NPRC deletion mitigated atherosclerosis by inhibiting oxidative stress, inflammation and apoptosis in ApoE knockout mice – Signal Transduction and Targeted Therapy

NPRC expression is increased in atherosclerotic lesions in vivo

In the first part of in vivo experiment, Western blot analysis demonstrated increased NPRC expression in the aortic tissues of ApoE^−/− mice fed with Western diet compared with those fed with chow diet (Fig. 1a, b). Similar results were observed in the aortic roots of the two groups of mice by immunofluorescence-staining of NPRC (Fig. 1c, d). The increased NPRC expression in atherosclerotic lesions suggests a potential role of NPRC in the regulation of atherosclerosis development.

NPRC deletion attenuates atherosclerotic lesions in vivo

To explore the role of NPRC in atherosclerotic lesion formation, we generated ApoE^−/−NPRC^−/− mice in the second part of in vivo experiments (Supplementary Fig. 2a). Deficient NPRC expression in aortas of ApoE^−/−NPRC^−/− mice was confirmed by Western blot and immunofluorescence (Fig. 1e–h). ApoE^−/−NPRC^−/− mice showed a bone-overgrowth phenotype, with a notable increase of body length (Fig. 1i, j) and aortic length (Fig. 2a) in comparison with littermate ApoE^−/− mice. There was no significant difference in body weight, heart rate, diastolic and mean blood pressure (Fig. 1j), and the serum levels of TC, LDL-C, HDL-C, and VLDL-C between ApoE^−/−NPRC^−/− and littermate ApoE^−/− mice (Fig. 1k). However, systolic blood pressure and serum levels of TG were lower in ApoE^−/−NPRC^−/− mice than their littermate ApoE^−/− mice, the latter of which was probably related to a thinner phenotype of ApoE^−/−NPRC^−/− mice (Fig. 1j, k). Systemic deletion of NPRC resulted in a 63% reduction of atherosclerotic lesion area (P < 0.001) as demonstrated by Oil-red O staining of the en face aorta versus that of ApoE^−/− mice (Fig. 2a). Similarly, the cross-sectional area of atherosclerotic lesions in the aortic root was decreased by 60% (P < 0.001) as displayed by H&E staining in ApoE^−/−NPRC^−/− mice versus ApoE^−/− mice (Fig. 2b, c). These results indicated that NPRC deletion attenuated atherosclerotic lesions in ApoE^−/−NPRC^−/− mice relative to ApoE^−/− mice.

Comparison of atherosclerotic lesions and inflammatory cytokine expression between ApoE^−/− and ApoE^−/−NPRC^−/− mice. a Representative images of Oil Red O staining of en face aorta (upper panel) and quantification of Oil red O positive staining area (lower panel) in ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 10 per group) (scale bar = 5 mm). b Representative images of H&E staining (scale bar = 200 μm), Oil Red O staining (scale bar = 50 μm), Sirius red staining (scale bar = 100 μm) and immunohistochemical staining for Moma2 (scale bar = 50 μm) and α-SMA (scale bar = 50 μm) in the aortic tissues of ApoE^−/− and ApoE^−/−NPRC^−/− mice. c Quantification of cross-sectional and en face aortic plaque area, and Moma2, α-SMA and collagen I positive staining area in ApoE^−/− and ApoE^−/− NPRC^−/− mice (n = 5 per group). d Representative images of immunohistochemical staining for TNFα, MCP1, and IL-6 in atherosclerotic lesions of ApoE^−/− and ApoE^−/−NPRC^−/− mice (scale = 50 μm). e Quantification of TNFα, MCP1, and IL-6 positive staining area in atherosclerotic lesions of ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 5 per group). f Representative images of immunohistochemical staining for ICAM1 and VCAM1 in atherosclerotic lesions of ApoE^−/− and ApoE^−/−NPRC^−/− mice (scale = 100 μm). g Quantification of ICAM1 and VCAM1 positive staining area in atherosclerotic lesions of ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 5 per group). h Serum levels of IL-6, MCP1, TNFα, ICAM1, and VCAM1 in ApoE^−/− and ApoE^−/−NPRC^−/− mice measured by MSD (n = 10 per group). Normal distributions were tested by Shapiro–Wilk method. Unpaired two-tailed Student’s t tests were applied in (a), (c), (e), (g), and (h)

