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Annals of Clinical & Laboratory Science 37:135-140 (2007)
© 2007 Association of Clinical Scientists

Role of a Molecular Variant of Rat Atrial Natriuretic Peptide Gene in Vascular Remodeling

Paola De Paolisa,b, Valerio Nobilia, Alessia Lombardic, David Tarasib, Daniela Barbatoc, Simona Marchittic, Ursula Gantend, Ercole Brunettia, Massimo Volpeb,c and Speranza Rubattub,c
a Research Center, San Pietro Hospital, Rome; b Cardiology Department, IInd School of Medicine, La Sapienza University, Saint Andrea Hospital, Rome; c IRCCS Neuromed, Pozzilli, Italy; and d MDC, Center for Molecular Medicine, Berlin, Germany.

Address correspondence to Speranza Rubattu, M.D., IRCCS Neuromed, Localita’ Camerelle, 86077 Pozzilli (Isernia), Italy; tel 0039 0865 915227; fax 0039 0865 927575; e-mail: rubattu.speranza{at}neuromed.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Aknowledgments
 References
 
Previous studies in a hypertensive animal model of stroke and in humans showed that mutations of the atrial natriuretic peptide (ANP) gene are associated with increased risk of stroke. To elucidate the vascular disease mechanisms that result from structural modifications of the ANP gene, we investigated a coding mutation of the ANP gene in stroke-prone spontaneously hypertensive rats (SHRsp). This mutation leads to a Gly/Ser transposition in the prosegment of ANP. We found that presence of this mutation is associated with increased immunostaining of ANP in the wall of SHRsp cerebral vessels. The mutation causes a major inhibitory effect on endothelial cell proliferation, as assessed by thymidine incorporation, and on angiogenesis, as determined by an endothelial cell tube formation assay, in human umbilical vein endothelial cells (HUVEC) exposed to ANP/SHRsp. These in vitro findings show that the SHRsp-derived form of ANP has an inhibitory effect on vascular remodeling and they provide further support for a role of the ANP gene in the pathogenesis of cerebrovascular disease in the animal model.

Keywords: atrial natriuretic peptide, gene mutation, endothelial proliferation, angiogenesis


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Aknowledgments
 References
 
The rat ANP gene, a member of the natriuretic peptide family, encodes a 151 amino acid (aa) peptide which, after removal of the signal peptide, is stored as a 126 aa pro-ANP (ANP1–126) in the atria [1]. The whole 126 aa pro-hormone is then cleaved by serum proteases into several circulating fragments. The COOH-terminal peptide {alpha}ANP (ANP1–28 or ANP99–126) was the first peptide to be discovered twenty years ago [2]. It contributes to the regulation of blood pressure and salt/water balance under normal and pathophysiological conditions [3]. Recent studies have underscored a role of {alpha}ANP on vascular remodeling by regulation of both apoptosis [4,5] and proliferation of cardiac fibroblasts [6] and endothelial cells after vascular injury [7–9]. The modulatory role of {alpha}ANP in these processes appears to be cell-type specific [10] and dose-dependent [7]. Little attention has been paid to the other product of cleavage, ANP1–98 (Nt-proANP), and its derivative peptides, which also circulate in humans [11] and have important physiological functions with diuretic, natriuretic, kaliuretic, and vasodilator effects [12]. These peptides appear to be released in response to atrial stretch, acute blood volume expansion, and high-salt diet [12].

Using a genetic linkage approach, we previously showed that the rat ANP gene is a strong candidate for causing stroke in the animal model of the SHRsp [13]. Furthermore, we demonstrated that the ANP gene is a direct contributor to stroke in 2 different human populations [14,15]. Structural alterations of this gene are associated with the increased risk of stroke in both rats and humans [14–17]. In particular, the following mutations were identified in the SHRsp ANP gene, as compared to the corresponding gene in control rats of the SHRsr strain: (a) a regulatory mutation, located within a PEA2 binding site with the role of enhancer, responsible for a lower degree of ANP gene transcription [16,17], and (b) a coding mutation on exon 2 responsible for an amino acid (Gly to Ser) trans-position at position 75 of proANP1–126, causing a differential pro-peptide processing [16].

