Introduction

Thickening of the arteries has been implicated in the development of hypertension.1, 2 Recently, the contributory roles of adventitia in artery remodeling have garnered increasing attention. For example, it has been well documented that adventitial fibroblasts (AFs) can be transformed into myofibroblasts (MFs) and migrate into the neointima of arteries after endoluminal injury in balloon injury models.3, 4, 5, 6 Our recent findings and those of others using a transverse aortic constriction (TAC) model collectively showed that the arteries under pressure overload experience dramatic remodeling processes including changes in morphology, vessel composition and mechanical properties.7, 8, 9 The induced pressure overload in the TAC model can mimic the status of arteries under volume-dependent hypertension in humans, although, unfortunately, an ideal animal model for adventitial remodeling is not yet available. Therefore, the elucidation of the underlying remodeling mechanisms remains a challenging task.

Transient receptor potential melastatin 7 (TRPM7), an Mg–ATP-regulated divalent cation channel with a unique bifunctional structure (viz. the ion channel and carboxyl-terminal α-kinase domain), was first identified in 2001.10, 11 The role of TRPM7 in regulating Mg2+ homeostasis has been well established in hypertension.12, 13 TRPM7 can mediate proinflammatory bradykinin signaling where annexin-1 has been activated.14, 15 As one of the identified substrates for TRPM7, annexin-1 executes its anti-inflammatory impacts by modulating the expression of inflammatory factors and the chemotaxis of inflammatory cells.16, 17, 18 In a recent study, we detected lower expression of annexin-1 in both AFs and vascular adventitia of spontaneously hypertensive rats (SHR) as compared with Wistar–Kyoto rats.19 A growing body of evidence suggests that TRPM7 variation, along with altered annexin-1, alters the function of the vascular endothelial and smooth muscle cells.14, 15, 20, 21, 22 Therefore, it is of added interest to test whether TRPM7, an upstream molecule of annexin-1, is associated with the remodeling process. To address these issues, we sought to examine whether TRPM7 can mediate the vascular remodeling process induced by pressure overload in the RCCA-B in TAC rats.

Methods

Animal model

Male Sprague–Dawley rats (270±20 g) were obtained from the Shanghai SLAC Laboratory Animal (Shanghai, China). TAC was induced as described previously.23 In brief, rats were anesthetized with an intraperitoneal injecting mixture of ketamine (100 mg kg−1) and xylazine (5 mg kg−1) and were connected to a volume-cycled rodent ventilator at a respiratory rate of 70 breaths per minute and a tidal volume of 2.5 ml. The thoracic cavity was opened, and the aorta was ligated between the origins of the innominate and left carotid arteries. In sham-operated rats, a suture was placed around the aorta but not tightened. To inhibit TRPM7, 2-aminoethoxydiphenyl borate (2-APB, 2.5 mg kg−1; Sigma-Aldrich, Shanghai, China) was administered to the rats by intraperitoneal injection once every 3 days. The first injection was administered at 1 h before the TAC operation. 2-APB was first dissolved in dimethyl sulfoxide to 50 μg μl−1 and then diluted with a saline solution to 0.5 μg μl−1 (1:100 dilution). All procedures were performed in accordance with the Care and Use Guide of Laboratory Animals of the National Institutes of Health with the approval (SYXK-2003-0026) of the Scientific Investigation Board of Shanghai Jiao Tong University School of Medicine.

Histological analysis

Rats were killed postoperatively at 14th day after perfusion with 4% paraformaldehyde under physiological pressure. The right carotid artery was carefully removed and embedded in optimum cutting temperature tissue-freezing medium (Sakura, Torrance, CA, USA) or in paraffin after formalin fixation for 24 h. For morphometric analysis, paraffin-embedded tissue was sectioned at 5 μm thickness and stained with hematoxylin and eosin followed by examination by light microscopy (Carl Zeiss, Jena, Germany). The medial and adventitial cross-section areas were measured using Axio Vision 4.8.1 software (Carl Zeiss).

