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Angiotensin II Type 2 Receptor Signaling Attenuates Aortic Aneurysm in Mice Through ERK Antagonism

April 2011
Science 332(6027):361-5

April 2011
332(6027):361-5

DOI:10.1126/science.1192152

Source
PubMed

Authors:

Jefferson J Doyle

Johns Hopkins Medicine

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Angiotensin II (AngII) mediates progression of aortic aneurysm, but the relative contribution of its type 1 (AT1) and type 2 (AT2) receptors remains unknown. We show that loss of AT2 expression accelerates the aberrant growth and rupture of the aorta in a mouse model of Marfan syndrome (MFS). The selective AT1 receptor blocker (ARB) losartan abrogated aneurysm progression in the mice; full protection required intact AT2 signaling. The angiotensin-converting enzyme inhibitor (ACEi) enalapril, which limits signaling through both receptors, was less effective. Both drugs attenuated canonical transforming growth factor-β (TGFβ) signaling in the aorta, but losartan uniquely inhibited TGFβ-mediated activation of extracellular signal-regulated kinase (ERK), by allowing continued signaling through AT2. These data highlight the protective nature of AT2 signaling and potentially inform the choice of therapies in MFS and related disorders.

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Angiotensin II Type 2 Receptor Signaling Attenuates Aortic

Aneurysm in Mice Through ERK Antagonism

Jennifer P. Habashi1,2,*, Jefferson J. Doyle1,*, Tammy M. Holm1, Hamza Aziz1, Florian

Schoenhoff1, Djahida Bedja3, YiChun Chen1, Alexandra N. Modiri1, Daniel P. Judge4, and

Harry C. Dietz1,2,4,†

1Howard Hughes Medical Institute and Institute of Genetic Medicine, Johns Hopkins University

School of Medicine, Baltimore, MD 21205, USA.

2Division of Pediatric Cardiology, Department of Pediatrics, Johns Hopkins University School of

Medicine, Baltimore, MD 21205, USA.

3Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of

Medicine, Baltimore, MD 21205, USA.

4Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD21205,

USA.

Abstract

Angiotensin II (AngII) mediates progression of aortic aneurysm, but the relative contribution of its

type 1 (AT1) and type 2 (AT2) receptors remains unknown. We show that loss of AT2 expression

accelerates the aberrant growth and rupture of the aorta in a mouse model of Marfan syndrome

(MFS). The selective AT1 receptor blocker (ARB) losartan abrogated aneurysm progression in the

mice; full protection required intact AT2 signaling. The angiotensin-converting enzyme inhibitor

(ACEi) enalapril, which limits signaling through both receptors, was less effective. Both drugs

attenuated canonical transforming growth factor–β (TGFβ) signaling in the aorta, but losartan

uniquely inhibited TGFβ-mediated activation of extracellular signal–regulated kinase (ERK), by

allowing continued signaling through AT2. These data highlight the protective nature of AT2

signaling and potentially inform the choice of therapies in MFS and related disorders.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder that includes

a predisposition for aortic root aneurysm and aortic rupture. MFS is caused by a deficiency

of the microfibrillar constituent protein fibrillin-1 that is imposed by heterozygous mutations

in FBN1. In prior work, we demonstrated that transforming growth factor–β (TGFβ)

signaling was elevated in affected tissues of mice heterozygous for a cysteine substitution in

an epidermal growth factor–like domain of fibrillin-1 (Fbn1C1039G/+), the most common

class of mutation in people with MFS (1-4). Many disease manifestations—including aortic

aneurysm (1), developmental emphysema (2), myxomatous degeneration of the

atrioventricular valves (3), and skeletal muscle myopathy (4)—are attenuated by systemic

†To whom correspondence should be addressed. hdietz@jhmi.edu.

*These authors contributed equally to this work.

Supporting Online Material

www.sciencemag.org/cgi/content/full/332/6027/361/DC1

Materials and Methods

Figs. S1 to S12

References

NIH Public Access

Author Manuscript

Science. Author manuscript; available in PMC 2011 May 19.

Published in final edited form as:

Science

. 2011 April 15; 332(6027): 361–365. doi:10.1126/science.1192152.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

administration of a pan-specific polyclonal TGFβ-neutralizing antibody (TGFβNAb) in

fibrillin-1–deficient mice. Similar protection was achieved by treating Fbn1C1039G/+ mice

with the angiotensin II (AngII) type 1 (AT1) receptor blocker (ARB) losartan (1, 4). ARBs

can attenuate TGFβ signaling in some tissues by lowering the expression of TGFβ ligands,

receptors, and activators (5-7). In this mouse model of MFS, losartan's protection correlated

with decreased phosphorylation and nuclear translocation of Smad2 (pSmad2), a direct

effector of canonical TGFβ signaling, and decreased expression of prototypical Smad-

dependent TGFβ-responsive gene products, such as connective tissue growth factor and

collagens.

The contribution of AT2 to aortic aneurysm progression remains controversial. AT2

signaling can oppose AT1-mediated enhancement of TGFβ signaling in some cell types and

tissues (Fig. 1A) (8, 9). It can also induce vascular smooth muscle cell (VSMC) apoptosis,

theoretically contributing to aortic wall damage. Apoptosis was observed in cultured cells

derived from end-stage aneurysms in people with MFS (10), but has not been found in early-

or intermediate-stage aortic wall lesions in MFS mice (1). Vascular expression of AT2 is

largely limited to prenatal life, but it may continue to be relevant postnatally in the context

of certain disease states, as evidenced by the acceleration of inflammatory aneurysms in

AngII-infused mice treated with an AT2 antagonist (11). In contrast, β-aminopropionitrile

monofumarate (BAPN)–induced aortic aneurysm and dissection in rats, which was

associated with increased expression of AT2 and VSMC apoptosis, was ameliorated by

limiting AngII production with angiotensin-converting enzyme inhibitor (ACEi) but not by

selective AT1 receptor blockade (12). AT2 signaling has the capacity to attenuate both

canonical (Smad-dependent) and noncanonical (mitogen-activated protein kinase or MAPK)

TGFβ signaling cascades, most notably the extracellular signal-regulated kinase (ERK), in

some tissues (13, 14). Thus, AT2 signaling can both augment and inhibit the pathogenesis of

aneurysm in pre-clinical models, and the mechanistic explanation for the discordance is

unclear. This has direct clinical relevance, as it leaves open to question the relative

therapeutic merits of selective AT1 blockade with ARBs versus limiting signaling through

both AT1 and AT2 with ACEi, despite small trials suggesting that either approach has

potential in MFS (15-17).

