Chronic angiotensin-(1–7) administration improves vascular remodeling after angioplasty through the regulation of the TGF-β/Smad signaling pathway in rabbits

doi:10.1016/j.bbrc.2009.08.112

Biochemical and Biophysical Research Communications

Volume 389, Issue 1, 6 November 2009, Pages 138-144

https://doi.org/10.1016/j.bbrc.2009.08.112 Get rights and content

Abstract

Objective

Angiotensin-(1–7) [ANG-(1–7)] has been reported to attenuate neointimal formation after vascular injury and stent implantation in rats, but the mechanism remains mostly unresolved. Interestingly, the levels of circulating transforming growth factor-beta1 (TGF-β1) after myocardial infarction were suppressed by ANG-(1–7), which suggests a possible downstream target for the anti-remodeling action of ANG-(1–7). Our study focused on the effects of ANG-(1–7) on vascular remodeling, including neointimal formation and collagen synthesis, and determining whether or not these effects were dependent upon the TGF-β signaling pathway.

Methods

Thirty-two New Zealand white rabbits underwent sham surgery or angioplasty in abdominal aorta. The animals were divided into four groups, which were sham, control, ANG-(1–7), and ANG-(1–7) + A-779. Subsequently, an osmotic minipump was implanted to deliver saline, ANG-(1–7) (576 μg kg⁻¹ d⁻¹) or ANG-(1–7) + A-779 (576 μg kg⁻¹ d⁻¹) for 4 weeks.

Results

The ANG-(1–7) group displayed a significant reduction in neointimal thickness (207.51 ± 16.70 μm vs. 448.08 ± 15.30 μm, P < 0.001), neointimal area (0.266 ± 0.009 mm² vs. 0.408 ± 0.002 mm², P < 0.001), and restenosis rate (28.13 ± 2.74% vs. 40.13 ± 2.74%, P < 0.001) when compared to the control group. ANG-(1–7) also inhibited collagen synthesis by significantly decreasing the mRNA expression of Collagen I and Collagen III (vs. Control group: 0.2190 ± 0.0036 vs. 0.3852 ± 0.0212, P < 0.001 and 1.1328 ± 0.0554 vs. 1.7378 ± 0.1164, P < 0.001, respectively). Furthermore, the expression of TGF-β1 and phosphor-Smad2 (p-Smad2) were significantly suppressed by ANG-(1–7) (vs. Control group: 1.21 ± 0.07 vs. 1.54 ± 0.08, P < 0.001 and 0.31 ± 0.01 vs. 0.43 ± 0.02, P < 0.001, respectively), but no effect on p38 phosphorylation was observed. [d-Ala⁷]-ANG-(1–7) (A-779), showed a tendency to attenuate the anti-remodeling effects of ANG-(1–7).

Conclusion

ANG-(1–7) decreases the amount of vascular remodeling, including a reduction in neointimal formation and collagen synthesis, after angioplasty in rabbits. The responsible mechanism may function through the possible down-regulation of TGF-β1 levels and inhibition of the Smad2 pathway.

Introduction

Coronary reperfusion treatment is widely used to restore blood flow to the ischemic myocardium. Vascular remodeling has been identified as the major cause for the delayed failure of angioplasty procedures [1]. Vascular remodeling may result from abnormal wound healing, including initial constriction, neointimal formation, and extracellular matrix (ECM) accumulation [2]. Experiments in humans and animal models have indicated that the renin-angiotensin system (RAS) plays a very important role in vascular remodeling [3]. RAS intervention, through Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor antagonist (ARB), has been proven to be a rather safe and effective method for the treatment of cardiovascular diseases [4], [5]. Therefore, targeting the RAS-axis may provide more potent therapies for the inhibition of vascular remodeling after angioplasty.

Angiotensin-(1–7) [ANG-(1–7)] is an endogenous bioactive peptide in the RAS. It is considered to have a counteracting effect to angiotensin II (ANG II) on the cardiovascular system by its inhibition of cell migration and proliferation, ECM deposition, inflammation, thrombosis [6]. Most effects are beneficial to the prevention of vascular remodeling. Furthermore, Langeveld et al. [7] and Strawn et al. [8] demonstrated that continuous ANG-(1–7) infusion attenuates neointimal formation after vascular injury and reduced in-stent restenosis (ISR) in a rat model, respectively. No further investigation has been undertaken to observe effects of ANG-(1–7) on collagen synthesis, which is another important aspect that affects vascular remodeling after angioplasty. In addition, the mechanism by which ANG-(1–7) prevents remodeling after vascular injury has not yet been studied in detail. Over the past few years, there has been an increased focus on the mechanism for the anti-remodeling action of ANG-(1–7) in heart. Iwata et al. [9] demonstrated that ANG-(1–7) could decrease transforming growth factor-beta 1 (TGF-β1) mRNA levels in cultured adult rat cardiac fibroblasts (ARCF). Grobe et al. [10] recently demonstrated that the plasma levels of TGF-β1 in myocardial infarction rat models were 40% lower in the group with ANGII co-infused with ANG-(1–7) than in the group with ANGII alone. Taken together, these observations reveal a trend towards a suppression in the expression levels of TGF-β1 by ANG-(1–7) during the remodeling phase in heart, which suggests a possible mechanism for the anti-remodeling activity of ANG-(1–7). The vascular response to angioplasty and the response of the myocardium to infarction are very similar, with an early accumulation of myofibroblasts and a subsequent loss of cells that results in an acellular matrix-rich structure. Given this similarity, we hypothesized that the anti-remodeling action of ANG-(1–7) during vascular remodeling after angioplasty may be associated with the TGF-β1 signaling pathway.