NPRC deletion enhances stability of atherosclerotic lesions in vivo

Next, we analyzed the cellular composition of the atherosclerotic lesions by immunostaining of the aortic root in the second part of in vivo experiments. The relative content of MOMA2⁺-macrophage/monocytes and Oil-red O positive staining area in these lesions were reduced by 59% (P < 0.001) and 43% (P < 0.01), respectively, in ApoE^−/−NPRC^−/− mice relative to the littermate ApoE^−/− mice, suggesting that the extent of inflammation and the lipid deposition in atherosclerotic lesions were substantially diminished by NPRC deletion. On the other hand, the relative content of smooth muscle cells as measured by alpha-smooth muscle actin positive staining area, and that of collagen as measured by Sirius-red positive stained area in atherosclerotic lesions were increased by 84% (P < 0.001) and 64% (P < 0.001), respectively, in ApoE^−/−NPRC^−/− mice in comparison with the littermate ApoE^−/− mice (Fig. 2b, c). Consequently, the vulnerability index of atherosclerotic lesions was reduced by 53% (P < 0.001) in ApoE^−/−NPRC^−/− mice versus the littermate ApoE^−/− mice (Fig. 2c). These results indicated that systemic NPRC deletion enhanced the stability of the aortic atherosclerotic lesions in ApoE^−/−NPRC^−/− mice versus ApoE^−/− mice.

NPRC deletion reduces pro-inflammatory cytokines in plasma and atherosclerotic lesions in vivo

To reveal the underlying molecular mechanisms of NPRC-deletion-mediated anti-atherosclerotic effects, we performed RNA high-throughput sequencing, using the whole aortas of ApoE^−/−NPRC^−/− and ApoE^−/− mice (n = 5 in each group, Supplementary Fig. 3a) in the second part of in vivo experiments. The results showed that the top 10 pathways exhibiting the most prominent difference between ApoE^−/−NPRC^−/− and ApoE^−/− mice in KEGG or GO pathway enrichment analysis were inflammation-related pathways (Supplementary Fig. 3a). In addition, the expression levels of pro-inflammatory cytokine including TNFα, MCP1, IL-6, ICAM1 and VCAM1 were lower in the aortic tissues of ApoE^−/−NPRC^−/− mice than ApoE^−/− mice, as demonstrated by qPCR (Supplementary Fig. 3b), Western blot (Supplementary Fig. 3c, d) and immunohistochemical staining (Fig. 2d to g). Furthermore, serum levels of TNFα, IL-6 and MCP1, ICAM1, and VCAM1 were significantly lower in ApoE^−/−NPRC^−/− mice than ApoE^−/− mice (Fig. 2h). These data indicated that NPRC deletion led to a substantial reduction of local and systemic inflammation in ApoE^−/− mice.

Endothelial cell-specific NPRC knockout attenuates the size and instability of atherosclerotic lesions in vivo

First, we compared the NPRC expression level in HAECs, VSMCs, and macrophages in vitro by qPCR, and found that NPRC expression was lower in VSMCs than in HAECs, and extremely low in macrophages (Supplementary Fig. 3e), suggesting that NPRC deletion in endothelial cells may be sufficient to achieve the beneficial effect. In addition, we compared the NPRC expression level between atherosclerotic lesions and lesion-free wall of the aortic root by inmmunofluoresence, and found that NPRC expression was upregulated primarily in endothelial cells and much less in smooth muscle cells (Supplementary Fig. 3f–g). For this reason, we crossed Tek-Cre mice with NPRC^flox/flox mice to derive NPRC^ecKO mice in the third part of in vivo experiments. NPRC^ecKO mice and their littermate NPRC^ecWT mice were fed with Western diet for 16 weeks after receiving an injection of PCSK9 AAV through tail veins. At the end of the experiment, no significant difference in the serum levels of TG, TC, LDL-C, HDL-C, and VLDL-C between NPRC^ecKO and littermate NPRC^ecWT mice (Supplementary Fig. 3h) was found. Deficient NPRC expression in aortas of NPRC^ecKO mice was confirmed by immunofluorescence (Supplementary Fig. 3i, j). Compared with NPRC^ecWT mice, NPRC^ecKO mice displayed 27% reduction of atherosclerotic lesion area (P < 0.01) as demonstrated by Oil-red O staining of the en face aorta (Fig. 3a). Similarly, the cross-sectional area of atherosclerotic lesions in the aortic root was decreased by 35% (P < 0.001) as displayed by H-&E staining in NPRC^ecKO mice versus NPRC^ecWT mice (Fig. 3b, c). These results indicated that endothelial cell-specific NPRC deletion attenuated atherosclerotic lesions, albeit to a less degree than systemic NPRC deletion, possibly due to the fact that NPRC is also expressed in VSMCs (Supplementary Fig. 3e–g). Furthermore, the relative content of MOMA2⁺-macrophage/monocytes and Oil-red O positive staining area in these lesions were reduced by 19% (P < 0.01) and 13% (P < 0.05), respectively, in NPRC^ecKO mice relative to the littermate NPRC^ecWT mice (Fig. 3b, c), suggesting that the extent of inflammation and the lipid deposition in atherosclerotic lesions were substantially mitigated by endothelial cell-specific NPRC deletion. In contrast, the relative content of smooth muscle cells as measured by alpha-smooth muscle actin positive staining area, and that of collagen as measured by Sirius-red positive stained area in atherosclerotic lesions were increased by 50% (P < 0.001) and 53% (P < 0.001), respectively, in NPRC^ecKO mice in comparison with the littermate NPRC^ecWT mice (Fig. 3b, c). Consequently, the vulnerability index of atherosclerotic lesions was reduced by 60% (P < 0.001) in NPRC^ecKO mice versus the littermate NPRC^ecWT mice (Fig. 3c). Consistently, the expression levels of pro-inflammatory cytokines including TNFα, MCP1, IL-6, ICAM1, and VCAM1 were significantly lower in the aortic tissues of NPRC^ecKO mice than NPRC^ecWT mice, as demonstrated by immunohistochemical staining (Fig. 3d, e). These data indicated that endothelial cell-specific NPRC deletion attenuated local and systemic inflammation and enhanced the stability of atherosclerotic lesions.