To assess the role of the AT2 receptor in MFS, we bred mice with a disrupted Agtr2 allele

(encoding AT2; AT2KO) (18, 19) with Fbn1C1039G/+ mice, our established model of MFS

(20). Agtr2 is encoded on the X chromosome in humans and mice, and the AT2KO allele

associates with loss of mRNA and protein expression, as assessed by radioligand binding, in

either homozygous females or hemizygous males. The AT2KO mice develop normally, with

no evidence of cardiovascular pathology or early mortality (21).

We followed the progression of aortic root aneurysm by echocardiogram until the mice were

killed at 12 months (Fig. 1B). There was a small difference in aortic root size between wild-

type (WT) and AT2KO mice (P < 0.05) at 2 months, but this difference was absent at all

future time points (P = 0.70). The aortic root diameter of AT2KO:Fbn1C1039G/+ mice was

significantly larger than that seen in Fbn1C1039G/+ mice at 2 months of age (P < 0.001), and

this difference was maintained through to 12 months of life (P <0.05). The postnatal aortic

root growth over 10 months was not different between Fbn1C1039G/+ mice with or without

AT2 expression (P = 0.80). This could reflect postnatal waning of AT2 receptor expression,

attainment of an absolute threshold of aortic root growth rate in AT2KO:Fbn1C1039G/+ mice,

and/or the accelerated death observed in AT2KO: Fbn1C1039G/+ mice that effectively

removed the most severely affected animals from later analyses. We found that 32% of

AT2KO:Fbn1C1039G/+ mice died before the scheduled killing, compared with 12% of

Fbn1C1039G/+ mice (P < 0.01) and 0% of AT2KO or WT mice (Fig. 1C). Growth of the

more distal ascending aorta over 10 months was significantly greater in

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AT2KO:Fbn1C1039G/+ mice compared with Fbn1C1039G/+ littermates (P < 0.05), whereas

there was no significant difference between WT, AT2KO, and Fbn1C1039G/+ mice (Fig. 1D).

Histological and morphometric analyses of the aortic media were performed at 12 months.

AT2KO:Fbn1C1039G/+ mice showed medial thickening, reduced elastin content, and

increased elastic fiber fragmentation (Fig. 2A and figs. S1 to S3) compared with

Fbn1C1039G/+ or AT2KO mice (P < 0.01 for all comparisons). These parameters were not

significantly different in AT2KO and WT mice (P = 0.07, P = 0.68, and P = 1.0,

respectively). Therefore, the histological changes in the aorta paralleled the

echocardiography findings, which supported the conclusion that AT2 receptor elimination

exacerbates aortic disease in MFS mice.

The potential for exacerbation of the MFS phenotype outside of the cardiovascular system

was also assessed. At 12 months, excised lungs were inflated with agar, sectioned, and

stained for histological and morphometric analyses (figs. S4 and S5). Increased distal

airspace caliber, a marker of impaired distal alveolar septation and emphasematous lung

disease, can be quantified by calculating a mean linear intercept (MLI). There was no

significant difference in MLI between WT and AT2KO mice (P = 1.0). Compared with WT

and AT2KO littermates, Fbn1C1039G/+ mice had a significant increase in MLI (P < 0.05),

whereas AT2KO:Fbn1C1039G/+ mice had a yet further increase in MLI (P < 0.05). This

confirms that AT2 receptor elimination can exacerbate the MFS phenotype outside of the

cardiovascular system.

We next performed a head-to-head comparison of ACEi versus ARBs. Fbn1C1039G/+ mice

and WT littermates were treated with hemodynamically equivalent doses (fig. S6) of either

the ACEi enalapril (10 to 15 mg/kg of body weight per day) or the ARB losartan (40 to 60

mg/kg per day) (22), beginning at 8 weeks of age, and were assessed with serial

echocardiograms. Aortic root growth over the 7 months of treatment was significantly

greater in placebo-treated Fbn1C1039G/+ mice compared with WT littermates (P < 0.01),

whereas losartan led to a significant regression in growth in Fbn1C1039G/+ mice (P <

0.0001), to rates that were significantly less than that seen in WT littermates (P < 0.0001)

(1). It is noteworthy that losartan reduced aortic root growth in Fbn1C1039G/+ mice, but had

no effect in WT littermates (P = 0.27). Enalapril treatment had significantly less effect than

losartan in Fbn1C1039G/+ mice (P < 0.0001); in fact, it was only marginally better than

placebo treatment (P = 0.05) (Fig. 2B). Enalapril was also no more beneficial than placebo

in improving aortic architecture score in Fbn1C1039G/+ mice (P = 0.19), whereas losartan

was significantly more beneficial than both placebo and enalapril treatment (P < 0.05 for

both) (fig. S7).

We next assessed whether AT2 signaling is needed to achieve losartan's full therapeutic

benefit. AT2KO:Fbn1C1039G/+ mice were treated with losartan from 8 weeks of age and

followed by serial echocardiography until they were killed at 9 months of age (Fig. 2C).

Although there was a trend for increased aortic root growth in AT2KO:Fbn1C1039G/+ mice

compared with Fbn1C1039G/+ littermates (P = 0.06), the decrease in aortic root growth seen

in AT2KO:Fbn1C1039G/+ mice treated with losartan was only 40% of that seen in

Fbn1C1039G/+ animals that expressed AT2 (P < 0.001), despite there being no difference in

blood pressure between the groups (Fig. 2C and fig. S6). The modest reduction in aortic root

growth seen in losartan-treated AT2KO:Fbn1C1039G/+ mice was comparable to that

previously observed with propranolol, and it may be similarly attributable to a decline in

blood pressure rather than a modulation of cytokine signaling (1).

Together, these experiments suggest that AT2 signaling protectively modifies MFS and that

the therapeutic effect of ACEi likely relates to AT1 receptor blockade or antihypertensive

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effects. In addition, we have shown that selective AT1 antagonism with the ARB losartan is

beneficial in Fbn1C1039G/+ mice and that AT2 signaling is needed to achieve the full

potential of ARBs. Both the canonical (Smad-dependent) and non-canonical (MAPK,

predominantly ERK1/2 but also JNK in some experimental contexts) TGFβ signaling

cascades are activated in Fbn1C1039G/+ mice in a TGFβ- and AT1 receptor–dependent

manner (23). To investigate the mechanism of protection by AT2 receptor signaling, we

monitored the status of both canonical and noncanonical TGFβ signaling in Fbn1C1039G/+

mice lacking the AT2 receptor or in response to losartan or enalapril treatment. Western blot

analysis showed that Smad2 activation was significantly greater in the aortic root and

proximal ascending aorta of Fbn1C1039G/+ mice compared with WT controls (P < 0.01) but

that there was no significant difference between AT2KO:Fbn1C1039G/+ and Fbn1C1039G/+

mice (P = 0.30). In contrast, ERK1/2 activation was significantly greater in Fbn1C1039G/+

mice compared with WT littermates (P < 0.01) and was further increased in

AT2KO:Fbn1C1039G/+ mice compared with Fbn1C1039G/+ (P < 0.01), AT2KO (P < 0.01),

and WT littermates (P < 0.001) (Fig. 3A). The difference in ERK1/2 activation was specific

to the aortic root and proximal ascending aorta, the areas most predisposed to aneurysm

formation in MFS, as there was no significant difference in the descending thoracic aortas of

the same animals (fig. S8). No significant differences in JNK1 or p38 activation were

observed (fig. S9).