TGF-β belongs to a superfamily of proteins that serve critical roles in ECM production and the regulation of cell growth, differentiation, migration, and apoptosis in different organ systems. TGF-β1, which is the most important isoform for the cardiovascular systemhas been reported to be a most potent profibrotic cytokine [11]. Moreover, many studies [12] found that TGF-β expression is increased in human restenotic lesions as well as in neointimal hyperplasia after balloon injury in animals. In experimental models [13], [14], [15], targeting TGF-β by antibody, soluble receptor or ribozyme oligonucleotides effectively reduced neointimal formation and the constrictive remodeling that are associated with angioplasty. The mechanisms involved in TGF-β-mediated vascular remodeling are complex, including the activation of Smad protein, protein kinases-production of mediators [16].

In the present study, ANG-(1–7) was given for 4 weeks to a rabbit abdominal aorta injury model to explore the effects of chronic ANG-(1–7) administration on vascular remodeling after angioplasty. Furthermore, the anti-remodeling response to ANG-(1–7) was characterized by examining collagen synthesis and the expression levels of the protein components of the TGF-β signaling pathway, including those in the Smad-dependent (Smad2) and Smad-independent (p38) pathway. The resulting data allowed us to determine that the mechanism of the anti-remodeling activity of ANG-(1–7) after angioplasty may depend on the inhibition of the TGF-β/Smad signaling pathway and ECM deposition.

Section snippets

Materials and methods

Animals. The experimental population consists 32 healthy, 12-week-old New Zealand white rabbits weighing 1.7–2.2 kg, which were derived from the Animal Experiment Centre of Guangdong Province, China and were raised in the Department of Animal Experiment Center of Sun Yat-sen University. All animals were provided with normal rabbit chow and tap water and housed in individual cages with a 12-dark/light cycle. All animal experimental procedures had ethical approval and followed the Guidelines for

Effects of chronic treatment with ANG-(1–7) on blood pressure and heart rate

Chronic ANG-(1–7) injection had no effect on BP or HR in rabbits after balloon injury. The systolic BP (SBP), diastolic BP (DBP) and HR (96 ± 9 mm Hg, 63 ± 7 mm Hg, and 343 ± 35 bpm, respectively, n = 8) in the ANG-(1–7) group rabbits were not different from those of the control group (93 ± 13 mm Hg, 62 ± 8 mm Hg, and 370 ± 29 bpm, respectively, n = 8) (Supplement 2).

Angiography result and effects of ANG-(1–7) on neointimal formation

After the intravenous infusion of ANG-(1–7) for four weeks, the vascular wall in the ANG-(1–7) group was smoother than that of the control group, as seen

Discussion

Following a four-week intravenous infusion ANG-(1–7) effectively reduced neointimal growth after angioplasty in a rabbit model. The abdominal angiography showed that the residual lumen was better preserved in the ANG-(1–7) group than in the control group. Furthermore, the HE and Weigert stains showed that ANG-(1–7) significantly reduced the neointimal thickness, neointimal area and restenosis rates when compared with those of the control group. The specific antagonist for ANG-(1–7), A-779,

Conclusion

In summary, this study is the first to our knowledge that provides evidence that ANG-(1–7) reduces vascular remodeling, by not only inhibiting neointimal formation but also decreasing collagen secretion. These effects are possibly mediated through the regulation of the TGF-β/Smad signaling pathway in an in vivo, rabbit abdominal aorta injury model. These findings may lead to a new therapy for diseases that involve vascular remodeling in the presence or absence of injury.

Acknowledgments

We thank Mr. Gang Dai and Dr. Ren Li for their technical assistance.

