Angiotensin II Induces a Region-Specific Hyperplasia of the Ascending Aorta Through Regulation of Inhibitor of Differentiation 3

Abstract

Rationale

Angiotensin II (AngII) has diverse effects on smooth muscle cells. The diversity of effects may relate to the regional location of this cell type.

Objective

The aim of this study was to define whether AngII exerted divergent effects on smooth muscle cells (SMC) in the aorta and determine the role of blood pressure and specific oxidant mechanisms.

Methods and Results

AngII (1,000 ng/kg/min) infusion for 28 days into mice increased systolic blood pressure (SBP) and promoted medial expansion of equivalent magnitude throughout the entire aorta. Both effects were ablated by AT1a receptor deficiency. Similar increases in blood pressure by administration of norepinephrine promoted no changes in aortic medial thickness. Increased medial thickness was due to SMC expansion attributable to hypertrophy in most aortic regions, with the exception of hyperplasia of the ascending aorta. Deficiency of the p47^phox component of NADPH oxidase ablated AngII-induced medial expansion in all aortic regions. Analysis of mRNA and protein throughout the aorta revealed a much higher abundance of the inhibitor of differentiation 3 (Id3) in the ascending aorta compared to all other regions. A functional role was demonstrated by Id3 deficiency inhibiting AngII-induced SMC hyperplasia of the ascending aorta.

Conclusions

In conclusion, AngII promotes both aortic medial hypertrophy and hyperplasia in a region-specific manner via an oxidant mechanism. The ascending aortic hyperplasia is dependent on Id3.

INTRODUCTION

Angiotensin II (AngII) has many physiological and pathological effects on smooth muscle cells (SMCs) of aortic tissue both in vitro and in vivo. Most studies on cultured aortic SMCs have isolated cells from the thoracic region of rats.^1–3 In these cells, AngII consistently promotes hypertrophy^1,2, with a lesser number of studies demonstrating a proliferation response.³ The relative effects of AngII on hypertrophy versus proliferation, in vitro, appear to depend on culture conditions, such as confluency and the co-incubation with specific cytokines.³ The expression of the cyclin-dependent kinase inhibitor p27^kip1, in cultured cells, is a factor determining the relative effect on hypertrophy versus proliferation.⁴

Many AngII-induced responses on SMCs are due to stimulation of NADPH oxidase with the subsequent generation of reactive oxygen species (ROS).^5–7 These include AngII-induced hypertrophy and proliferative responses. NADPH oxidase is a multimeric complex. SMCs express specific isoforms of gp91^phox, with nox1 being the principal regulator of AngII-induced responses.^8–10 The activity of nox1 is regulated by the presence of p47^phox.11 Consistent with the nox1-p47^phox axis, deficiency of p47^phox blunts AngII-induced increases in systolic blood pressure.¹² More recently, the dominant-negative helix-loop-helix protein (dnHLH), inhibitor of differentiation 3 (Id3), has been invoked as a regulator of redox-mediated AngII induced proliferation.^13,14

AngII effects on aortic contractile responses have provided insight that this octapeptide has region-specific effects. AngII does not induce contractions of mouse aortic rings isolated from thoracic tissue, but generates a large transient contraction in abdominal tissue in vitro.^15,16 Contractions of aortic rings are mediated by AT1b receptors despite the presence of AT1a receptors, which may be indicative of divergent signaling mechanisms stimulated by these subtypes.^15,16 Region-specific effects on aorta have been noted in response to other bio-activators such as transforming growth factor-beta.^17,18 These region-specific effects may be due to the phenotypic diversity of aortic SMCs that arise as a consequence of heterogeneity of embryological origin.¹⁹

AngII infusion, in vivo, is well known to promote changes in medial aortic SMCs via mechanisms that are independent of increased SBP.²⁰ The aim of the present study was to determine whether AngII infusion in vivo promoted heterogeneous effects on SMC growth and/or proliferation throughout the entire aorta. Despite previously described differences in contractile activity of AngII on thoracic versus abdominal aorta^15,16, we were unable to demonstrate morphological differences in these two regions during AngII infusion. However, the ascending aorta has a different response than the rest of the aorta with a medial expansion that was attributable to hyperplasia, versus hypertrophy in other regions. Aortic medial expansion throughout the aorta was ablated by deficiency of p47^phox, while deficiency of Id3 inhibited the hyperplasia induced by AngII in the ascending aorta.