In our comparison of ARBs versus ACEi, Smad2 activation was significantly greater in

Fbn1C1039G/+ mice compared with WT controls (P < 0.05), and losartan treatment

significantly decreased Smad2 activation in Fbn1C1039G/+ mice (P < 0.05) to levels

indistinguishable from WT (P = 0.31) (Fig. 3B). Enalapril reduced Smad2 activation in

Fbn1C1039G/+ mice significantly more than losartan (P < 0.01), a finding that did not parallel

the therapeutic effects of these agents (Fig. 2B). ERK1/2 activation was significantly greater

in Fbn1C1039G/+ mice compared with WT controls (P < 0.01), and treatment with losartan

reduced it to WT levels (P = 0.80). In contrast, enalapril treatment had significantly less

effect on ERK1/2 activation than losartan (P < 0.001); in fact, it was no more effective than

placebo (P = 0.50). JNK1 and p38 activation was similar in Fbn1C1039G/+ and WT mice;

both losartan and enalapril caused a modest reduction in JNK1 activation (P < 0.01), but

neither had any effect on p38 activation (fig. S10). Thus, the biochemical status of the

noncanonical ERK1/2, but not the canonical Smad, TGFβ signaling cascade correlated with

the therapeutic effects of these agents. In keeping with this finding, losartan had a reduced

ability to lower ERK1/2 activation in Fbn1C1039G/+ mice lacking the AT2 receptor (P <

0.01) (Fig. 3C). By contrast, there was no significant difference in Smad2, JNK1, or p38

activation in losartan-treated Fbn1C1039G/+ mice that did or did not express AT2 (fig. S11).

To assess for a contribution of other components of the renin-angiotensin-aldosterone

system, we treated Fbn1C1039G/+ mice with the aldosterone receptor antagonist

spironolactone (24). We found no significant inhibition of aortic root growth over 7 months

time (P = 0.23) (fig. S12).

In conclusion, dual blockade of AT1 receptor–mediated ERK1/2 activation and AT2

receptor–mediated ERK1/2 inhibition, as occurs either with the use of ACEi in

Fbn1C1039G/+ mice or the use of losartan in AT2KO:Fbn1C1039G/+ mice, results in no net

change in ERK1/2 activation status and adds a very modest therapeutic benefit. By contrast,

losartan reduces ERK1/2 phosphorylation through a combination of both inhibiting AT1

receptor–mediated ERK activation and by shunting AngII signaling through the AT2

receptor. This indicates that, in the presence of AT1 receptor blockade, ongoing AT2

receptor signaling is required for the attenuation of ERK phosphorylation and that enalapril's

lack of effect on ERK is attributable to the loss of AT2 receptor signaling potential with this

agent (Fig. 3D). Given that the small reduction in aortic root growth in Fbn1C1039G/+ mice

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achieved by enalapril in this study was comparable to that achieved previously by

propanolol (1), this suggests that its small beneficial effect may well have been mediated by

blood pressure reduction. Although the concordant effects of prior manipulations and

therapies on canonical and noncanonical TGFβ signaling made it impossible to dissect their

relative contributions, the differential effects of enalapril treatment suggest that TGFβ-

mediated ERK1/2 activation is the predominant driver of aneurysm progression in MFS. In

light of this, analysis of ERK1/2 activation status may permit the optimization of dosing

regimens for losartan or other ARBs in ongoing or future clinical trials in people with MFS.

Furthermore, focused attention on the ERK1/2 signaling cascade may unveil new

therapeutic targets in the treatment of aortic aneurysm disease.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

Supported by the NIH (H.C.D., D.P.J.); Howard Hughes Medical Institute (H.C.D.); the National Marfan

Foundation (H.C.D., J.P.H., J.J.D.); the Cellular and Molecular Medicine Training Program, Johns Hopkins School

of Medicine (J.J.D.); and the Smilow Center for Marfan Syndrome Research (H.C.D.). We thank T. Inagami for the

AT2KO mice. Johns Hopkins University and the authors (H.C.D., J.P.H., D.P.J.) have filed a patent relating to the

use of TGFβ antagonists, including AT1 receptor blockers, for the treatment of Marfan syndrome.

References and Notes

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Fig. 1.

The role of AngII in the aorta. (A) AngII acts on the AT1 receptor causing increased cellular

proliferation, fibrosis, and matrix metalloproteinase–2and/or −9 (MMP2/9) activity while

decreasing apoptosis. Conversely, the AT2 receptor is thought to decrease proliferation,

fibrosis, and MMP activity, while increasing apoptosis. ACEi's block the conversion of

AngI to AngII, limiting signaling through both AT1 and AT2 receptors, whereas ARBs

selectively block AT1. (B) Average absolute aortic root diameter (±2SEM) measured

serially by echo-cardiogram over the first year of life. Note that AT2KO:Fbn1C1039G/+ mice

have a significantly larger aortic root diameter than Fbn1C1039G/+ mice at each time point.

AT2KO:Fbn1C1039G/+ as compared with Fbn1C1039G/+ mice. (D) Ascending aortic growth

from 2 to 12 months of age. Note the increased rate of ascending aortic growth in

AT2KO:Fbn1C1039G/+ mice. Final absolute ascending aortic diameter: WT (1.41 ± 0.07

mm), AT2KO (1.40 ± 0.07 mm), Fbn1C1039G/+ (1.42 ± 0.20 mm), AT2KO:Fbn1C1039G/+

(1.72 ± 0.42 mm). (B to D) WT (n = 5), AT2KO (n =10), Fbn1C1039G/+ (n =17),

AT2KO:Fbn1C1039G/+ (n =19). *P < 0.05; †P < 0.001; ††P < 0.0001; NS, not significant.

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Fig. 2.