The aim of the present study was to explore the possible functional consequences of the SHRsp ANP gene coding mutation. In particular, we analyzed the in vitro effects of the mutant Nt-proANP on endothelial cell proliferation and on the process of angiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Aknowledgments
 References
 
Preparation of stably transfected AtT20 clones.  The full-length SHRsr and SHRsp proANP cDNAs were isolated from atria of both strains and cloned into the Eco RI site of PCR TM3 vector (InVitrogen) containing the gene for genectin resistance. ProANPSHRsr and proANPSHRsp plasmids were transfected into AtT20 mouse pituitary corticotropic tumor cells using the lipofectamine method (InVitrogen). Positive clones were isolated and subsequently tested for proANP secretion through an immunoenzymatic assay (see below). For each experiment the proANPSHRsr and proANPSHRsp clones were plated and 24 hr later the medium containing the relative proANP peptides (conditioned medium) was collected and used as a stimulus for endothelial cells. Nt-proANP and {alpha}ANP levels in the media of AtT20 clones were quantified by immunoenzymatic assays (Pantec and Alpha Pharmaceutical, respectively). Conditioned medium derived from AtT20 transfected with the empty vector was used as control.

Testing of the efficiency of lipofection was carried out in a separate set of transient transfections with both GFP (green fluorescence protein) and either SHRsr or SHRsp ANP construct (used at identical concentrations). The number of cells expressing GFP was counted by flow activated cell sorting (FACS).

In order to assess only the levels of proANP and {alpha}ANP released into the media, 2 additional cell lines, COS-7 and 293, were transiently transfected with either proANPSHRsr or proANPSHRsp construct and the cell media were analysed as mentioned above.

Nt-proANP and {alpha}ANP levels from brain tissue.  Whole brains from SHRsr and SHRsp rats (6 brains for each strain) were frozen and stored at –70°C until protein extraction. For this purpose, tissues were homogenized by use of a polytron (Tekmar Co.) with 50 mmol/L Tris-HCl buffer (pH 7.5) containing 150 mmol/L NaCl and 1 mmol/L EDTA. During homogenization, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml of aprotinin, leupeptin, and pepstatin each were added. Measurements of Nt-proANP and {alpha}ANP levels in the protein extracts were performed as described for the cell media. The experimental protocol was approved by the institutional review board.

Immunohistochemical analysis.  Hypothalamus from both SHRsr and SHRsp strains (6 animals each) was isolated and frozen. Thick sections (20 µm) were fixed in paraformaldehyde (PFA) 4%, washed in PBS 1x, and treated with BSA (3%, w/v) and Triton X100 (0.1%) for 1 hr. Sections were then incubated with a polyclonal anti-{alpha}ANP antibody (1:500; Bachem) overnight at 4°C followed by 3 washes in PBS with Tween 20 (0.2%). The secondary antibody (1:400; rhodamine-conjugated sheep anti-rabbit; Sigma) was incubated for 1 hr at room temperature. Double hybridization with a monoclonal anti-von Willebrand antibody (1:500; Dako) was performed for 1 hr at room temperature. Immunoreactivity was detected by incubating sections with a secondary antibody (1:400; FITC-conjugated horse anti-mouse; Dako). Localization and intensity of fluorescence were observed with a fluorescent microscope. Images were captured by a digital camera and processed by Adobe Photoshop software. Negative controls including omission of the primary antibody were also performed (data not shown).

In vitro functional studies.  (a) cGMP assay.  HUVEC (Cambrex) were grown in basic media (EBM-2; Cambrex) containing growth supplements (EGM-2; Cambrex). The experiments (n = 6) were performed using the cells within 4 passages. HUVEC were seeded in 6-well plates (2 x 105 cells/well). At 24 hr later, cells were pretreated for 30 min with 0.1 mM isobutylmethylxanthine (IBMX; Sigma) and then exposed for 30 min to the conditioned medium. cGMP was collected as described previously [11]. Finally, cGMP radio-immunoassay was performed according to the manufacturer’s instructions (Amersham). A total of 6 experiments was carried out for each clone.

(b) Endothelial cell proliferation study.  HUVEC were seeded in 24-well plates (8 x 104 cells/well), cultured in EGM-2 (Cambrex) for 24 hr, subsequently starved, and stimulated with either conditioned media for 24 hr in the presence of 10% FCS. During the last 18 hr, the cells were pulsed with 3H-thymidine (0.5 mCi/well, Amersham). The cells were then treated with 10% trichloroacetic acid and solubilized in 0.5 N NaOH. The incorporated 3H-thymidine was measured by {gamma}-counter (Packard). A total of 6 experiments was carried out with each clone.

(c) Endothelial cell tube formation assay.  Each well of a 24-well plate was coated with 0.3 ml of Matrigel (8 mg/ml; BD Bioscences) and allowed to harden at 37°C for at least 30 min. HUVEC were removed by trypsin-EDTA, washed, and resuspended in conditioned media derived from each clone at 2.5 x 104 cell/ml density. Then, 0.3 ml was gently added to each plate in duplicate. The plates were monitored up to 24 hr and photographed with a microscope fitted with a digital camera. Six experiments were carried out with each clone.