MATERIALS AND METHODS

AT1a receptor deficient mice (B.129P2-Agtr1a^tm1UNC), C57BL/6, and p47^phox mutant mice (B6(Cg)-Ncf1^m1J/J, C57^p47/p47), were purchased from the Jackson Laboratory. Id3 deficient mice were a generous gift from Dr. Zhang (Duke University). All studies were conducted utilizing male mice with littermate controls.

All materials and methods are described in detail in the online data supplement, available online at http://circres.ahajournals.org. (See “Supplement Material”)

RESULTS

AngII infusion uniformly increased aortic thickening via AT1a receptors, independent of pressure

AngII infusion (1,000 ng/kg/min) into C57BL/6 mice led to a sustained increase in SBP (~40 mmHg) during the 28 days of delivery (Figure 1A). AngII infusion also led to increased medial thickness and area to a similar percent in all aortic regions (Figure 1B; Online Table I). To determine the contribution of pressure per se to medial expansion, NE was administered to male C57BL/6 mice at doses that increased systolic blood pressure (SBP) by the same magnitude as AngII (approximately 40–50 mmHg). Contrary to AngII infusion, NE administration led to no significant changes in medial thickness compared to age-, gender-, and strain-matched mice infused with saline. Equivalent data were also obtained by increasing blood pressure through administration of N^ϖ-nitro-L-arginine methyl ester (L-NAME, data not shown).

Figure 1. AngII induced uniform medial expansion throughout the aorta independent of increased SBP.

(A) SBP was measured prior to pump implantation and every week during drug administration. Points represent the mean of weekly observations (n=5) and bars represent SEM. * Denotes P < 0.0001 for AngII and NE versus saline using repeated measures with Bonferroni post-hoc. # Denotes P < 0.003 for AngII versus NE at week 4. (B) Aortic medial thickness measured in arch, thoracic, supra-, and infrarenal sections of aorta from C57BL/6 mice infused with saline, AngII, or NE. Histobars are means of 5 – 10 mice ± SEM. * Denotes P < 0.001 in AngII infused mice versus the other 2 groups (one-way ANOVA with Holm Sidak post hoc). -

To define whether AngII-induced medial thickening was mediated through AT1a receptors, similar experiments were performed in AT1a receptor −/− mice. Plasma renin concentrations were markedly increased in saline-infused AT1a receptor −/− mice compared to C57BL/6 wild type controls (Online Table I). Interestingly, AngII infusion reduced plasma renin concentrations in both C57BL/6 and AT1a receptor −/− mice. To determine if AT1b receptors mediated effects of AngII to reduce plasma renin concentrations in AT1a receptor −/− mice, we co-infused AngII and losartan. AngII-mediated reductions in plasma renin concentrations were abolished in AT1a receptor −/−mice administered losartan. AngII infusion failed to change SBP in AT1a receptor −/−mice (Figure 2A). Moreover, infusion of AngII did not change aortic medial thickness or area in AT1a receptor −/− mice (Figure 2B, Online Table I). Infusion of NE increased SBP in AT1a receptor −/− mice (Figure 2A). However, NE infusion had no effect on plasma renin concentrations or medial dimensions in any aortic region of AT1a receptor −/− mice (Figure 2B and Online Table I).

Figure 2. AngII infusion induced medial thickening via interaction with AT1a receptors.

(A) SBP was measured prior to pump implantation and every week during drug administration in AT1a receptor −/− mice. Points represent the mean of weekly observations (n=5) and bars represent SEM. * Denotes P < 0.0001 for NE versus saline or AngII using repeated measures with Bonferroni post-hoc. (B) Aortic medial thickness measured in arch, thoracic, supra-, and infrarenal sections of aorta from AT1a receptor −/− mice. Histobars represent means ± SEM.