Frozen arteries embedded in the optimum cutting temperature compound were sectioned at 10 μm and mounted on glass slides. The slides were air-dried, acetone-fixed and blocked with 5% bovine serum albumin followed by incubation with primary antibodies to CD68 (1:200; Serotec, Oxford, UK), TRPM7 (1:100; Abcam, Cambridge, UK), annexin-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), SM-22-α (1:200; Abcam), monocyte chemotactic protein-1 (MCP-1; 1:100; Santa Cruz Biotechnology) and collagen I (1:100; Santa Cruz Biotechnology), and then secondary antibodies conjugated with fluorescein isothiocyanate or tetrametrylrhodarnine isothiocyante (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 4′-6-diamidino-2-phenylindole (Sigma-Aldrich, Saint Louis, MO, USA). Sections were mounted in fluorescence-mounting medium (Dako, Copenhagen, Danmark). Laser scanning confocal microscopy was performed using a Zeiss epifluorescence microscope system.

Cell culture

The methods used for the isolation and culture of AFs from thoracic aorta of 6–8-week-old male Sprague–Dawley rats were similar to previously described techniques.24 Briefly, adventitia of thoracic aortas were separated and cut into sections, which were incubated on dishes with Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal calf serum (Gibco, Grand Island, NY, USA), 100 U ml−1 penicillin and streptomycin. Subconfluent cells were made quiescent by incubation in DMEM supplemented with 0.1% bovine serum albumin for 24 h before stimulation. Cells were used from passages 4–6.25 The mouse promyelocytic leukemia cell line RAW264.7 was purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells were grown in DMEM containing with 10% FBS at 37 °C in 5% CO2. Subconfluent cells were incubated with serum-free DMEM medium, and the conditioned medium was collected after 48 h.

AFs were treated with 100% macrophage-conditioned medium for 6, 12, 24 and 48 h and were incubated for 24 h in 1–100% macrophage-conditioned medium. To inhibit TRPM7, AFs were pretreated with 100-μM 2-APB for 1 h and then stimulated with 100% RAW264.7-conditioned medium for an additional 24 h. 2-APB was first dissolved in dimethyl sulfoxide to 0.2 M and then diluted with DMEM- or RAW264.7-conditioned medium to 100 μM (1:2000 dilution). It has been reported that a dimethyl sulfoxide level of <0.1% does not have any effect of its own,26 and thus solvent control was not performed in this present study.

siRNA and transfection

The anti-TRPM7 small interfering RNA (siRNA; 5′-UUCUCCGAACGUGUCACGUTT-3′, corresponding to coding region 1881–1901 of Rattus norvegicus TRPM7 mRNA (GeneID 679906) and a non-targeting siRNA control (5′-CCGGAGGUCAGGUCGAAAUTT-3′) were synthesized by Genepharma (Shanghai, China). The AFs seeded in 12-well plates were transfected for 24 h with 50 nM of each siRNA using X-tremeGENE siRNA Transfection Reagent (Roche, Branchburg, NJ, USA) according to the manufacturer’s instructions and were then stimulated with macrophage-conditioned medium for an additional 24 h.

Western blotting

AFs were harvested and protein was isolated using cell lysis buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 2% SDS; 5 mM dithiothreitol; and 10 mM phenylmethanesulfonyl fluoride) containing freshly added protease inhibitor cocktail (Merck, Darmstadt, Germany). Protein concentrations were determined using the Bradford protein assay. The soluble lysates (20 μg per lane) were subjected to 8% SDS-PAGE for TRPM7, 12% SDS-PAGE for SM-22-alpha and 10% SDS-PAGE for annexin-1 and β-actin. Primary antibodies for TRPM7 (1:1000; Abcam), annexin-1 (1:1000; Santa Cruz Biotechnology) and SM-22-α (1:5000; Sigma-Aldrich, Shanghai, China) were incubated in 5% nonfat milk for 1 h at room temperature. The levels of β-actin (1:6000; Sigma-Aldrich, St Louis, MO, USA) were analyzed to confirm equal loading. Relative protein levels were visualized using an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA).

Statistical analyses

Data are presented as the mean±s.d. Continuous variables were compared by either Student’s t-test or an analysis of variance followed by the Student–Newman–Keuls post hoc test SPSS software, version 11.5 (SPSS Inc., Chicago, IL., USA). P<0.05 was considered statistically significant.

Results

Vascular remodeling in RCCA-B

Histomorphometric analysis revealed that pressure overload induced dramatic vessel thickening in RCCA-B 14 days after TAC compared with the sham-operated group (Figures 1a and b; Supplementary Table 1. The medial cross-section area was increased by ∼50% (P<0.01, Figure 1c). In addition, the adventitial cross-section area was increased by ∼125% (P<0.01, Figure 1d). However, there was no significant difference in the left common carotid arteries compared with the sham group (data not shown).