Therapeutic effects in the aorta. (A) WT (n = 5), AT2KO (n = 4), Fbn1C1039G/+ (n = 7), and

AT2KO:Fbn1C1039G/+ (n = 7) mice. Verhoeff–Van Gieson (VVG) stain reveals diffuse

fragmentation of elastic fibers and thickening of the media in Fbn1C1039G/+ mice; these

findings are exaggerated in AT2KO:Fbn1C1039G/+ mice. (B) Average aortic root growth

(T2SEM) over 7 months of treatment in placebo- (n = 13) orlosartan- (n = 7) treated WT

mice and placebo- (n = 17), losartan- (n = 5), or enalapril- (n = 15) treated Fbn1C1039G/+

mice, as measured by echocardiography. Note the regression in aortic size observed in

losartan-treated Fbn1C1039G/+ mice and the marginal (P = 0.05) decrease in growth in the

enalapril-treated cohort. Final absolute aortic root diameter: WT (1.74 ± 0.10 mm), losartan-

treated WT (1.77 ± 0.15 mm), Fbn1C1039G/+ (2.19 ± 0.19 mm), losartan-treated

Fbn1C1039G/+ (1.96 ± 0.09 mm), and enalapril-treated Fbn1C1039G/+ (2.18 ± 0.18 mm). (C)

Average aortic root growth (±2SEM) over 7 months of treatment in WT (n = 8), placebo- (n

= 22), and losartan- (n = 11) treated Fbn1C1039G/+ mice and placebo- (n = 19) and losartan-

(n = 6) treated AT2KO:Fbn1C1039G/+ mice. Note the diminished effectiveness of losartan

treatment in AT2KO:Fbn1C1039G/+ mice, as compared with losartan treatment in

Fbn1C1039G/+ mice. Final absolute aortic root diameter: WT (1.77 ± 0.10 mm),

Fbn1C1039G/+ (2.13 ± 0.16 mm), AT2KO:Fbn1C1039G/+ (2.34 ± 0.13 mm), losartan-treated

Fbn1C1039G/+ (1.96 ± 0.09 mm), and losartan-treated AT2KO:Fbn1C1039G/+ (2.06 ± 0.07

mm). *P < 0.05; **P < 0.01; †P < 0.001; ††P < 0.0001; NS, not significant.

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Fig. 3.

Mechanism of protection by AT2 signaling. (A) Western blot analysis of ERK1/2 and

Smad2 activation in the aortic root and proximal ascending aorta of four mice of each

genotype. Note that Smad2 activation is increased equally in AT2KO:Fbn1C1039G/+ and

Fbn1C1039G/+ mice, compared with WT littermates. ERK1/2 activation is significantly

increased in Fbn1C1039G/+ mice when compared with WT littermates and is further

increased in AT2KO:Fbn1C1039G/+ mice. (B) Western blot analysis of ERK1/2 and Smad2

activation in the aortic root and proximal ascending aortas of three each of WT and

placebo-, losartan- or enalapril-treated Fbn1C1039G/+ mice. Note that Smad2 activation is

decreased in both losartan- and enalapril-treated Fbn1C1039G/+ mice when compared with

placebo-treated animals, with a more pronounced effect in enalapril-treated animals. In

contrast, enalapril treatment failed to reduce ERK1/2 activation, whereas losartan reduced

ERK1/2 activation to levels indistinguishable from WT littermates. (C) Western blot

analysis of ERK1/2 activation in the aortic root and proximal ascending aorta of three WT,

AT2KO, and placebo- or losartan-treated AT2KO:Fbn1C1039G/+ mice. Note that losartan

loses its ability to decrease ERK1/2 activation in AT2KO:Fbn1C1039G/+ mice, demonstrating

that the inhibition of ERK1/2 activation is mediated by the AT2 receptor. (D) Summary of

the effects of AngII receptors on both canonical and noncanonical TGFβ signaling. AT1

receptor stimulation drives ERK1/2 activation, whereas AT2 receptor stimulation inhibits it.

Losartan attenuates ERK1/2 activation by blocking the AT1 cascade while simultaneously

shunting signaling through the AT2 receptor. *P <0.05; **P <0.01; †P < 0.001; NS, not

significant.

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Investigation of Strategies to Block Downstream Effectors of AT1R-Mediated Signalling to Prevent Aneurysm Formation in Marfan Syndrome

Article

Full-text available

May 2024
INT J MOL SCI

Cardiovascular outcome in Marfan syndrome (MFS) patients most prominently depends on aortic aneurysm progression with subsequent aortic dissection. Angiotensin II receptor blockers (ARBs) prevent aneurysm formation in MFS mouse models. In patients, ARBs only slow down aortic dilation. Downstream signalling from the angiotensin II type 1 receptor (AT1R) is mediated by G proteins and β-arrestin recruitment. AT1R also interacts with the monocyte chemoattractant protein-1 (MCP-1) receptor, resulting in inflammation. In this study, we explore the targeting of β-arrestin signalling in MFS mice by administering TRV027. Furthermore, because high doses of the ARB losartan, which has been proven beneficial in MFS, cannot be achieved in humans, we investigate a potential additive effect by combining lower concentrations of losartan (25 mg/kg/day and 5 mg/kg/day) with barbadin, a β-arrestin blocker, and DMX20, a C-C chemokine receptor type 2 (CCR2) blocker. A high dose of losartan (50 mg/kg/day) slowed down aneurysm progression compared to untreated MFS mice (1.73 ± 0.12 vs. 1.96 ± 0.08 mm, p = 0.0033). TRV027, the combination of barbadin with losartan (25 mg/kg/day), and DMX-200 (90 mg/kg/day) with a low dose of losartan (5 mg/kg/day) did not show a significant beneficial effect. Our results confirm that while losartan effectively halts aneurysm formation in Fbn1C1041G/+ MFS mice, neither TRV027 alone nor any of the other compounds combined with lower doses of losartan demonstrate a notable impact on aneurysm advancement. It appears that complete blockade of AT1R function, achieved by administrating a high dosage of losartan, may be necessary for inhibiting aneurysm progression in MFS.

Transforming Growth Factor β Signaling Pathway as a Potential Drug Target in Treating Aortic Diseases

Article

Full-text available

Mar 2024

Review Transforming Growth Factor β Signaling Pathway as a Potential Drug Target in Treating Aortic Diseases Zijie Liu 1,2, Tianyu Song 3, and Liping Xie 1,2,3, * 1 Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China 2 School of Basic Medical Sciences, Nanjing Medical University, Nanjing 211166, China 3 ‍Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Nanjing Medical University, Nanjing 211166, China * Correspondence: lipingxie@njmu.edu.cn Received: 16 October 2023 Accepted: 20 November 2023 Published: 6 March 2024 Abstract: The transforming growth factor β (TGF-‍β) signaling pathway is crucial for preserving the structural homeostasis of the aorta and promoting aortic development. This pathway encompasses both SMAD-dependent canonical pathway and SMAD-independent non-canonical signaling pathway. Heritable thoracic aortic aneurysms and dissection are highly correlated with genetic alterations in TGF-‍β canonical signaling-related genes. However, depending on the stage of the disease, the TGF-‍β signaling pathway can have either inhibitory or aggravation effects, making its roles in aortic disease complex and occasionally contradictory. This review aims to elucidate the biological mechanisms underlying the TGF-‍β signaling pathway in the most common aortic diseases, namely acute aortic syndromes and aortic aneurysms, and to evaluate the potential clinical application of TGF-β-targeting therapies in aortic diseases.