The basis of AngII-induced medial thickening differed among aortic regions

Medial thickening with AngII infusion was associated with a marked expansion of the intra-laminar space in all aortic regions (Figure 3). This expansion may potentially be attributable to contributions from extracellular matrix expansion, and SMC hypertrophy or hyperplasia. To determine the contribution of extracellular matrix (ECM), aortas were stained with Movat’s pentachrome to permit visualization of elastin, collagen, and proteoglycans (Online Figure III). There were no overt differences in staining for ECM components. Serial sections from all regions were immunostained for α-actin or stained with propidium iodide to access expansion of cell size and number, respectively (Figure 3). Immunostaining for α-actin demonstrated uniform reactivity throughout the intra laminar spaces (Figure 3A). Nuclei counts were normalized to a standard section length, since the medial expansion confounded the utilization of vessel area. AngII infusion did not significantly affect nuclei density in both thoracic and abdominal aortic regions (Figure 3B). When combined with data from immunostaining, this was consistent with the medial expansion being due to increased volume of SMCs. Unexpectedly, AngII promoted a marked increase in nuclei density in tissue sections from the ascending aorta (Figure 3B). Since this entire region immunostained positively for SMCs, this was consistent with medial expansion due to hyperplasia. AngII also exerted region-specific effects on cultured SMCs. In agreement with the observations in vivo, AngII promoted a proliferation of SMCs cultured from ascending aortas, but not in SMCs isolated from other aortic regions (Online Figure IV). PDGF-BB promoted similar proliferation in SMCs cultured from all aortic regions. Therefore, the basis for AngII-induced medial expansion differed among aortic regions.

Figure 3. AngII infusion induced medial thickness by either hypertrophy or hyperplasia.

(A) Representative C57BL/6 aortic sections immunostained for α-actin. Tissue sections from ascending, thoracic, suprarenal, and infrarenal aortas of mice infused with either saline or AngII for 28 days were immunostained with a rabbit anti-α-actin (1:200 dilution). (B) Nuclei density are represented by normalization to aortic section length (0.25 mm). Circles represent means of individual mice, diamonds represent means of 5–10 mice in each group ± SEM. * Denotes P < 0.001 for differences between saline and AngII-infused mice using a Student’s t test. Examples of aortic arch sections from mice infused with saline (C) or AngII (D) and stained with propidium iodide.

AngII-induced growth suppression is unchanged among aortic regions

The previous data demonstrates AngII induction of vascular growth and proliferation. However, AngII can also act as a potent growth suppressor. Therefore, we evaluated apoptotic events via Tdt-mediated dUTP nick end labeling (TUNEL). SMCs cultured from different aortic regions significantly increased apoptotic events with the addition of AngII versus saline (~10 fold), however, there was no difference between the four regions (Online Figure V). These data do not support differences in AngII-induced growth suppression among the aortic regions.

p47^phox deficiency ablated AngII-induced medial thickness

Given the role of AngII-induced ROS on SMC hypertrophy and hyperplasia, and the above region-specific data, we examined whether concentrations of superoxide (O₂ ^·−) and hydrogen peroxide (H₂O₂) differed along the aorta. In SMCs cultured from different aortic regions, there was uniform expression of ROS in all aortic regions examined (Online Figure VI). Furthermore, in vivo and ex vivo measurement of aortic O₂ ^·− demonstrated no regional differences throughout the aorta (Online Figure VII). To determine whether inability to generate ROS resulted in functional changes in vivo, saline, AngII, or NE was infused into mice with a spontaneous loss of functional mutation for p47^phox. Basal blood pressure was not different between p47^phox deficient mice compared to wild type (C57BL/6). AngII infusion did not increase blood pressure initially in p47^phox functionally deficient mice (Figure 4), but did result in a belated increase during week 4 of infusion. Plasma renin concentrations were unchanged in saline-infused p47^phox deficient compared to wild type mice, and infusion of AngII led to similar reductions in plasma renin concentrations in p47^phox deficient mice (Online Table I). The functional deficiency of p47^phox had no effect on the ability of NE infusion to increase SBP (Figure 4A). The absence of functional p47^phox abolished AngII-induced increases in medial thickness for all regions of the aorta (Figure 4B).