Figure 1
figure 1

Histomorphometric analysis of the right common carotid artery proximal to the band (RCCA-B) in the sham-operated (SHAM) and transverse aortic constriction (TAC) groups 14 days after the procedure. (a,b) Hematoxylin and eosin staining revealed increased medial and adventitial thicknesses in RCCA-B from the TAC group. a, adventitia; m, media. Bar, 100 μm. Total magnification, × 100. (c,d) Quantification data showing increased folds of the medial and adventitial cross-section areas in the TAC group. Six sections were used in the quantification. Results are expressed as the mean±s.d., n=6 for each group; *P<0.05 compared with the SHAM group. A full color version of this figure is available at Hypertension Research online.

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TAC induced macrophage accumulation and TRPM7 upregulation in RCCA-B adventitia

An elevated expression of CD68, a specific phenotype marker for rat macrophages,27, 28 was observed in the adventitia of RCCA-B (Figures 2a and b), suggesting the accumulation of macrophages in TAC rats. In addition, TRPM7 was found to be upregulated in RCCA-B adventitia where macrophages accumulated in TAC rats (Figures 2d and e). In this process, the expression of the TRPM7 downstream target, annexin-1, was diminished in RCCA-B (Figures 2g and h).

Figure 2
figure 2

Immunofluorescence staining of CD68, transient receptor potential melastatin 7 (TRPM7) and annexin-1 in the SHAM (a,d,g), transverse aortic constriction (TAC; b,e,h) and TAC treated with 2-aminoethoxydiphenyl borate (2-APB; c,f,i) groups (n=6). CD68 and TRPM7 were significantly increased in the adventitia of the right carotid arteries after TAC and were attenuated after treatment with 2-APB. Annexin-1 expression was markedly reduced and was subsequently rescued after 2-APB treatment. Nuclei were stained with 4′,6-diamidino-2-phenylindole. a, adventitia; L, lumen; m, media. Bar, 100 μm. Results are expressed as the mean±s.d., *P<0.05 compared with the SHAM group. **P<0.05 compared with the TAC group. A full color version of this figure is available at Hypertension Research online.

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Vascular inflammation and remodeling alterations by TRPM7 blocking

To investigate the in vivo role of TRPM7 in vascular remodeling after TAC, the effects of 2-APB intraperitoneal injection on vascular adventitial inflammation in RCCA-B were examined. As shown in Figures 2e and f, 2-APB treatment inhibited the upregulation of TRPM7 in TAC rats. Further, CD68, annexin-1 and MCP-1 levels were determined to test whether the blockade of TRPM7 regulated vascular adventitial inflammation in RCCA-B. Macrophage infiltration was significantly attenuated after 2-APB pretreatment compared with the TAC group (Figures 2b and c), and the downregulation of annexin-1 by TAC was also rescued by 2-APB (Figures 2h and i). The expression of another inflammatory factor, MCP-1, was induced by pressure overload in RCCA-B (Figures 3a and b) and reduced by 2-APB treatment (Figure 3c). SM-22-α and collagen I levels were measured by immunostaining to further determine the effects of 2-APB on vascular remodeling process in RCCA-B. The blockade of TRPM7 attenuated the expression of MF phenotypic marker SM-22-α (Shiz et al.29) under pressure overload in the adventitia of RCCA-B (Figures 3d–f), indicating that 2-APB treatment inhibited the transformation of AFs to MFs. In addition, abundant collagen I expression induced by TAC was significantly attenuated in the adventitia of RCCA-B after 2-APB treatment (Figures 3g–i).

Figure 3
figure 3

Immunoflurescence staining of monocyte chemotactic protein-1 (MCP-1), SM-22-α and collagen I in the SHAM (a,d,g), transverse aortic constriction (TAC; b,e,h) and TAC treated with 2-aminoethoxydiphenyl borate (2-APB; c,f,i) groups (n=6). MCP-1, SM-22-α and collagen I expression were significantly increased in the adventitia of the right carotid arteries proximal to the band after TAC compared with the SHAM groups. 2-APB treatment inhibited MCP-1, SM-22-α and collagen I upregulation in TAC groups. Nuclei were stained with 4′,6-diamidino-2-phenylindole. a, adventitia; L, lumen; m, media. Bar, 100 μm. Results are expressed as the mean±s.d., *P<0.05 compared with the SHAM group. **P<0.05 compared with the TAC group. A full color version of this figure is available at Hypertension Research online.