Uncovering Druggable Targets in Aortic Dissection: An Association Study Integrating Mendelian Randomization, pQTL, and Protein–Protein Interaction Network

Article

Full-text available

May 2024

Aortic dissection (AD) is a life-threatening acute aortic syndrome. There are limitations and challenges in the discovery and application of biomarkers and drug targets for AD. Mendelian randomization (MR) analysis is a reliable analytical method to identify effective therapeutic targets. We aimed to identify novel therapeutic targets for AD and investigate their potential side-effects based on MR analysis. Data from protein quantitative trait loci (pQTLs) were used for MR analyses to identify potential therapeutic targets. We probed druggable proteins involved in the pathogenesis of aortic dissection from deCODE. In this study, a two-sample MR analysis was conducted, with druggable proteins as the exposure factor and data on genome-wide association studies (GWAS) of AD as the outcome. After conducting a two-sample MR, summary data-based Mendelian randomization (SMR) analysis and colocalization analysis were performed. A protein–protein interaction (PPI) network was also constructed to delve into the interactions between identified proteins. After MR analysis and the Steiger test, we identified five proteins as potential therapeutic targets for AD. SMR analysis and colocalization analysis also confirmed our findings. Finally, we identified ASPN (OR = 1.36, 95% CI: 1.20, 1.54, p = 4.22 × 10⁻⁵) and SPOCK2 (OR = 0.57, 95% CI: 0.41, 0.78, p = 4.52 × 10⁻⁴) as the core therapeutic targets. Through PPI network analysis, we identified six druggable targets, enabling the subsequent identification of six promising drugs from DrugBank for treating AD. This discovery of specific proteins as novel therapeutic targets represents a significant advancement in AD treatment. These findings provide more effective treatment options for AD.

Novel Insights into the Aortic Mechanical Properties of Mice Modeling Hereditary Aortic Diseases

Article

Jul 2024

Objective Hereditary aortic diseases (hADs) increase the risk of aortic dissections and ruptures. Recently, we have established an objective approach to measure the rupture force of the murine aorta, thereby explaining the outcomes of clinical studies and assessing the added value of approved drugs in vascular Ehlers–Danlos syndrome (vEDS). Here, we applied our approach to six additional mouse hAD models. Material and Methods We used two mouse models (Fbn1C1041G and Fbn1mgR ) of Marfan syndrome (MFS) as well as one smooth-muscle-cell-specific knockout (SMKO) of Efemp2 and three CRISPR/Cas9-engineered knock-in models (Ltbp1, Mfap4, and Timp1). One of the two MFS models was subjected to 4-week-long losartan treatment. Per mouse, three rings of the thoracic aorta were prepared, mounted on a tissue puller, and uniaxially stretched until rupture. Results The aortic rupture force of the SMKO and both MFS models was significantly lower compared with wild-type mice but in both MFS models higher than in mice modeling vEDS. In contrast, the Ltbp1, Mfap4, and Timp1 knock-in models presented no impaired aortic integrity. As expected, losartan treatment reduced aneurysm formation but surprisingly had no impact on the aortic rupture force of our MFS mice. Conclusion Our read-out system can characterize the aortic biomechanical integrity of mice modeling not only vEDS but also related hADs, allowing the aortic-rupture-force-focused comparison of mouse models. Furthermore, aneurysm progression alone may not be a sufficient read-out for aortic rupture, as antihypertensive drugs reducing aortic dilatation might not strengthen the weakened aortic wall. Our results may enable identification of improved medical therapies of hADs.

Pharmacological treatments, mouse models, and the aorta

Chapter

Jan 2024

J.D. Humphrey

A novel genetic mouse model of fatal aortic dissection reveals massive inflammatory cell infiltration in the thoracic aorta

Preprint

Full-text available

May 2024

Background: Aortic dissection (AD) is the separation of medial layers of the aorta and is a major cause of death in patients with connective tissue disorders such as Marfan syndrome. However, molecular triggers instigating AD, its temporospatial progression, and how vascular cells in each vessel layer interact and participate in the pathological process remain incompletely understood. To unravel the underlying molecular mechanism of AD, we generated a spontaneous AD mouse model. Methods: We incorporated a novel missense variant (p.G234D) in FBN1, the gene for fibrillin-1, identified in a non-syndromic familial AD patient into mice using CRISPR/Cas9 system. We performed histopathological analyses of the aortic lesions by histology, immunofluorescence staining, electron microscopy, synchrotron-based imaging and single-cell (sc)RNA-sequencing. Biochemical analysis was performed to examine the binding capacity of mutant human FBN1G234D protein to latent Tgfbeta binding proteins (LTBPs), and signaling pathways in the mutant aortic wall were examined by western blot analysis. Results: 50% of the Fbn1G234D/G234D mutant mice died within 5 weeks of age from multiple intimomedial tears that expanded longitudinally and progressed to aortic rupture accompanied by massive immune cell infiltration. scRNA-sequencing, validated by immunostaining, revealed a significant increase in MHC class II-positive pro-inflammatory macrophages and monocytes at the site of intima tears with upregulation of MMP2/9 and marked disruption of elastic lamina. Subendothelial matrices, such as type IV collagen and laminin, expanded into the medial layer, where fibronectin expression was highly upregulated. Fbn1G234D/G234D endothelial cells exhibited altered mechanosensing with loss of parallel alignment to blood flow and upregulation of VCAM-1 and ICAM-1, all of which likely contributed to the infiltration of immune cells. Biochemically, FBN1G234D lost the ability to bind to latent TGFbeta binding protein (LTBP)-1, -2, and -4, resulting in the downregulation of TGFbeta signaling in the aortic wall. Conclusions: We show that dynamic interactions involving endothelial cells (ECs) and macrophages/monocytes in the intima, where the ECM microenvironment is altered with the reduced TGFbeta signaling, contributes to the initiation of AD. Our novel AD mouse model provides a unique opportunity to identify target molecules involved in the intimomedial tears that can be utilized for development of therapeutic strategies.