Figure 4. Functional deficiency of p47^phox attenuated both AngII-induced hypertrophy and hyperplasia.

(A) SBP was measured prior to pump implantation and every week during infusion with saline, AngII, or NE. Points represent means of weekly observations (n=5) and bars represent SEM. * Denotes P < 0.0001 for NE versus saline or AngII using repeated measures with Bonferroni post-hoc. # Denotes P < 0.01 for AngII versus saline infused mice. (B) Effects of infusions on medial thickness in p47^phox deficient mice in 4 aortic regions. Histobars are means of 5 mice ± SEM.

AngII initiated Id3 translocation to nuclei

To provide a basis for AngII-induced medial SMC hyperplastic response that was limited to the ascending portion of aortas, we defined the expression of Id3. Real-time PCR analysis of Id3 mRNA demonstrated significantly increased (3.5 fold) abundance in ascending aortas versus other aortic regions (Figure 5A). Western blotting of Id3 protein in cultured SMCs revealed similar regional differences in the aorta (Figure 5B–D. Online Figure VIII). As noted previously²¹, Id3 abundance fluctuated over time in the presence of serum, but was always greater in cells isolated from the ascending region. Furthermore, AngII incubation of SMCs isolated from ascending aortas resulted in Id3 colocalization within nuclei at 1 and 24 hours, with cycling to the cytoplasmic compartment at 12 hours (Online Figures IX–XI). This effect was not observed in other aortic regions (Online Figure XI). These data demonstrate AngII initiates cyclic Id3 localization to the nuclei compartment of SMC in the ascending aorta, suggesting novel mechanisms whereby id3 mediates the regional hyperplastic effects of AngII.

Figure 5. Id3 was most abundant in SMCs of the ascending aortic region.

(A) Id3 mRNA abundance was quantified by real-time PCR. Histobars are means of 3 mice and bars are SEM. * Denotes P < 0.001 for abundance in ascending aorta versus all other regions (one-wy ANOVA). Ascending, thoracic, suprarenal, or infrarenal SMCs were serum-starved for 72 hours and incubated with either saline or AngII (1 μM) for 1 (B), 12 (C), or 24 (D) hours. Histobars represent Id3 protein abundance normalized to β-actin and are means of 3 individual experiments ± SEM. * Denotes P < 0.001 for ascending aortic SMCs versus all other regions, while ** denotes P < 0.05 for ascending aortic SMCs incubated with AngII versus saline at the 12 hour interval (one-way ANOVA with Holm Sidak post hoc).

Id3 deficiency ablated ascending aortic hyperplasia

To determine the in vivo contribution of Id3 to AngII-induced ascending aortic hyperplasia, Id3−/− mice were infused with either saline or AngII. AngII markedly increased SBP in Id3−/− mice (Figure 6A). Furthermore, Id3 deficiency did not abolish AngII-induced increases of medial thickness in the ascending and abdominal regions (Figure 6B). Similar to Id3+/+ mice, immunostaining of aortas from Id3−/− mice with α-actin demonstrated uniform reactivity throughout the intralaminar spaces and no overt matrix deposition by Movats Pentachrome staining (data not shown). Moreover, plasma renin concentrations were similarly reduced by AngII infusion in Id3−/− mice, compared to other mouse strains (Online Table I). SMC nuclei density was unchanged in other aortic regions compared to wild type mice (data not shown). However, AngII-induced increases in ascending aortic SMC nuclei density were strikingly reduced by Id3 deficiency (Figure 7).

Figure 6. Id3 deficiency did not attenuate AngII-induced increases in SBP and medial thickness.