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Role of TRPM7 in the transformation from AFs to MFs

As mentioned above, TRPM7 was found to be related with the recruitment of macrophages after the TAC operation; however, the role of macrophages in vascular remodeling remains unclear. TRPM7 and macrophages mainly existed in the adventitia during remodeling, and thus the expression of SM-22-α was determined in primary rat vascular AFs pretreated with RAW264.7 macrophage-conditioned medium by western blot analysis. The use of RAW264.7-conditioned medium increased the level of SM-22-α expression in a time- and dose-dependent manner (Figures 4 and 5). Interestingly, the expression of TRPM7 was increased, although the annexin-1 level was attenuated after pretreatment with macrophage-conditioned medium. TRPM7 blocker 2-APB was used in AFs to elucidate the role of TRPM7 in phenotypic modulation. It was observed that SM-22-α expression decreased after 2-APB pretreatment relative to incubation with RAW264.7-conditioned medium (Figure 4b). In addition to attenuating TRPM7, 2-APB lowered the expression of annexin-1 induced by macrophage-conditioned medium (Figure 4b).

Figure 4
figure 4

Western blot analysis of protein expression in adventitial fibroblasts. (a) Rat adventitial fibroblasts were treated with macrophage-conditioned medium for the indicated durations. (b) Adventitial fibroblasts were treated for 24 h with the indicated concentrations of macrophage-conditioned medium. To inhibit transient receptor potential melastatin 7 (TRPM7), adventitial fibroblasts were pretreated with 100 μM 2-aminoethoxydiphenyl borate (2-APB) for 1 h and then stimulated with 100% macrophage-conditioned medium for 24 h. The upper panel shows representative immunoblots of TRPM7, annexin-1 and SM-22-α expression in adventitial fibroblasts. β-actin served as the internal control. The lower panels show the quantification of bands by densitometry. Results are shown as fold increases relative to the control for three independent experiments. Data are the mean±s.d. *P<0.05 compared with the control. **P<0.05 compared with 100% macrophage-conditioned medium.

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Figure 5
figure 5

Small interfering RNA (siRNA)-mediated repression of transient receptor potential melastatin 7 (TRPM7) induced adventitial fibroblasts (AFs) transformation and increased expression of annexin-1. The adventitial fibroblasts were transfected with siRNA for 24 h and then stimulated with macrophage-conditioned medium for an additional 24 h. The upper panel shows representative immunoblots of TRPM7, annexin-1 and SM-22-α expression in adventitial fibroblasts. β-actin served as an internal control. The lower panels show the quantification of bands by densitometry. The results are shown as fold increases relative to the control for three independent experiments. Data are the mean±s.d. *P<0.05 compared with the non-targeting siRNA control (NC). **P<0.05 compared with NC treated with macrophage-conditioned medium.

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Further, cultured AFs were treated with specific TRPM7 siRNA or non-targeting siRNA control, and the expression of SM-22-α and annexin-1 was examined after RAW264.7-conditioned medium treatment for 24 h. TRPM7 was shown to be required for the macrophage-conditioned medium-triggered phenotypic modulation of AFs and downregulation of annexin-1 (Figure 5).

Discussion

Our findings demonstrated that in vivo macrophages accumulated in the remodeled adventitia of RCCA-B under pressure overload, and in vitro they precipitated the transformation of AFs to MFs. Strikingly, a novel molecule related to pressure overload was discovered in our study; TRPM7 can mediate the pressure overload-induced macrophage accumulation and affect the expression of MCP-1 and annexin-1 in RCCA-B. Further, TRPM7 participated in vascular AFs transformation and increased collagen I formation in vivo. Macrophages further led to AFs transformation, elevated TRPM7 and reduced annexin-1, all of which were normalized by the addition of 2-APB and anti-TRPM7 siRNA.

It was once believed that adventitia is a contributor to vascular function as a support tissue; however, there is increasing evidence that adventitia has a crucial role in several vascular diseases, such as hypertension, atherosclerosis and restenosis.3 The phenotypic transformation of AFs to MFs contributes to neointimal formation in injured porcine coronary arteries30 and adventitial remodeling in thickened aorta and the right carotid in the TAC model.8, 9 In view of the similarity between arteries proximal to the band in the TAC model and arteries under pressure overload in human hypertension, as well as the widespread application of the TAC model in left ventricular hypertrophy and heart failure,23, 31 we suppose that the model presented here is appropriate. As expected, greater than onefold thickening of adventitia was detected at 14 days post TAC operation. Moreover, in agreement with previous reports,8, 9 we observed increases in macrophage infiltration as well as SM-22-α, MCP-1 and collagen I levels in the TAC model, indicating the reliability of our experiments.