Western blotting for VE-cadherin.  HUVEC grown to 80% confluence were exposed for 6 and 18 hr, respectively, to conditioned medium derived from each clone. Cellular extracts were prepared in radioimmunoprecipation assay (RIPA) buffer and equal amounts of lysates were separated on 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham). The filter was then incubated with an anti-VE-cadherin antibody (1 µg/ml; Santa Cruz Labs). Results were visualized with an enhanced chemiluminescence detection system (ECL, Amersham). VE-cadherin levels were corrected using HSP72/73 as the housekeeping gene. The experiment was performed in triplicate.

Statistical analyses.  Values are expressed as mean ± SE. Comparisons between groups was carried out by one-way ANOVA, followed by a non-parametric post-hoc test; p <0.05 was considered the threeshold for significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Aknowledgments
 References
 
Biochemical studies.  The level of atrial natriuretic peptide was undetectable in the media from cells transfected with the empty vector. As shown in Table 1Go, levels of Nt-proANP were 10 times higher in media of proANPSHRsp clones, compared to media of proANPSHRsr clones. Higher levels of Nt-proANPSHRsp were measured even when the cell density of proANPSHRsp clones was lower than that of proANPSHRsr clones. Furthermore, the transfection efficiency (70%) was identical with the 2 ANP constructs (data not shown).


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  		Table 1. Nt-ProANP levels in the conditioned media of different cell lines transfected with either the proANPSHRsr or proANPSHRsp construct and in brain protein extracts from the SHRsr and SHRsp strains.
 
Increased levels of Nt-proANPSHRsp were observed in 2 additional cell lines (COS-7 and 293) transiently transfected with either construct, as well as in brain extracts of SHRsp, compared to the SHRsr strain (see Table 1Go). In contrast, low levels of {alpha}ANP (close to undetectable) were found and no differences were detected either in the media from transfected cells or in brain protein extracts from either strain (data not shown).

Immunohistochemical stains of hypothalamic sections from brains of both strains revealed marked increase of ANP staining in the vasculature of SHRsp, compared to the SHRsr strain (Fig. 1Go). Double hybridization with anti-von Willebrand antibody showed that synthesis of proANP takes place in the endothelium.


Figure 1
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  		Fig. 1. Immunohistochemical stains of ANP in brain vessels of SHRsr and SHRsp strains. Hypothalamic sections from SHRsr and SHRsp strains were incubated with a polyclonal anti-{alpha}ANP antibody. Subsequently, the sections were stained with an anti-von Willebrand antibody. (original magnification = 20x; representative images for 6 animals of each strain).

 
Biological studies.  To verify the biological activity of wild type and mutant Nt-proANP, HUVEC were exposed to conditioned media from cells transfected with either ANP construct and cGMP production was measured. The mutant SHRsp peptide induced a much lower cGMP response, compared to cells stimulated with conditioned medium from proANPSHRsr (23 ± 5 vs 51 ± 7 fmol/ml/105 cells, p <0.05).

HUVEC exposed to conditioned medium from proANPSHRsr transfected cells showed 20% inhibition of DNA synthesis, compared to control FCS-treated cells (p <0.01). On the other hand, HUVEC exposed to conditioned medium derived from proANPSHRsp transfected cells showed much greater inhibition of DNA synthesis (60% inhibition vs control; p <0.0001) (Fig. 2Go).


Figure 2
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  		Fig. 2. Proliferation of HUVEC exposed for 24 hr to conditioned medium derived from either proANPSHRsr or proANPSHRsp transfected cells (mean of 6 experiments). Results are expressed as percent of inhibition vs control (**p <0.001 vs control; ***p <0.0001 vs both control and SHRsr).

 
To test the involvement of wild type and mutant proANP peptides on angiogenesis, an in vitro qualitative assay of tube formation was used. Microscopy of random fields showed that endothelial cells grown in the presence of FCS were able to induce new tube formation. A slight reduction of tube formation was observed in cells exposed to proANPSHRsr conditioned medium, whereas cells exposed to proANPSHRsp conditioned medium showed marked inhibition of vascular reendothelialization (Fig. 3Go). To support these data, we characterized VE-cadherin by Western blotting and observed that this adhesion molecule was significantly suppressed in the presence of the SHRsp mutant peptide (Fig. 4Go).


Figure 3
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  		Fig. 3. Effects of SHRsr and SHRsp proANP constructs vs controls on the process of tube formation in vitro (original magnification = 10x; representative images of 6 experiments).

