefficient. Lack of viremia and reduced coexpression and colocalization of placental angiotensin-converting enzyme 2 and transmembrane serine protease 2 may serve as protective mechanisms against vertical transmission.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a crucial proangiogenic component in pancreatic ductal adenocarcinoma (PDA), and its high expression levels have been correlated with poor prognosis and early postoperative recurrence. Angiotensin II (AngII), which has been shown to increase VEGF production in a variety of cancers, is actively generated in the pancreas. We hypothesized that AngII plays a crucial role in PDA-associated angiogenesis.

METHODS

We analyzed the expression and localization of AngI converting enzyme (ACE) and AngII type 1 receptor (AT1R) in relation to VEGF in matched invasive human PDA (n=25) and surrounding nonmalignant tissues using real-time polymerase chain reaction, Western immunoblotting, and immunohistochemistry. VEGF levels in conditioned media of HS766T and PK9 PDA cells treated with or without AngII (10(-7) mol/L) were measured by ELISA. The effects of an AT1R blocker (losartan) and an ACE inhibitor (captopril) on VEGF production and cellular proliferation were also examined.

RESULTS

ACE and AT1R mRNA and protein levels were significantly upregulated in 19 of the 25 neoplastic tissues examined (approximately 75%), when compared with matching controls. VEGF expression was significantly higher in tissues that expressed high levels of AT1R and ACE (n=19), compared with low levels (n=4) or negative (n=2) cases. ACE protein collocalized with AT1R and VEGF in the malignant ducts and in the stromal cells. Addition of AngII significantly enhanced VEGF mRNA production and protein secretion, an effect that was prevented when cells were preincubated with captopril or losartan. Blocking endogenous AngII by captopril or losartan significantly suppressed cell proliferation.

CONCLUSIONS

Both ACE and AT1R are functionally expressed in PDA and may be involved in tumor angiogenesis. Because AT1R blockers and ACE inhibitors are already widely used clinically, they may represent a potential novel and promising strategy for controlling angiogenesis, prevention of metastasis, and prolongation of survival in patients with primary or metastatic PDA.

BACKGROUND

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have effects that extend beyond cholesterol reduction. We used an angiotensin (Ang) II-dependent model to test the hypothesis that cerivastatin ameliorates cardiac injury.

RESULTS

We treated rats transgenic for human renin and angiotensinogen (dTGR) chronically from weeks 4 to 7 with cerivastatin (0.5 mg/kg by gavage). We used immunohistochemistry, electrophoretic mobility shift assays, and reverse transcription-polymerase chain reaction techniques. Compared with control dTGR, dTGR treated with cerivastatin had reduced mortality, blood pressure, cardiac hypertrophy, macrophage infiltration, and collagen I, laminin, and fibronectin deposition. Basic fibroblast growth factor mRNA and protein expression were markedly reduced, as was interleukin-6 expression. The transcription factors NF-kappaB and AP-1 were substantially less activated, although plasma cholesterol was not decreased.

CONCLUSIONS

These results suggest that statins ameliorate Ang II-induced hypertension, cardiac hypertrophy, fibrosis, and remodeling independently of cholesterol reduction. Although the clinical significance remains uncertain, the results suggest that statins interfere with Ang II-induced signaling and transcription factor activation, thereby ameliorating end-organ damage.