Comparison of atherosclerotic lesions, inflammatory cytokine expression, and oxidative stress between NPRC^ecWT and NPRC^ecKO mice. a Representative images of Oil Red O staining of en face aorta (upper panel) and quantification of Oil red O positive staining area (lower panel) in NPRC^ecWT and NPRC^ecKO mice (n = 5 per group) (scale bar = 5 mm). b Representative images of HE staining (scale bar = 200 μm), Oil Red O staining (scale bar = 50 μm), Sirius red staining (scale bar = 100 μm), and immunohistochemical staining for Moma2 (scale bar = 50 μm) and α-SMA (scale bar = 50 μm) in the aortic tissues of NPRC^ecWT and NPRC^ecKO mice. c Quantification of cross-sectional and en face aortic plaque area, and MOMA2, α-SMA and collagen I positive staining area in NPRC^ecWT and NPRC^ecKO mice (n = 5 per group). d Representative images of immunohistochemical staining for TNFα, MCP1, IL-6, ICAM1, and VCAM1 in atherosclerotic lesions of NPRC^ecWT and NPRC^ecKO mice (scale = 50 μm). e Quantification of TNFα, MCP1, IL-6, ICAM1, and VCAM1 positive staining area in atherosclerotic lesions of NPRC^ecWT and NPRC^ecKO mice (n = 5 per group). f Representative Western blot images of eNOS expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 5 per group). g Quantification of eNOS expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 5 per group). h Quantification of mRNA level of eNOS in aorta from ApoE^−/− mice and ApoE^−/−NPRC^−/− mice (n = 5 per group). i Quantification of oxLDL level in serum and aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice by ELISA (n = 5 per group). j Representative DHE staining of ROS in the aortic root from ApoE^−/− and ApoE^−/−NPRC^−/− mice (scale = 50 μm). k Quantification of mean fluorescence intensity of ROS in the aortic root from ApoE^−/− and ApoE^−/−NPRC^−/− mice. l Quantification of oxLDL level in serum and aorta from NPRC^ecWT and NPRC^ecKO mice by ELISA (n = 5 per group). m Representative DHE staining of ROS in the aortic root from NPRC^ecWT and NPRC^ecKO mice (scale = 50 μm). n Quantification of mean fluorescence intensity of ROS in the aortic root from NPRC^ecWT and NPRC^ecKO mice. Normal distributions were tested by Shapiro–Wilk method. Unpaired two-tailed Student’s t tests were applied in (a), (c), (e), (g), (h), (i), (l), and (n)

NPRC defection alleviates oxidative stress in the aortic tissues and HAECs by rescuing eNOS expression