Cyclic strain alters the transcriptional and migratory response of scleral fibroblasts to TGFβ

Article

Apr 2024
EXP EYE RES

Exploration of the role of elastic system ﬁbers in the periodontal ligament歯根膜における弾性線維の役割の探索

Article

Jan 2018

Elastic system ﬁbers in humans are present in various tissues throughout the human body. These ﬁbers exhibit extremely slow turnover；thus, their repair is delayed even during aging-related deterioration, leading to reduced tissue elasticity associated with disease. Deterioration of elastic system fibers contributes to pathological conditions, such as arteriosclerosis in the arteries and pulmonary emphysema in the lungs. Improper tooth alignment and periodontal disease are induced more frequently in the oral cavity area during the aging process；a major cause of these pathologies is the decline in homeostasis related to deterioration of elastic system ﬁbers in the periodontal ligament. Marfan syndrome (MFS) is caused by mutations in the gene encoding ﬁbrillin, the main constituent of elastic system fibers. Patients exhibit tall stature, long fingers, eye abnormalities (including crystal subluxation), heart valve abnormalities and aortic aneurysms. The relationship between fibrillin abnormalities and MFS pathology is attracting increasing attention, and there has been a variety of research on heart and lung tissue in MFS patients. The periodontal ligament contains two major fiber components：collagen ﬁbers and elastic system ﬁbers—most of these elastic system ﬁbers are oxytalan ﬁbers. However, there have been few research studies on deterioration of the elastic system ﬁbers in the periodontal ligament and resultant eﬀects on periodontal ligament homeostasis. In this report, we discuss the role of elastic system ﬁbers in the periodontal ligament based on previously reported ﬁndings.

Topical Losartan for Treating Corneal Haze After Ultraviolet-A/Riboflavin Collagen Cross-Linking

Article

Mar 2024
CORNEA

Purpose The purpose of this study was to report our first clinical experience using topical losartan for the treatment of severe corneal haze after epithelium-off corneal cross-linking (CXL). Methods A 20-year-old man presented with clinically significant corneal haze in the right eye 1 month following Ultraviolet-A/Riboflavin Epithelium-off Collagen CXL. Haze progressed to a deep stromal scar, and vision was 20/150 with no improvement on refraction, 60 days after CXL. After unsuccessful treatment with topical corticosteroids, the patient elected to start off-label treatment with topical losartan 0.8 mg/mL, administered 6 times per day. Results After 3 months of initiating topical losartan, the right eye vision improved to preoperative vision of 20/40-1. Corneal haze was significantly reduced as observed on slitlamp examination and on Scheimpflug corneal tomography (Pentacam; OCULUS, Inc. Arlington, WA). Conclusions Topical losartan, a transforming growth factor-β inhibitor, is a potential treatment in clinically significant corneal haze following epithelium-off corneal CXL. This clinical experience highlights the potential efficacy of topical losartan as a novel therapeutic option in such cases, but further clinical studies are needed.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan

Article

Apr 2024
EXP EYE RES

Steven E. Wilson

Noncanonical TGF Signaling Contributes to Aortic Aneurysm Progression in Marfan Syndrome Mice

Article

Full-text available

Apr 2011
SCIENCE

Transforming growth factor-β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal-regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase-1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Expression of the AT2 receptor developmentally programs extracellular signal-regulated kinase activity and influences fetal vascular growth

Article

Full-text available

Feb 1999

Angiotensin II type 2 (AT2) receptor is abundantly expressed in vascular smooth muscle cells (VSMC) of the fetal vasculature during late gestation (embryonic day 15-20), during which the blood vessels undergo remodeling. To examine directly the influence of AT2 receptor expression in the developmental biology of VSMC, we studied cultures of VSMC from fetal and postnatal wild-type (Agtr2(+)) and AT2 receptor null (Agtr2(-)) mice. Consistent with in vivo data, AT2 receptor binding in cultured Agtr2(+) VSMC increased by age, peaking at embryonic day 20, and decreased dramatically after birth. Angiotensin II-induced growth in Agtr2(+) VSMC (embryonic day 20) was increased by the AT2 receptor blocker PD123319, indicating that the AT2 receptors are functional and exert an antigrowth effect in Agtr2(+) VSMC. Growth of VSMC in response to serum decreased age dependently and was higher in Agtr2(-) than in Agtr2(+), inversely correlating with AT2 receptor expression. However, serum-induced growth in Agtr2(+) and Agtr2(-) VSMC and the exaggerated Agtr2(-) VSMC growth was maintained even in the presence of PD123319 or losartan, an AT1 receptor blocker. Moreover, Agtr2(-) VSMC showed greater growth responses to platelet-derived growth factor and basic fibroblast growth factor, indicating that Agtr2(-) cells exhibit a generalized exaggerated growth phenotype. We studied the mechanism responsible for this phenotype and observed that extracellular signal-regulated kinase (ERK) activity was higher in Agtr2(-) VSMC at baseline and also in response to serum. ERK kinase inhibitor PD98059 inhibited both growth and ERK phosphorylation dose-dependently, while the regression lines between growth and ERK phosphorylation were identical in Agtr2(+) and Agtr2(-) VSMC, suggesting that increased ERK activity in Agtr2(-) VSMC is pivotal in the growth enhancement. Furthermore, the difference in ERK phosphorylation between Agtr2(+) and Agtr2(-) was abolished by vanadate but not by okadaic acid, implicating tyrosine phosphatase in the difference in ERK activity. These results suggest that the AT2 receptor expression during the fetal vasculogenesis influences the growth phenotype of VSMC via the modulation of ERK cascade.

Angiotensin II upregulates transforming growth factor- type I receptor on rat vascular smooth muscle cells

Article

Full-text available

Mar 2000

Angiotensin II (Ang II) and transforming growth factor-beta (TGF-beta) modulate cell growth and metabolism. Our objective was to evaluate the effect of Ang II on the characteristics and expression of TGF-beta receptors on vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats. The addition of TGF-beta1 elicited a biphasic response on DNA synthesis in cultured VSMC in the absence of Ang II, but TGF-beta1 did not stimulate DNA synthesis in the presence of Ang II. TGF-beta binding data showed that Ang II increased the specific binding of 125I-TGF-beta1 by enhancing the expression of lower affinity receptors and increasing the number of binding sites. Ang II alone did not stimulate DNA synthesis in these cultures. However, Ang II significantly stimulated DNA synthesis after the inhibition of endogenous TGF-beta with a neutralizing antibody. The DNA synthesis stimulated by phorbol ester milisterol (PMA) was not affected by the TGF-beta neutralizing antibody. Affinity labeling data revealed receptor-ligand complexes of 280, 85, and 70 kDa, corresponding to TGF-beta type III, II, and I receptors, respectively. Incubation of VSMC with Ang II but not with PMA markedly increased the expression of the TGF-beta type I receptor. Reverse transcription and polymerase chain reaction data also indicated that Ang II, but not PMA, significantly increased the expression of TGF-beta type I receptor mRNA. Results suggest that Ang II increases the binding of TGF-beta with upregulation of TGF-beta type I receptor via a C-kinase-independent pathway. The enhanced expression of the TGF-beta type I receptor may counteract Ang II-promoted growth of VSMC.

TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome

Article

Dec 2004

Mitral valve prolapse (MVP) is a common human phenotype, yet little is known about the pathogenesis of this condition. MVP can occur in the context of genetic syndromes, including Marfan syndrome (MFS), an autosomal-dominant connective tissue disorder caused by mutations in fibrillin-1. Fibrillin-1 contributes to the regulated activation of the cytokine TGF-β, and enhanced signaling is a consequence of fibrillin-1 deficiency. We thus hypothesized that increased TGF-β signaling may contribute to the multisystem pathogenesis of MFS, including the development of myxomatous changes of the atrioventricular valves. Mitral valves from fibrillin-1–deficient mice exhibited postnatally acquired alterations in architecture that correlated both temporally and spatially with increased cell proliferation, decreased apoptosis, and excess TGF-β activation and signaling. In addition, TGF-β antagonism in vivo rescued the valve phenotype, suggesting a cause and effect relationship. Expression analyses identified increased expression of numerous TGF-β–related genes that regulate cell proliferation and survival and plausibly contribute to myxomatous valve disease. These studies validate a novel, genetically engineered murine model of myxomatous changes of the mitral valve and provide critical insight into the pathogenetic mechanism of such changes in MFS and perhaps more common nonsyndromic variants of mitral valve disease. Histologic and morphometric assessment of mitral valve architecture in P6.5 mice. Fbn1 genotypes are indicated as follows: +/+ (Fbn1+/+), +/− (Fbn1C1039G/+), and −/− (Fbn1C1039G/C1039G). (A) Representative mitral valve sections from each genotype at P6.5 showing increased length and thickness in mutant valves as compared to wild-type littermates. Magnification, ×20. Scale bars: 100 μm. (B) Morphometric analysis of mitral valve length during the first week of postnatal life. Fbn1C1039G/C1039G valves were significantly longer by P6.5 when compared with those of Fbn1+/+ animals (–;P –; 0.05). (C) Morphometric analysis of mitral valve thickness during the first week of postnatal life. Increased valve thickness in Fbn1C1039G/C1039G versus Fbn1+/+ mice was significant by P4.5 (*P –; 0.005 vs. Fbn1+/+), and by P6.5, differences between all genotypes were statistically significant (*P –; 0.005 vs. Fbn1+/+; –;P –; 0.05 vs. Fbn1C1039G/+). Error bars indicate 95% confidence intervals. (D) Echocardiographic parasternal long axis views of 9-month Fbn1C1039G/+ and Fbn1+/+ mouse hearts. The anterior mitral valve leaflet (arrowhead) shows increased length and thickening, as well as systolic prolapse into the LA in fibrillin-1–;deficient mice. AoR, aortic root; AscAo, ascending aorta; LA, left atrium.

An angiotensin-converting enzyme inhibitor, not an angiotensin II type-1 receptor blocker, prevents β-aminopropionitrile monofumarate-induced aortic dissection in rats

Article

Oct 2002
J VASC SURG

H Nagashima

Objective: Cystic medial degeneration (CMD) is a histologic abnormality that is common in aortic diseases such as aortic dilation, aneurysm, or dissection. Although little is known about the mechanism underlying CMD, we have previously demonstrated that angiotensin II signaling via angiotensin II type 2 receptor (AT2R) plays a central role in apoptosis of vascular smooth muscle cells (VSMCs) occurring in CMD associated with Marfan syndrome. The aim of this study is to elucidate the role of angiotensin II signaling in THE pathogenesis of aortic diseases associated with CMD. Method: We investigated the effects of angiotensin-converting enzyme inhibitor (ACEI), temocapril (n = 15), angiotensin II receptor type-1 (AT1R) blocker, CS-866 (n = 15), and vehicle control (n = 17) on 0.25% β-aminopropionitrile monofumarate (BAPN)-induced aortic dissection and histopathologic findings in a rat model. Results: Temocapril significantly prevented aortic dissection (P < .05), CMD (P < .01), and VSMC apoptosis (P < .01) compared with vehicle control in BAPN-fed rats. However, CS-866 did not show any preventive effect. Reversed transcriptase-polymerase chain reaction demonstrated that expression of both AT1R and AT2R was detected in control rat aortas, and that AT2R expression was significantly upregulated in the aortas of BAPN-fed rats (P < .01). Blood pressure was significantly and equally lowered in both temocapril and CS-866 groups compared with control. Conclusions: Differential expression of angiotensin II receptors and AT2R signaling are involved in the pathogenesis of CMD and aortic dissection in BAPN-fed rats. ACEIs might be of clinical value for the prevention and treatment of aortic diseases related to CMD. (J Vasc Surg 2002;36:818-23.)

Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor

Article

Oct 1995

THERE are two major angiotensin II receptor isoforms, AT1 and AT2. AT1 mediates the well-known pressor and mitogenic effects of angiotensin II (refs 1-5), but the signalling mechanism and physiological role of AT2 (refs 6-11) has not been established. Its abundant expression in fetal tissues12 and certain brain nuclei13 suggest possible roles in growth, development and neuronal functions. Here we report the unexpected finding that the targeted disruption of the mouse AT 2 gene resulted in a significant increase in blood pressure and increased sensitivity to the pressor action of angiotensin II. Thus AT2 mediates a depressor effect and antagonizes the AT1-mediated pressor action of angiotensin II. In addition, disruption of the AT 2 gene attenuated exploratory behaviour and lowered body temperature. Our results show that angiotensin II activates AT1 and AT2, which have mutually counteracting haemo-dynamic effects, and that AT2 regulates central nervous system functions, including behaviour.

Aldosterone blockade by Spironolactone improves the hypertensive vascular hypertrophy and remodeling in angiotensin II overproducing transgenic mice

Article

Mar 2009

Recent evidence has revealed that aldosterone (ALDO) is produced in the vasculature, and acts directly in the cardiovascular system. This study was designed to examine the role of ALDO in the process of long-term renin-angiotensin system (RAS) induced vascular remodeling. Hypertensive transgenic mice that overproduce angiotensin II (AngII), i.e., Tsukuba-Hypertensive-Mice (THM), were given tap water or 1% salt water and treated with or without Spironolactone (SPRL: 20mg/kg/day) for 4 weeks. We also employed A7r5 cells and investigated the effect of SPRL on the AngII mediated signal transduction in the vascular smooth muscle cells. Intimal hyperplasia, medial hypertrophy and degradation of medial elastic laminae were observed in the abdominal aorta, independent of blood pressure. Taking 1% salt water markedly enhanced these changes. In contrast, SPRL-treated THM showed almost complete disappearance of these intimal hyperplasia and medial hypertrophy. Osteopontin (OPN) was markedly up-regulated in the intima and media. However, it was inhibited by SPRL treatment in spite of high level of AngII. In A7r5 cells, AngII (10(-7)muM) induced OPN expression and pretreatment with MEK, PI3K, and EGFR inhibitor suppressed it. SPRL pretreatment also inhibited AngII-induced ERK and AKT phosphorylation, and resulted in the suppression of AngII-induced OPN expression. ALDO blockade by SPRL restores the vascular remodeling caused by the long-term RAS enhancement even in the high level of AngII, independent of blood pressure. Blocking AngII alone may not be sufficient, and direct ALDO blockade is also important to prevent vascular disease.