TRPM7 has been implicated in the dysfunction of endothelial cells and vascular smooth muscle cells,12, 22, 32, 33, 34 although its expression and function remain poorly understood in vascular adventitia. It is noteworthy that reduction of annexin-1, a substrate of TRPM7, was observed in pressure overload-induced adventitial remodeling as well as in MFs transformation caused by macrophage-conditioned medium. We also observed this reduction in AFs and the aorta of spontaneously hypertensive rats in a previous study.19 Thus, we further established the pathophysiological significance of TRPM7 in TAC rats by demonstrating an increased expression of TRPM7 in vascular adventitia, where inflammation and remodeling occurred. Further treatment with TRPM7 inhibitor 2-APB35, 36 was observed to reverse the changes in inflammation factors and molecules relevant to vascular remodeling under pressure overload. Taken together, these findings suggest that TRPM7 could mediate pathological alteration in the vascular adventitia. It has been reported that the TRPM7 current can be activated by stretching or shear stress in vascular smooth muscle cells and other cells, supporting its role in response to vessel injury.37, 38, 39 Therefore, we present the hypothesis that the activation of TRPM7 by pressure overload-induced stretching can recruit macrophages into thickened adventitia and increase remodeling in a mechanosensitive manner.

To determine whether inflammation and remodeling in vascular adventitial are correlated, we explored the role of macrophage-conditioned medium in the phenotypic modulation of AFs. We found that macrophages were involved in vascular remodeling via the triggering of MF differentiation. Interestingly, exposure to macrophage-conditioned medium led to an upregulation of the expression of TRPM7, whereas this process companied with MF differentiation was inhibited by the addition of 2-APB. 2-APB is typically reported to inhibit the TRPM7 channel activity. In this present study, we found that 2-APB suppressed the expression of TRPM7 triggered by pressure overload and exposure to the macrophage-conditioned medium, which is consistent with the observations of Liu H et al.35 As a blocker of store-operated Ca entry,40 2-APB may regulate TRPM7 expression by altering Ca storage and/or release, which are involved in processes related to the regulation of gene expression. Otherwise, to examine a more specific effect of TRPM7 on AFs phenotype switching in vitro, we pretreated the cultured AFs with anti-TRPM7 siRNA before being stimulated with RAW264.7-conditioned medium. We found that the suppression of TRPM7 partly normalized the changes in AF gene expression (SM-22-α and annexin-1) initiated by the macrophage-conditioned medium, further supporting the hypothesis that TRPM7 is required for cell phenotype switching in AFs exposing to the macrophage-conditioned medium. On the basis of this line of evidence, it is reasonable to speculate that by recruiting inflammation cells, TRPM7 might interact with macrophages to participate in vascular remodeling.

TRPM7 is vital for embryonic development, and its global disruption in prenatal mice is lethal.41 The siRNA of TRPM7 can inhibit its activities in vitro but not in vivo (data not shown). Considering the similar effects between 2-APB and the siRNA of TRPM7 on in vitro phenotypic modulation of AFs and as suggested by other in vivo studies,20, 42, 43 we employed 2-APB to block the effect of TRPM7 in vivo in this study. However, on a micromolar scale, 2-APB can increase TRPM6 to a maximal extent but significantly inhibit TRPM7 channel activities, whereas on a millimolar scale, it can potentiate TRPM6/7 and TRPM7 channel activities.44 Here, we acknowledge that 2-APB is a nonspecific inhibitor of TRPM7, which may present a limitation of this study in vivo.

In summary, our findings demonstrate that TRPM7 modulates the infiltration of macrophages and acts synergistically to promote collagen accumulation and vascular adventitial MF formation during pressure overload in TAC rats. To the best of our knowledge, this is the first description of the potential regulatory roles of TRPM7 in vascular inflammation and pressure overload-mediated vascular adventitial collagen accumulation and cell phenotypic transformation. These data suggest that the targeting of TRPM7 has potential therapeutic importance for vascular diseases.