As nitric oxide (NO), synthesized and released by vascular endothelial cells, activates the PKG signaling pathway in VSMCs to keep vessel dilated, and endothelial NO synthase (eNOS) is the dominant enzyme to catalyze the formation of NO in endothelial cells, eNOS plays a key role in maintaining endothelial function and vascular homeostasis.²⁹ In the aorta of ApoE^−/−NPRC^−/− mice in the second part of in vivo experiments, we found enhanced expression of eNOS compared to ApoE^−/− mice by Western blot and qPCR (Fig. 3f–h). In addition, the expression of p-eNOS in the aortic root was upregulated in ApoE^−/−NPRC^−/− mice versus ApoE^−/− mice, and in NPRC^ecKO mice versus NPRC^ecWT mice by inmmunofluoesence (Supplementary Fig. 4a–d). As endothelium-derived NO also acts as an antioxidant to inhibit LDL oxidation and peroxidases, we measured the oxidized LDL level in serum and aortic tissues and ROS level in the aortic root of mice. We found that the oxidized LDL level in serum and aortic tissues and ROS level in the aortic root assayed by DHE staining were reduced in ApoE^−/−NPRC^−/− mice versus ApoE^−/− mice (Fig. 3i–k). These results were further verified by the evidence that the oxidized LDL level in the aortic tissues and ROS level in the aortic root were also attenuated in NPRC^ecKO mice compared with the littermate NPRC^ecWT mice in the third part of in vivo experiments (Fig. 3l–n). Similarly, oxLDL stimulation time-dependently downregulated expression of eNOS in HAECs in vitro, whereas knockdown of NPRC rescued substantially eNOS expression in HAECs treated with oxLDL (Fig. 4a–d). Moreover, ROS production induced by oxLDL in HAECs was alleviated by knockdown of NPRC (Fig. 4e, f). These results suggested that NPRC deletion protected endothelial cells from oxidative stress by rescuing eNOS expression.

Loss of NPRC increased eNOS expression and alleviated endothelial oxidative stress and inflammation. a Representative Western blot images of NPRC expression in si-NC (Negative Control) and si-NPRC (NPRC-knockdown) HAECs. b Quantification of NPRC expression in si-NC and si-NPRC HAECs (n = 5 per group). c Representative Western blot images of eNOS expression in oxLDL-stimulated si-NC and si-NPRC HAECs. d Quantification of eNOS expression in oxLDL-stimulated si-NC and si-NPRC HAECs (n = 5 per group). e Representative DHE staining of ROS in oxLDL-stimulated si-NC and si-NPRC HAECs. f Quantification of mean fluorescence intensity of ROS in oxLDL-stimulated si-NC and si-NPRC HAECs (n = 5 per group). g Representative Western blot images of VCAM1 and ICAM1 expression in oxLDL-stimulated si-NC and si-NPRC HAECs. h Quantification of VCAM1 and ICAM1 expression in oxLDL-stimulated si-NC and si-NPRC HAECs (n = 5 per group). i Representative images of Raw 264.7 migration from oxLDL-stimulated si-NC and si-NPRC HAECs (scale bar = 50 μm). j Quantification of crystal violet positive cells in Raw 264.7 stimulated by medium from si-NC and si-NPRC HAECs (n = 5 per group). k Quantification of mRNA expression levels of MCP1, TNFα, and IL-6 in si-NC and si-NPRC HAECs after oxLDL stimulation at indicated time points (n = 5 per group). l Representative images of phagocytosis of Raw 264.7 stimulated by medium from si-NC and si-NPRC HAECs after oxLDL stimulation (scale bar = 50 μm). m Quantification of relative positive staining area of Oil Red O (n = 5 per group). Normal distributions were tested by Shapiro–Wilk method. Unpaired two-tailed Student’s t tests were applied in (b), (f), (j), and (m). Two-way ANOVA was utilized in (d), (h), and (j)

NPRC knockdown inhibits macrophage migration, cytokine expression, and phagocytosis via effects on HAECs in vitro

Previous studies reported that endothelial cells are activated during the early phase of atherosclerosis, with resultant secretion of adhesive molecules and chemokines to recruit macrophages into the vascular wall. Thus, we examined whether knockdown of endothelial NPRC affects expression of adhesive molecules and chemokines in HAECs, and the results showed that expression of pro-inflammatory adhesive molecules, such as ICAM1 and VCAM1, was upregulated time-dependently after oxLDL stimulation, whereas this trend was markedly attenuated by NPRC knockdown (Fig. 4g, h) in HAECs. We found that Raw 264.7 cells exhibited less migration stimulated by the medium of NPRC-knockdown HAECs than that by the medium of control HAECs (Fig. 4i, j). In addition, qPCR demonstrated increased expression of TNFα, IL-6, and MCP-1 in peritoneal macrophages stimulated by the medium from control HAECs after oxLDL treatment. In contrast, expression of these cytokines was downregulated in peritoneal macrophages stimulated by the medium from NPRC-knockdown HAECs after oxLDL treatment (Fig. 4k). Finally, macrophages stimulated by the medium from NPRC-knockdown HAECs showed less phagocytosis of lipids than those stimulated by the medium from control HAECs (Fig. 4l, m). Taken together, these results demonstrated that NPRC knockdown inhibited pro-inflammatory cytokine expression in HAECs, which in turn suppressed macrophage migration, cytokine expression, and phagocytosis.

