Natriuretic peptide receptor‐C‐mediated attenuation of vascular smooth muscle cell hypertrophy involves Gqα/PLCβ1 proteins and ROS‐associated signaling

Abstract Hypertension is associated with vascular remodeling due to hyperproliferation and hypertrophy of vascular smooth muscle cells (VSMC). Recently, we showed the implication of enhanced expression of Gqα and PLCβ1 proteins in hypertrophy of VSMCs from 16‐week‐old spontaneously hypertensive rats (SHR). The aim of this study was to investigate whether C‐ANP4‐23, a natriuretic peptide receptor‐C (NPR‐C) ligand that was shown to inhibit vasoactive peptide‐induced enhanced protein synthesis in A10 VSMC could also attenuate hypertrophy of VSMC isolated from rat model of cardiac hypertrophy and to further explore the possible involvement of Gqα/PLCβ1 proteins and ROS‐mediated signaling in this effect. The protein synthesis and cell volume, markers of hypertrophy were significantly enhanced in VSMC from 16‐week‐old SHR compared with age‐matched WKY rats and C‐ANP4‐23 treatment attenuated both to WKY levels. In addition, C‐ANP4‐23 treatment also attenuated the enhanced expression of AT1 receptor, Gqα, PLCβ1, Nox4, and p47phox proteins, the enhanced activation of EGFR, PDGFR, IGF‐1R, enhanced phosphorylation of ERK1/2/AKT and c‐Src in VSMC from SHR. Furthermore, the enhanced levels of superoxide anion and NADPH oxidase activity exhibited by VSMC from SHR were also attenuated to control levels by C‐ANP4‐23 treatment. These results indicate that C‐ANP4‐23 via the activation of NPR‐C attenuates VSMC hypertrophy through decreasing the overexpression of Gqα/PLCβ1 proteins, enhanced oxidative stress, increased activation of growth factor receptors, and enhanced phosphorylation of MAPK/AKT signaling pathways. Thus, it can be suggested that C‐ANP4‐23 may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis.


| Cell culture and incubation
Aortic VSMCs from 16-week-old SHR and age-matched WKY rats were cultured as described previously 21 and contained high levels of smooth-muscle-specific actin. 7,21,22 The cells after incubation at 37°C in 95% air and 5% CO 2 humidified atmosphere in Dulbecco's modified Eagle's medium (DMEM) (with glucose, L-glutamine, and sodium bicarbonate) containing 1% antibiotics (containing penicillin, streptomycin, and amphoterecin B) and 10% heat-inactivated fetal bovine serum (FBS) were passaged upon reaching confluence with 0.5% trypsin containing 0.2% EDTA and utilized between passages 2 and 8. Confluent cells from SHR and WKY rats after starving for 24 hours in DMEM without FBS at 37°C were further incubated for 24 hours in the absence or presence of 0.1 lmolÁL À1 C-ANP 4-23 .

| Western blotting
The levels of protein expression and phosphorylation were determined by Western blotting as described previously. 6,23 After SDS-PAGE, the proteins were transferred to a nitrocellulose membranes and the blots were washed with PBS containing 0.

| Cell volume measurement
Cell volume measurement was performed as described earlier. 7 VSMCs from 16-week-old SHR and age-matched WKY rats were grown to 50% confluence in cell imaging dish (35 9 10 mm).

| Determination of superoxide anion production
Basal superoxide anion production in VSMC was measured using the lucigenin-enhanced chemiluminescence method with low concentration (5 lmol/L) of lucigenin as previously described. 7,23,25 VSMC from SHR and WKY rats were incubated in the absence and pres-

| NADPH oxidase activity determination
The activation of NADPH oxidase activity in the samples was assessed by adding 10 À4 mol/L NADH (Sigma-Aldrich) to the vials before counting. Luminescence induced by basal O 2 was then subtracted from the luminescence value induced by NADH. 7

| Statistical analysis
The number of independent experiments is reported. Each experiment was conducted at least 4 times using separate cell population.
All data are expressed as the mean AE SD. Comparisons between groups were made with one way analysis of variance (ANOVA) followed by Dunnett tests using GraphPad Prism5 software. Results were considered significant at a value of P < .05.