(A) SBP was measured during AngII infusion in Id3 −/− mice. Points represent means of weekly observations (n = 5) ± SEM. * Denotes P < 0.0001 for saline versus AngII infused mice using repeated measures with Bonferroni post-hoc. (B) Aortic medial thickness was measured in ascending and suprarenal aortic sections of Id3 deficient mice infused with saline or AngII. Histobars are means of 6–7 mice ± SEM. * Denotes P < 0.001 for AngII versus saline infusion (one-way ANOVA with Holm Sidak post hoc).

Figure 7. Deficiency of Id3 ablated AngII-induced hyperplasia in ascending aortas.

Nuclei density in ascending aortas from Id3 +/+ and −/− mice. Circles represent means of individual mice, diamonds represent means of 4 – 5 mice in each group ± SEM. * Denotes P < 0.001 for ascending aortas from Id3 +/+ mice infused with AngII versus saline by Student’s t test.

DISCUSSION

This study demonstrated that AngII promoted a uniform medial expansion throughout the aorta. Despite the uniformity of the expansion, the underlying cause differed in a region-specific manner. SMC hyperplasia occurred in the ascending aorta, but SMC’s hypertrophied in all other aortic regions. This effect was due to stimulation of AT1a receptors, but independent of increases in SBP. AngII-induced medial expansion was ablated in all aortic regions in mice with functional deficiency of p47^phox, while the hyperplastic response localized to the ascending aorta was inhibited by deficiency of Id3.

AngII-induced medial expansion has the potential to be attributable to both cellular and extracellular components. While AngII is known to increase several ECM proteins, including collagen^22,23 and proteoglycans²⁴, these did not appear to provide a major contribution to medial expansion. Therefore, this study focused on the contributions of increases in cellular components to medial expansion. Many studies have demonstrated that AngII promotes hypertrophy^1,2 of cultured aortic SMCs, with a lesser number demonstrating proliferation.³ Most of these studies have been performed with SMCs that have been derived from rat thoracic aorta. A lesser number of studies have chronically infused AngII in vivo to determine effects on medial dimensions.^25–27 SMC proliferative responses to AngII infusion have been demonstrated in rat carotid and mesenteric arteries.^20,28–30 In contrast, the predominance of studies have demonstrated AngII increased medial thickness in vivo through hypertrophy of rat^25,26,28 and mouse^10,27,31 aortas. The location of aortic sections used in these studies has not been commonly stated, but is likely to be the thoracic region. Thus, the description of medial expansion being attributable to hypertrophy in the thoracic aorta is consistent with the present study. Our results are the first to describe heterogeneity of AngII-induced responses within the aorta. Results from this study demonstrated that AngII-induced proliferation was limited to SMCs cultured from the ascending aorta versus other aortic regions. However, the present study did not discern whether the in vivo increase in nuclei was attributable to proliferation or polypoidization, although the magnitude of the increase in nuclei in the ascending aorta is much larger than has been described for polypoidization.^25,32–34

The basis for the heterogeneous cause of AngII-induced medial expansion in different regions of the aorta may be attributable to the diversity of embryonic origin of SMCs with potential functional differences.¹⁹ SMCs in the ascending aortic regions are primarily of neural crest origin, which extends from the aortic root to just distal of the subclavian artery.³⁵ This same lineage also extends to the carotid arteries. Conversely, thoracic and abdominal aortic SMCs are derived from somite and splanchnic mesoderm lineages, respectively. Regional differences in aortic SMCs have been described previously.¹⁹

Deficiency of AT1a receptors ablated AngII-induced medial expansion in all regions. Previous studies have highlighted the role of the AT1b receptor subtype in aortic function.^15,36 Although both “a” and “b” subtypes of AT1 receptors are expressed in the mouse aorta, deficiency of the “b” subtype substantially reduced AngII-induced contractions that are restricted to the abdominal aorta.¹⁶ Although the two subtypes are highly homologous, the differences are predominantly in the final intracellular loop and the cytoplasmic tail of this seven membrane spanning protein.³⁷ These amino acid substitutions have predictive differences on intracellular signaling.^38,39 Thus, in future studies, the relative differences between the subtypes will provide insight into the definition of the intracellular pathway that leads to medial expansion.