NPRC knockdown attenuates apoptosis of HAECs via enhancing AKT1 phosphorylation in vitro

In the process of atherosclerosis, increased apoptosis of vascular endothelial cells may destruct endothelial integrity and permit pro-inflammatory cells to migrate into the arterial wall.³ In this study, we first examined the apoptotic status of HAECs in vitro by TUNEL after stimulation of oxLDL, and found that oxLDL increased the number of apoptotic HAECs, whereas NPRC knockdown in HAECs effectively attenuated apoptosis induced by oxLDL (Fig. 5a, b). In addition, we measured the expression levels of several apoptosis-related proteins, such as cleaved-caspase3 and cleaved-caspase7, which were decreased in NPRC-knockdown HAECs versus control HAECs (Fig. 5c, d). As p-AKT1 plays an important role in protecting endothelial cells from apoptosis,³⁰ we further examined the phosphorylation levels of AKT1, which were found to be time-dependently declined in control HAECs after oxLDL stimulation while this trend was partially rescued in NPRC-knockdown HAECs receiving the same oxLDL treatment (Fig. 5c, d). These results demonstrated that NPRC knockdown prevented endothelial cell apoptosis induced by oxLDL via enhancing phosphorylation of AKT1.

Loss of NPRC increased AKT1 phosphorylation, mitigated oxidative stress, inflammation, and apoptosis. a Representative images of TUNEL assay in si-NC and si-NPRC HAECs stimulated by oxLDL (scale bar = 100 μm). b Quantification of number of TUNEL-positive cells in si-NC and si-NPRC HAECs stimulated by oxLDL (n = 5 per group). c Representative Western blot images of p-AKT1, AKT1, Cleaved-caspase 3, Caspase 3, Cleaved-caspase 7, and Caspase 7 expression in oxLDL-stimulated si-NC and si-NPRC HAECs. d Quantification of p-AKT1, AKT1, Cleaved-caspase 3, Caspase 3, Cleaved-caspase 7, and Caspase 7 expression in oxLDL-stimulated si-NC and si-NPRC HAECs (n = 5 per group). e Representative Western blot images of IL-6, TNFα, and MCP1 in oxLDL-stimulated si-NC and si-NPRC HAECs. f Quantification of IL-6, TNFα, and MCP1 expression in oxLDL-stimulated si-NC and si-NPRC HAECs (n = 5 per group). g Representative Western blot images of p-Iκκα/β, Iκκα, p-P65, and P65 expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice. h Quantification of p-Iκκα/β/Iκκ and p-P65/P65 expression in aorta from ApoE^−/− and ApoE^−/− NPRC^−/− mice (n = 5 per group). i Representative Western blot images of p-Iκκα/β, Iκκα, Iκκβ, p-P65, and P65 expression in oxLDL-stimulated si-NC and si-NPRC HAECs. j Quantification of p-Iκκα/β, Iκκα, Iκκβ, p-P65, and P65 expression in oxLDL-stimulated si-NC and si-NPRC HAECs (n = 5 per group). k Representative immunofluorescence images of p-P65 in si-NC and si-NPRC HAECs stimulated by oxLDL (scale bar = 50 μm). l Quantification of p-P65 immunofluorescent intensity in nuclei of si-NC and si-NPRC HAECs stimulated by oxLDL (n = 5 per group). Normal distributions were tested by Shapiro–Wilk method. Unpaired two-tailed Student’s t tests were applied in (b), (g), and (l). Two-way ANOVA was used in (d), (f), and (j)

NPRC knockdown suppresses inflammation via downregulating NF-κB pathway in vivo and in vitro