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Third, Ang-(1⿿7) attenuated the mitogen-stimulated increase in cyclooxygenase 2 and prostaglandin E synthase in cardiac fibroblasts to reduce the production of proliferative prostaglandins. [72]. Other studies support an inhibitory effect of Ang-(1⿿7) on the TGF-β pathway in cardiovascular tissues [73⿿75]. Ang-(1⿿7) inhibited collagen synthesis and reduced mRNA expression for TGF-β in adult rat cardiac fibroblasts [73].
The Renin Angiotensin System (RAS) is a pivotal physiological regulator of heart and kidney homeostasis, but also plays an important role in the pathophysiology of heart and kidney diseases. Recently, new components of the RAS have been discovered, including angiotensin converting enzyme 2 (ACE2), Angiotensin(Ang)-(1⿿7), Mas receptor, Ang-(1⿿9) and Alamandine. These new components of RAS are formed by the hydrolysis of Ang I and Ang II and, in general, counteract the effects of Ang II. In experimental models of heart and renal diseases, Ang-(1⿿7), Ang-(1⿿9) and Alamandine produced vasodilation, inhibition of cell growth, anti-thrombotic, anti-inflammatory and anti-fibrotic effects. Recent pharmacological strategies have been proposed to potentiate the effects or to enhance the formation of Ang-(1⿿7) and Ang-(1⿿9), including ACE2 activators, Ang-(1⿿7) in hydroxypropyl β-cyclodextrin, cyclized form of Ang-(1⿿7) and nonpeptide synthetic Mas receptor agonists. Here, we review the role and effects of ACE2, ACE2 activators, Ang-(1⿿7) and synthetic Mas receptor agonists in the control of inflammation and fibrosis in cardiovascular and renal diseases and as counter-regulators of the ACE⿿Ang II-AT₁ axis. We briefly comment on the therapeutic potential of the novel members of RAS, Ang-(1⿿9) and alamandine, and the interactions between classical RAS inhibitors and new players in heart and kidney diseases.
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The renin–angiotensin system (RAS) is thought to contribute significantly to inflammation and fibrosis. The classical axis of the RAS, formed by angiotensin-converting enzyme (ACE), angiotensin (Ang) II, and Ang receptor type 1, activates several cell functions and molecular signaling pathways related to tissue injury, inflammation, and fibrosis. In sharp contrast, the RAS axis composed by angiotensin-converting enzyme 2 (ACE2), Ang-(1-7), and receptor Mas exerts in general opposite effects with reported anti-inflammatory and antifibrogenic responses. In this chapter, we briefly report recent findings on the anti-inflammatory and antifibrogenic role of ACE2/Ang-(1-7)/Mas axis in the context of experimental human diseases.
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Finally but interestingly, we found that incubating HMCs with Ang-(1-7) alone increased TGF-β1 expression. It is beyond our expectation and seems contrary to many other studies [37–41]. Nevertheless, in previous experiments we found the regulatory effect of Ang-(1-7) on LDLr–SREBP2–SCAP negative feedback system in human mesangial cells.
Hyperlipidemia is an independent risk factor for renal disease, and lipid deposition is associated with glomerulosclerosis. The angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis (ACE2-Ang-(1-7)-Mas axis) has been reported to participate in lipid metabolic regulation but its mechanism remains unclear. We hypothesized Ang-(1-7) would reduce lipid uptake in human mesangial cells (HMCs) by regulating the low density lipoprotein receptor–sterol regulatory element binding proteins 2–SREBP cleavage activating protein (LDLr–SREBP2–SCAP) negative feedback system, and improve glomerulosclerosis by regulating the transforming growth factor-β1 (TGF-β1). In this study we found that ACE2 was undetected in HMCs. The administration of LDL caused normal LDLr–SREBPs–SCAP negative feedback effect. Exogenous Ang-(1-7) enhanced this negative feedback effect via down-regulating LDLr, SREBP2, and SCAP expression, and effectively inhibited LDL-induced lipid deposition and cholesterol increases. This enhanced inhibitory effect was reversed by the Mas receptor antagonist A-779. Meanwhile, Ang-(1-7) significantly decreased the high LDL-induced production of TGF-β1, an effect blocked by A-779. Interestingly, HMCs treated with Ang-(1-7) alone activated the TGF-β1 expression. Our results suggested that Ang-(1-7) inhibits LDL accumulation and decreases cholesterol levels via modulating the LDLr–SREBPs–SCAP negative feedback system through the Mas receptor. Moreover, Ang-(1-7) exhibits a dual regulatory effect on TGF-β1 in HMCs.

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^☆: This study was financially supported by the Science and Technology Program of Guangdong Province [Grant 2005B10401020].

View full text

Chronic angiotensin-(1–7) administration improves vascular remodeling after angioplasty through the regulation of the TGF-β/Smad signaling pathway in rabbits☆

Abstract

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Materials and methods

Effects of chronic treatment with ANG-(1–7) on blood pressure and heart rate

Angiography result and effects of ANG-(1–7) on neointimal formation

Discussion

Conclusion

Acknowledgments

Am. J. Cardiol.

Lancet

J. Biol. Chem.

J. Invest. Dermatol.

Plaque area increase and vascular remodeling contribute to lumen area change after percutaneous transluminal angioplasty of the femoropopliteal artery: an intravascular ultrasound study

J. Vasc. Surg.

Renin-angiotensin system intervention to prevent in-stent restenosis: an unclosed chapter

J. Cardiovasc. Pharmacol.

Effects of combined treatment with angiotensin II type 1 receptor blocker and statin on stent restenosis

J. Cardiovasc. Pharmacol.

Angiotensin-(1–7): blood, heart, and blood vessels

Curr. Med. Chem. Cardiovasc. Hematol. Agents

Angiotensin-(1–7) attenuates neointimal formation after stent implantation in the rat

Hypertension

Angiotensin-(1–7) reduces smooth muscle growth after vascular injury

Hypertension

Angiotensin-(1–7) binds to specific receptors on cardiac fibroblasts to initiate antifibrotic and antitrophic effects

Am. J. Physiol. Heart Circ. Physiol.