As with previous studies, absence of AT1a receptors had no effect on basal SBP in mice on a C57BL/6 background.^40–42 Moreover, AT1a receptor deficiency increased plasma renin concentrations, demonstrating removal of AngII-mediated negative feedback on renin synthesis and secretion.⁴⁴ This maintenance of pressure is probably due to the continued presence of AT1b receptors, since compound deficiency of both subtypes promotes a severe phenotype as occurs with deficiency of angiotensinogen, renin, or ACE.⁴³ The presence of functional AT1b receptors can be inferred in the present study by the downregulation of plasma renin concentrations during AngII infusion into AT1a receptor deficient mice and its reversal during concomitant infusion of the AT1 receptor antagonist, losartan.

Numerous studies have demonstrated a functional role of ROS, specifically O₂ ^·− and H₂O₂, in both hypertrophic and hyperplastic responses of SMCs.^13,14,31,44 AngII-induced production of O₂ ^·− and H₂O₂ has the potential to increase redox signaling pathways. However, we were unable to demonstrate any regional differences in ROS production. A major source of ROS during AngII stimulation of SMCs is via augmentation of NADPH oxidase activity as demonstrated by studies in cultured SMCs^5,44,45 and in vivo.^10,27,31 NADPH oxidases are multimeric complexes with components that differ in a cell-specific manner. In regard to AngII-induced responses in SMCs, these are mediated by a complex containing nox1 in which p47^phox is a critical component for activation.¹¹ The importance of this coupling has been shown using gp91ds-tat which is a peptide that inhibits the assembly of Nox1 with p47^phox.⁴⁶ Gp91ds-tat inhibits AngII-induced increases in SBP and medial expansion of carotid arteries. Conversely, deficiency of p47^phox attenuates AngII-induced increases in SBP.^12,47 In contrast to results from the present study, a previous study demonstrated that p47^phox deficiency increased basal blood pressure, even though AngII-induced increases in SBP were attenuated.⁴⁸ Moreover, p47^phox deficient mice showed basal increases in renin⁴⁸, which we were not able to demonstrate. Differences in results may arise from the mode of interference with p47^phox, since the previous study used genetically engineered p47^phox deficient mice, while the present study used p47^phox deficient mice that arose from a spontaneous mutation.⁴⁹ Although the spontaneous and engineered mutations are on exon 8 and 7, respectively, there is no obvious basis for these mice exhibiting different manifestations of deficiency. In contrast to the spontaneous mutation, engineered p47^phox deficient mice have been shown previously to exhibit no change in basal SBP. However, other studies have demonstrated a greater effect on SBP during AngII infusion than in the current study.^12,50 This greater efficacy of AngII-induced increases in SBP may be a consequence of the use of the [Val5] variant of AngII which has a greater efficacy at stimulating AT1 receptors compared to the AngII sequence of human and mouse peptides. Irrespective of these differences among studies, the current study demonstrated that deficiency of p47^phox ablated aortic medial expansion in all regions.

AngII-induced proliferation of cultured SMCs occurs via activation of NADPH oxidase with generation of superoxide production that subsequently induces the expression of the dnHLH protein, Id3.^13,14 Id3 dimerizes with the bHLH factor E47, inhibits E47-induced activation of expression of cyclin dependent kinase inhibitor p21^WAF1/Cip1, and promotes SMC proliferation.²¹ This mitotic effect is mediated via nuclei translocation of Id3 from the cytoplasm via an E47 nuclear localization signal (NLS).^51,52 Id3 has also been implicated in proliferative responses to carotid artery injury.⁵³ As noted earlier, the carotid artery shares common embryonic origin with cells of the ascending aorta.^19,35 In agreement with the disparity of the ascending aorta versus other aortic regions, Id3 mRNA and protein abundance was much greater in this region. In contrast to previous reports using rat thoracic SMCs, in this study AngII did not upregulate Id3 protein levels in mouse SMCs.¹³ In contrast, we demonstrate that AngII signaling can initiate Id3 localization to the nuclei compartment at 1 and 24 hours. Previous data demonstrated Id3 was upregulated and phosphorylated at these intervals in correlation with cell cycle progression.²¹ Furthermore, direct evidence of a role of Id3 in the hyperplastic response to AngII was derived from mice with genetic deficiencies of this protein. Although there is the potential for Id3 deficiency to lead to compensatory increases of other Id proteins, the total ablation of AngII-induced hyperplasia of the ascending aorta provided a striking demonstration of the specific requirement for Id3. Moreover, effects of Id3 deficiency on AngII-induced proliferative responses were observed despite a marked increase in the blood pressure response to AngII, supporting pressure-independent effects of AngII on SMC proliferation.