Previous studies showed that activated NF-κB signaling pathway induces transcription of cell adhesion molecules, such as ICAM1, VCAM1, E-selectin and P-selectin, and pro-inflammatory chemokines and cytokines including MCP1, IL-6, and TNFα, in endothelial cells.³¹ In the present study, NPRC-knockdown HAECs exhibited a markedly reduction of MCP1, TNFα, and IL-6 expression displayed by Western blot (Fig. 5e, f) and qPCR (Supplementary Fig. 4e). In addition, the levels of MCP1, TNFα and IL-6 in the liquid supernatant of control HAECs were remarkably elevated after oxLDL stimulation whereas these cytokine levels in the liquid supernatant of NPRC-knockdown HAECs were much lower after oxLDL stimulation (Supplementary Fig. 4f). More importantly, we found a much lower phosphorylation level of P65 and IKKα/β in the aortic tissues from ApoE^−/−NPRC^−/− mice than ApoE^−/− mice in the second part of in vivo experiments (Fig. 5g, h). In addition, we found a significant and time-dependent increase in the phosphorylation level of P65 and IKKα/β after oxLDL stimulation in control HAECs whereas the phosphorylation level of p65 and IKKα/β after oxLDL stimulation was significantly attenuated in NPRC-knockdown HAECs (Fig. 5i, j). We also found less translocation of p-P65 protein into nucleus in NPRC-knockdown HAECs than in control HAECs (Fig. 5k, l). These results suggested that knockdown of NPRC in HAECs alleviated inflammation via inhibiting pro-inflammatory NF-κB signaling pathway.

NPRC deletion activates cAMP/PKA and cGMP/PKG pathways in vivo

In addition to the role as a clearance receptor for ANP, BNP and CNP, NPRC has been found to inhibit guanine nucleotide regulatory protein (Gi) via its catalytic activity, which may decrease cyclic adenosine monophosphate (cAMP) levels. To explore the molecular mechanism of attenuated inflammation in ApoE^−/−NPRC^−/− mice, we examined the changes of cAMP/PKA signaling pathway in the second part of in vivo experiments. First, we measured the expression level of cAMP by chemiluminescent assay, and the phosphorylation level of phospho-PKA substrate (RRXS*/T*) and phosphorylated cAMP response element binding protein (CREB) in the mouse aorta by Western blot, and found that cAMP level and phosphorylation level of RRXS*/T* and CREB in aortic tissues were significantly increased in ApoE^−/−NPRC^−/− mice compared with ApoE^−/− mice, suggesting a considerable activation of cAMP/PKA/p-CREB pathway in the former group (Fig. 6a–c). We then examined the relation between NPRC knockdown and activation of PKA in endothelial cells and found that phospho-PKA substrates (RRXS*/T*) increased markedly in NPRC-knockdown HAECs relative to control HAECs, as shown by Western blot (Fig. 6d, e), which was similar to PKA activation in vivo. Thereafter, we examined the expression level of downstream proteins of PKA, such as PPARγ and PGC1α, and found that expression of these proteins was upregulated in ApoE^−/−NPRC^−/− mice versus ApoE^−/− mice as demonstrated by Western blot (Fig. 6f, g) and qPCR (Fig. 6h). Similarly, the expression level of PPAR-γ and PGC1α was higher in the aortic tissues of ApoE^−/−NPRC^−/− mice than ApoE^−/− mice as shown by immunohistochemical staining (Fig. 6i, j). In addition, we found that the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) was upregulated in ApoE^−/−NPRC^−/− mice relative to ApoE^−/− mice, suggesting that cGMP/PKG/p-VASP pathway was activated as well (Fig. 6f, g), possibly due to a defect of clearance for natriuretic peptides, especially CNP, in ApoE^−/−NPRC^−/− mice. To test this hypothesis, we measured the serum levels and protein expression levels of ANP, BNP, and CNP in the two groups of mice. The serum levels of ANP and CNP were substantially higher in ApoE^−/−NPRC^−/− mice than ApoE^−/− mice (Supplementary Fig. 5), while no difference was detected in the serum levels of BNP between the two groups of mice, likely due to the fact that the physiological level of BNP is much lower than ANP and CNP because secretion of BNP is mainly driven by atrial dilatation. As expected, the protein expression of NPRA and NPRB in the aortic tissues was similar between the two groups of mice (Fig. 6k, l). Similarly, the expression levels of ANP and BNP in the aortic tissues showed no difference between ApoE^−/−NPRC^−/− and ApoE^−/− mice while CNP exhibited a much higher expression level in the former than the latter group, probably because ANP and BNP are secreted by cardiomyocytes while CNP is secreted by vascular endothelial cells (Fig. 6k, l). Taken together, these results indicated that NPRC deletion activated both cAMP/PKA and cGMP/PKG pathways in ApoE^−/−NPRC^−/− mice.