Protective role of angiotensin II type 2 receptor signaling in a mouse model of pancreatic fibrosis

Article

Dec 2008

The renin-angiotensin system contributes to pathological processes in a variety of organs. In the pancreas, blocking the angiotensin II (AII) type 1 receptor (AT1) attenuates pancreatic fibrogenesis in animal models of pancreatitis. Because the role of the AII type 2 receptor (AT2) in modulating pancreatic injury is unknown we investigated the role of AT2 in pancreatic injury and fibrosis. Pancreatic fibrosis was induced by repetitive cerulein administration in C57BL/6 wild-type (WT) or AT2-deficient (AT2-/-) mice and assessed by morphology and gene expression at 10 days. There was no difference between WT and AT2-/- mice in the degree of acute pancreatic injury as assessed by amylase release at 9 and 12 h and by histological examination of the pancreas at 12 h. In contrast, parenchymal atrophy and fibrosis were more pronounced in AT2-/- mice compared with WT mice at 10 days. Fibrosis was accompanied by activation of pancreatic stellate cells (PSC) evaluated by Western blot analysis for alpha-smooth muscle actin and by immunocytochemistry; PSC activation was further increased in AT2-/- mice compared with WT mice. The level of pancreatic transforming growth factor-beta1 mRNA and protein after repetitive cerulein treatment was higher in AT2-/- mice than in WT mice. Our results demonstrate that, in contrast to AT1 receptor signaling, AT2 receptor signaling modulates protective antifibrogenic effects in a mouse model of cerulein-induced pancreatic fibrogenesis. We propose that the effects of AII on injury-induced pancreatic fibrosis may be determined by the balance between AT1 and AT2 receptor signaling.

Sustained Hypersensitivity to Angiotensin II and Its Mechanism in Mice Lacking the Subtype2 (AT2) Angiotensin Receptor

Article

Jun 1999

The vast majority of the known biological effects of the renin-angiotensin system are mediated by the type-1 (AT1) receptor, and the functions of the type-2 (AT2) receptor are largely unknown. We investigated the role of the AT2 receptor in the vascular and renal responses to physiological increases in angiotensin II (ANG II) in mice with targeted deletion of the AT2 receptor gene. Mice lacking the AT2 receptor (AT2-null mice) had slightly elevated systolic blood pressure (SBP) compared with that of wild-type (WT) control mice (P < 0.0001). In AT2-null mice, infusion of ANG II (4 pmol/kg/min) for 7 days produced a marked and sustained increase in SBP [from 116 +/- 0.5 to 208 +/- 1 mmHg (P < 0.0001) (1 mmHg = 133 Pa)] and reduction in urinary sodium excretion (UNaV) [from 0.6 +/- 0.01 to 0.05 +/- 0.002 mM/day (P < 0.0001)] whereas neither SBP nor UNaV changed in WT mice. AT2-null mice had low basal levels of renal interstitial fluid bradykinin (BK), and cyclic guanosine 3', 5'-monophosphate, an index of nitric oxide production, compared with WT mice. In WT mice, dietary sodium restriction or ANG II infusion increased renal interstitial fluid BK, and cyclic guanosine 3', 5'-monophosphate by approximately 4-fold (P < 0.0001) whereas no changes were observed in AT2-null mice. These results demonstrate that the AT2 receptor is necessary for normal physiological responses of BK and nitric oxide to ANG II. Absence of the AT2 receptor leads to vascular and renal hypersensitivity to ANG II, including sustained antinatriuresis and hypertension. These results strongly suggest that the AT2 receptor plays a counterregulatory protective role mediated via BK and nitric oxide against the antinatriuretic and pressor actions of ANG II.

Angiotensin II stimulates expression of transforming growth factor ?? receptor type II in cultured mouse proximal tubular cells

Article

Aug 1999

Tubulointerstitial fibrosis is a common end-point of many chronic renal diseases and contributes to the permanent loss of renal function. There is increasing evidence that the profibrogenic cytokine transforming growth factor TGF-beta plays an essential role in this process by inducing the production of extracellular matrix proteins by tubular cells through an autocrine mechanism. We have previously demonstrated that the vasopeptide angiotensin (ANG) II induces TGF-beta transcription and synthesis in cultured murine proximal tubular cells (MCT cell line). Since the overall effects of TGF-beta on a distinct target cell may also depend on the expression of specific cell surface receptors, the present study was undertaken to test the hypothesis that ANG II modulates expression of TGF-beta receptors in MCT cells. ANG II stimulated protein expression of TGF-beta receptor type II, but not that of type I, in MCT cells as detected by immunofluorescence and western blotting of cell lysates. This stimulated receptor expression was also reflected in an overall increase in specific binding of 125I-labeled TGF-beta1 to intact MCT cells. Coincubation with ANG II and an AT1 receptor antagonist abolished this increase in 125I-labeled TGF-beta1 binding. Furthermore, ANG II also increased steady-state mRNA expression for TGF-beta receptor type II. This stimulation was transduced through AT1 receptors and was independent of TGF-beta released into the culture medium. Transient transfection studies using various length enhancer/promoter elements of the human TGF-beta receptor type II linked to the CAT gene revealed that AP1 sites are a necessary prerequisite for ANG II induced transcriptional activity. ANG II had no effect on TGF-beta receptor types I or II protein or on mRNA expression in syngeneic mesangial cells. Our results provide for the first time convincing evidence that ANG II upregulates TGF-beta receptor type II expression on proximal tubular cells. Since this subtype of receptor is primarily engaged in the initial binding of TGF-beta, an increased receptor expression may result in amplification of the TGF-beta effects on tubular cells. Interference with an activated renin-angiotensin system could therefore counteract the profibrogenic effects of TGF-beta by abolishing ANG II induced expression of TGF-beta receptor type II.

Angiotensin II Type 2 Receptor Signaling Attenuates Aortic Aneurysm in Mice Through ERK Antagonism

Abstract and Figures

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