| C-ANP 4-23 attenuates the enhanced expression of AT1 receptor in VSMC from SHR
Angiotensin II (Ang II) has been shown to induce VSMC hypertrophy. 6,20 In addition, we recently showed that the enhanced levels of endogenous Ang II through the activation of AT1 receptors contribute to the enhanced expression of Gqa and PLCb1 proteins as well as VSMC hypertrophy in SHR. 6 To examine if C-ANP 4-23mediated attenuation of VSMC hypertrophy is due to the inhibition of enhanced expression of AT1 receptor, we examined the effect of C-ANP 4-23 treatment on the expression of AT1 receptor in VSMC from SHR and WKY rats. Results shown in Figure 4, indicate that the expression of AT1 receptor was significantly augmented by F I G U R E 2 Effect of C-ANP 4-23 treatment on enhanced levels of Gqa and PLCb1 in VSMC from SHR. Confluent VSMC from 16-week-old SHR and age-matched WKY rats were incubated in the absence or presence of C-ANP 4-23 (0.1 lmolÁL À1 ) for 24 hours. The cell lysates were prepared and subjected to Western blotting using specific antibodies against Gqa (A) and PLCb1 (B) as described in "Materials and Methods." Dynein was used as a loading control.  and AKT (B) were examined in VSMCs from SHR and WKY rats and the results are shown in Figure 9. The phosphorylation levels of ERK1/2 and AKT that were enhanced by about 120% and 50%, respectively, in VSMC from SHR as compared to WKY rats were completely abolished by C-ANP 4-23 treatment, however, this treatment, did not affect the phosphorylation of ERK1/2 and AKT in VSMCs from WKY rats.

| DISCUSSION
We earlier showed that VSMC from 16-week-old SHR exhibit enhanced expression of Gqa and PLCb1 proteins that contribute to VSMC hypertrophy. 6,7 We also showed that small peptide fragments of the cytoplasmic domain of NPR-C and C-ANP 4-23, an agonist of NPR-C, attenuated the vasoactive peptide-induced hypertrophy of A10 VSMC. 20 However, in this study, we report for the first time  33 On the other hand, the activation of NPR-C by C-ANP 4-23 and resultant decreased levels of intracellular cAMP 14,15 may not be the underlying mechanism contributing to the antihyper- oxidase activity, and the enhanced expression of Nox4, p47phox in aorta, heart as well as in kidney 41 and suggest that C-ANP 4-23 -induced inhibition of oxidative stress may also play a role in the antihypertrophic effect of C-ANP 4-23 .
The role of growth factor receptors in VSMC hypertrophy has been demonstrated by several studies. [42][43][44] We earlier showed the implication of growth factor receptor activation in enhanced expression of Gqa and PLCb1 proteins and VSMC hypertrophy in SHR. 22,36,45 In this study, we demonstrate for the first time that treatment of VSMC from SHR with C-ANP 4-23 attenuated the enhanced phosphorylation of EGFR, PDGFR, and IGF-1R and suggest that the antihypertrophic effect of C-ANP 4-23 may also be attributed to its ability to attenuate the enhanced activation of growth factor receptors.
We earlier showed the role of c-Src in the increased expression of Gqa and PLCb1 proteins and enhanced protein synthesis in VSMC from SHR. 7 The implication of c-Src in high glucose-induced overexpression of Gqa and PLCb1 in A10 VSMCs has also been reported. 46 Furthermore, c-Src has also been shown as the intervening molecule between oxidative stress and growth factor receptor transactivation because N-acetylcysteine, a scavenger of O 2 À inhibited the enhanced phosphorylation of c-Src, 7 and c-Src inhibitor PP 2 , inhibited the enhanced phosphorylation of PDGFR and IGFR in VSMC from SHR. 7 In this study, we showed that C-ANP 4-23 also attenuated the In conclusion, this study shows for the first time that C-ANP [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] through the activation of NPR-C-attenuates overexpression of AT1 receptor and all the signaling molecules including oxidative stress, c-Src and growth factor receptor activation as well as MAPK/AKT that were shown to be implicated in the enhanced expression of Gqa and PLCb1 proteins in VSMC from SHR and VSMC hypertrophy. 6,7 Thus, it may be suggested that C-ANP 4-23 -induced attenuation of the increased expression of Gqa and PLCb1 proteins and hypertrophy of VSMC from SHR may be attributed to its ability to inhibit the enhanced expression of AT1 receptor, enhanced oxidative stress and downstream signaling pathways ( Figure 10) and that C-ANP 4-23 may have protective effect against oxidative stress-induced vascular complications of hypertension and could be used as a potential therapeutic agent in the treatment of vascular complications associated with hypertension and other cardiovascular diseases.

ACKNOWLEDG EMENT
This project was supported by funds from the Heart and Stroke Foundation of Canada (HSFC) [G-15-0009078].

DISCLOSURES
No conflicts of interest.

AUTHOR CONTRI BUTIONS
Jain and Anand-Srivastava participated in research design, performed data analysis, wrote or contributed to the manuscript. Jain also conducted experiments for the manuscript.