Loss of NPRC activated cAMP/PKA and cGMP/PKG pathways in vivo and in vitro. a Quantification of cAMP level in aorta from ApoE^−/− and ApoE^−/− NPRC^−/− mice (n = 5 per group). b Representative Western blot images of p-PKA-substrate, p-CREB and CREB expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice. c Quantification of p-PKA-substrate and p-CREB/CREB expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 5 per group). d Representative Western blot images of p-PKA-substrate expression in si-NC and si-NPRC HAECs. e Quantification of p-PKA-substrate expression in si-NC and si-NPRC HAECs (n = 5 per group). f Representative Western blot images of p-VASP, VASP, PGC1α, and PPAR-γ expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice. g Quantification of p-VASP/VASP, PGC1α, and PPAR-γ expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 5 per group). h Quantification of mRNA levels of PGC1α and PPAR-γ in aorta of ApoE^−/− and ApoE^−/− NPRC^−/− mice (n = 5 per group). i Representative images of immunohistochemical staining of PGC1α, PPAR-γ, and p-CREB in aorta of ApoE^−/− mice and ApoE^−/−NPRC^−/− mice (scale bar = 50 μm). j Quantification of relative positive staining area of PGC1α, PPAR-γ, and p-CREB (n = 5 per group). k Representative Western blot images of NPRA, NPRB, ANP, BNP, and CNP expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice. l Quantification of NPRA, NPRB, ANP, BNP, and CNP expression in aorta from ApoE^−/− and ApoE^−/−NPRC^−/− mice (n = 5 per group). Normal distributions were tested by Shapiro–Wilk method. Unpaired two-tailed Student’s t tests were applied in (a), (c), (e), (g), (h), (j), and (l)

NPRC overexpression in endothelium aggravates atherosclerotic lesions in vivo

To further verify the role of NPRC in atherosclerotic lesion formation, we overexpressed NPRC gene in vascular endothelial cells of mice by injecting AAV9 containing ICAM-2 promotor-NPRC-EGFP-3flag-WPRE-SV40_polyA into ApoE^−/− or ApoE^−/−NPRC^−/− mice to generate ApoE^−/−OE or ANDK (ApoE and NPRC Double Knockout)^OE mice, and injecting AAV9 containing ICAM-2 promotor-EGFP-3flag-WPRE-SV40_polyA into ApoE^−/− or ApoE^−/−NPRC^−/− mice to generate control mice in the fourth part of in vivo experiments. Enhanced NPRC expression in aortas of ApoE^−/−OE and ANDK^OE mice was confirmed by immunofluorescence (Supplementary Fig. 6a, b). There was no significant difference in the serum levels of TG, TC, LDL-C, HDL-C, and VLDL-C between ApoE^−/−OE and littermate ApoE^−/− mice or between ApoE^−/−NPRC^−/− mice and littermate ANDK^OE mice (Supplementary Fig. 6c). Endothelial overexpression of NPRC resulted in an increase of 18% (P < 0.05) in atherosclerotic lesion area as demonstrated by Oil-red O staining of the en face aorta versus that of ApoE^−/− mice (Fig. 7a). The cross-sectional area of atherosclerotic lesions in the aortic root increased by 23% (P < 0.05) as displayed by H&E staining in ApoE^−/−OE mice versus ApoE^−/− mice (Fig. 7b, c). More strikingly, compared with ApoE^−/−NPRC^−/− mice, the en face atherosclerotic lesion area was increased by 82% (P < 0.01) in ANDK^OE mice (Fig. 7b, c). Similarly, the cross-sectional area of atherosclerotic lesions in the aortic root increased by 44% (P < 0.05) as displayed by H&E staining in ANDK^OE mice versus ApoE^−/−NPRC^−/− mice (Fig. 7b, c). These results indicated that NPRC overexpression in vascular endothelium aggravated atherosclerotic lesions on the background of ApoE^−/− or ApoE^−/−NPRC^−/−.