 

Figure 4
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  		Fig. 4. HUVEC were stimulated for 6 and 18 hr. VE-cadherin levels were reduced in HUVEC stimulated with Nt-proANPSHRsp (white bars), vs controls (black bars) and HUVEC exposed to Nt-proANPSHRsr (gray bars), (mean of 3 experiments; *p = <0.05, **p = <0.01 vs controls and SHRsr). The panel at the top of this figure shows a corresponding Western blot for VE-cadherin, representative of 3 experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Aknowledgments
 References
 
Atrial natriuretic peptide is an important regulator of blood pressure homeostasis and of cardiovascular remodeling. Evidence obtained during the last few years implicates ANP as a strong factor in the pathogenesis of cardiovascular diseases [3]. In fact, epidemiological studies have demonstrated that ANP is an independent predictor of left ventricular hypertrophy in the general population [18] and that plasma proANP levels predict the risk of cardiovascular events and death in apparently healthy persons [19]. We have previously shown that structural abnormalities of the ANP gene are associated with an increased risk of stroke in an animal model, the stroke-prone spontaneously hypertensive rat, and in 2 different human populations [14–17].

Based on this evidence, and in an attempt to elucidate possible mutation-dependent vascular disease mechanisms, we have undertaken over the last few years a series of in vitro studies with rat ANP constructs containing the known mutations. In a previous study we showed that a regulatory mutation that falls within an enhancer regulatory binding site, defined PEA2, is responsible for altered basal and inducible ANP gene transcription in cardiovascular cells [17]. In the present study we demonstrate that a coding mutation, responsible for a Gly/Ser transposition within the Nt-proANP of SHRsp strain and for a differential pro-peptide processing [16], influences the prohormone release and plays an important modulatory role in vascular remodeling.

The consistent detection of higher Nt-proANP levels in the media from all cells transfected with the proANPSHRsp construct, and the increased ANP immunostaining in the vasculature of SHRsp rats, as well as the presence of increased Nt-proANP levels in protein extracts from the SHRsp whole brain, strongly support an increased stability of the mutant SHRsp Nt-proANP.

Based on our functional data, the more stable Nt-proANP, specific to the SHRsp strain, appears to be responsible, mainly through increased tissue accumulation, for significant effects on vascular remodeling. In this regard, our experiments, performed with a ten-fold higher concentration of Nt-proANPSHRsp, compared to Nt-proANPSHRsr, as documented by the immunoenzymatic assay, were able to reproduce closely the in vivo condition. On the other hand, no differences in functional effects were observed when equimolar amounts of proANP from both strains were used in the experimental setting. Therefore, it is likely that the functional effects induced by the SHRsp mutant peptide are mainly dose-dependent and that the amino acid change, per se, does not alter the peptide functions. Interestingly, our findings are consistent with the well known antiproliferative action of high concentrations of ANP [20]. The contribution of {alpha}ANP to the effects described in the current experiments is clearly ruled out by its short half life and by the very low levels detected in our experimental conditions.

The mechanism of action of the Nt-proANP derivative peptides is similar to that of {alpha}ANP. In particular, a vasodilatory effect has been previously associated with increased cyclic GMP production [21,22]. Furthermore, specific binding sites for these peptides were identified and they appear to be different from the {alpha}ANP binding site [23]. In our studies the cGMP response was induced by both proANP constructs. cGMP production was higher when the cells were stimulated with conditioned medium derived from the AtT20 proANPSHRsr clones. The lower production of cGMP induced when HUVEC cells were exposed to conditioned medium derived from AtT20 proANPSHRsp clones may be explained by the interaction of the mutant, differently processed Nt-proANP with a different receptor. Due to the lack of precise knowledge about the receptor used by the final post-translational product of the mutant Nt-proANP, studies with specific antagonists could not be performed.

In conclusion, our data support a significant biological relevance of the Gly to Ser transposition detected in the Nt-proANP of the SHRsp strain. This mutation causes alterations of pro-peptide processing and release, leading to increased accumulation of the Nt-proANP peptide and/or its derivatives in the brain tissue and vasculature of this rat strain. Remarkably, a major impact on the processes of vascular remodeling could be detected in vitro only in the presence of the mutant SHRsp Nt-proANP peptide. A major limitation of the present study is the paucity of information about the translation product of the SHRsp mutant prohormone. However, our findings support the pathophysiological implications of mutant ANP in cerebrovascular disease and they help to identify mechanisms for vasculopathies that result from alterations of the ANP gene.


   Aknowledgments
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Aknowledgments
References
 
This work was supported by grants from the Italian Ministry of Health (IRCCS) to SR and MV and by a grant from MURST 2004 to SR. PDP is the recipient of a fellowship from the Italian Society of Hypertension.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Aknowledgments
 References
 

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