In summary, this study demonstrated that AngII infusion promotes aortic medial expansion by disparate mechanisms in a region-specific manner. A major difference was the demonstration of the disparity of responses in the ascending aorta compared to other regions. Unique responses of this region in aortic luminal expansion have also been demonstrated in mice harboring fibrillin-1 mutations that were infused with AngII.⁵⁴ Although this study defined AngII-induced changes in SMCs, this effect could be directly on SMCs or indirectly from another cell type. For example, endothelial cells secrete a wide range of products that directly affect SMC function. Recent data implicate PDGF-DD secretion from endothelial cells as a moderator of SMC phenotypic modulation, particularly in areas of disturbed blood flow such as the aortic arch.⁵⁵ Thus, subsequent studies will determine the contribution of SMC and endothelial cells to the observed phenotypes by using Cre-Lox technology to promote cell-specific AT1a receptor deficiency.

NOVELTY AND SIGNIFICANCE

What is known?

• In cultured cells, AngII predominantly promotes hypertrophy of SMCs, while some studies demonstrate proliferation.

• AngII induces the generation of ROS.

• Id3 promotes SMC proliferation.

What new information does this article contribute?

• AngII induced both aortic SMC hypertrophy and hyperplasia in a region-specific manner, in vivo, independent of blood pressure.

• AngII-mediated hypertrophy and hyperplasia were dependent upon AT1a receptors and ROS.

• AngII-induced hyperplasia in the ascending aorta was inhibited by Id3-deficiency.

Angiotensin II (AngII) infusions induce several distinct vascular pathologies that are region-specific in the aorta. In vitro experiments have implicated smooth muscle cells (SMCs) as having a role in these pathologies. The objective of this study was to define region-specific effects of AngII infusion in the aortic media, and define mechanisms for the differences in vivo. Infusions of AngII into normolipidemic mice for 28 days promoted equivalent medial thickening throughout the aorta due to stimulation of AT1a receptors, but was independent of blood pressure changes. This thickening was attributable to increased SMC size; except in the ascending aortic region where there was a profound increase in SMC-associated nuclei. The medial changes, in all regions, were ablated by the inability to form reactive oxygen species (ROS) by the NAPDH oxidase system, as demonstrated in p47^phox deficient mice. However, the AngII-induced increases in numbers of ascending aortic nuclei was ablated by the deficiency of inhibitor of differentiation 3 (Id3). This is the first study to demonstrate that AngII induces SMC hypertrophy with Id3-dependent, region-specific proliferation in the aortic arch. These studies provide a basis for defining the role of Id3 in the AngII-induced pathology of the ascending aorta.

Non-standard Abbreviations and Acronyms

AngII

Angiotensin II

DCF-DA

Dichlorodihydrofluorescein diacetate

DHE

Dihydroethidium

dnHLN

Dominant-negative Helix-Loop-Helix

DMEM

Dulbecco’s Modified Eagle’s Medium

ECM

Extracellular Matrix

Id3

Inhibitor of Differentiation 3

L-NAME

N^ϖ-nitro-L-arginine methyl ester

NE

Norepinephrine

ROS

Reactive Oxygen Species

SBP

Systolic Blood Pressure

SMC

Smooth Muscle Cell

SOD

Superoxide Dismutase

TUNEL

Tdt-mediated dUTP nick end labeling

O₂^·−

Superoxide

2-OH-E⁺

2-hydroxyethidium