Comparison of atherosclerotic lesions, inflammatory cytokine expression, and oxidative stress between ApoE^−/− and ApoE^−/−OE mice and between ApoE^−/−NPRC^−/− and ANDK^OE mice. a Representative images of Oil Red O staining of en face aorta (upper panel) and quantification of Oil red O positive staining area (lower panel) in ApoE^−/−, ApoE^−/−OE, ApoE^−/−NPRC^−/−, and ANDK^OE mice (n = 5 per group) (scale bar = 5 mm). b Representative images of HE staining (scale bar = 200 μm), Oil Red O staining (scale bar = 50 μm), Sirius red staining (scale bar = 100 μm), and immunohistochemical staining for Moma2 (scale bar = 50 μm) and α-SMA (scale bar = 50 μm) in the aortic tissues of ApoE^−/−, ApoE^−/−OE, ApoE^−/−NPRC^−/− and ANDK^OE mice. c Quantification of cross-sectional and en face aortic plaque area, and Moma2, α-SMA, and collagen I positive staining area in ApoE^−/−, ApoE^−/−OE, ApoE^−/−NPRC^−/−, and ANDK^OE mice (n = 5 per group). d Representative images of immunohistochemical staining for TNFα, MCP1, IL-6, ICAM1, and VCAM1 in atherosclerotic lesions of ApoE^−/−, ApoE^−/−OE, ApoE^−/−NPRC^−/−, and ANDK^OE mice (scale = 50 μm). e Quantification of TNFα, MCP1, IL-6, ICAM1, and VCAM1 positive staining area in atherosclerotic lesions of ApoE^−/−, ApoE^−/−OE, ApoE^−/−NPRC^−/−, and ANDK^OE mice (n = 5 per group). f Representative Western blot images of eNOS expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs. g Quantification of eNOS expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs (n = 5 per group). h Quantification of oxLDL level in serum and aorta from ApoE^−/− mice and ApoE^−/−oe mice by ELISA (n = 5 per group). i Representative images of DHE staining of ROS in the aortic root from ApoE^−/−, ApoE^−/−OE, ApoE^−/−NPRC^−/−, and ANDK^OE mice (scale = 50 μm). j Quantification of mean fluorescence intensity of ROS in the aortic root from ApoE^−/−, ApoE^−/−OE, ApoE^−/−NPRC^−/−, and ANDK^OE mice. Normal distributions were tested by Shapiro–Wilk method. One-way ANOVA tests were applied in (a), (c), (e). Unpaired two-tailed Student’s t tests were applied in (h) and (j). Two-way ANOVA was used in (f)

Next, we analyzed the cellular composition of the atherosclerotic lesions by immunostaining of the aortic root. The relative content of MOMA2⁺-macrophage/monocytes and Oil-red O positive staining area in these lesions increased by 11% (P < 0.01) and 12% (P < 0.05), respectively, in ApoE^−/−OE mice relative to the littermate ApoE^−/− mice, and enlarged by 28% (P < 0.05) and 15% (P < 0.05), respectively, in ANDK^OE mice relative to the littermate ApoE^−/−NPRC^−/− mice, suggesting that the extent of inflammation and the lipid deposition in atherosclerotic lesions were substantially aggravated by NPRC overexpression (Fig. 7b, c). In contrast, the relative content of smooth muscle cells as measured by alpha-smooth muscle actin positive staining area, and that of collagen as measured by Sirius-red positive stained area in atherosclerotic lesions decreased by 42% (P < 0.05) and 19% (P < 0.05), respectively, in ApoE^−/−OE mice in comparison with the littermate ApoE^−/− mice, and declined by 52% (P < 0.001) and 14% (P < 0.05), respectively, in ANDK^OE mice relative to the littermate ApoE^−/−NPRC^−/− mice (Fig. 7b, c). Consequently, the vulnerability index of atherosclerotic lesions was increased by 39% (P < 0.01) in ApoE^−/−OE mice versus the littermate ApoE^−/− mice and by 43% (P < 0.01) in ANDK^OE mice versus the littermate ApoE^−/−NPRC^−/− mice (Fig. 7b, c). These results indicated that endothelial NPRC overexpression weakened the stability of the aortic atherosclerotic lesions on the background of ApoE^−/− or ApoE^−/−NPRC^−/−.

NPRC overexpression increases pro-inflammatory cytokine expression in atherosclerotic lesions in vivo

In the fourth part of in vivo experiments, expression levels of pro-inflammatory cytokines and adhesive molecules including TNFα, MCP1, IL-6, ICAM1, and VCAM1 were upregulated in the aortic tissues of ApoE^−/−OE mice relative to ApoE^−/− mice, as demonstrated by immunohistochemical staining. Similarly, increased expression of TNFα, MCP1, IL-6, ICAM1, and VCAM1 were observed in the aortic tissues of ANDK^OE mice versus ApoE^−/−NPRC^−/− mice (Fig. 7d, e). These data indicated that endothelial NPRC overexpression led to a remarkable aggravation of inflammation in ApoE^−/− mice.

NPRC overexpression aggravates oxidative stress in vivo and in vitro by downregulating eNOS expression

To further elaborate the mechanism of NPRC in regulating the function of endothelial cells, NPRC was overexpressed in HAECs, which was confirmed by Western blot (Supplementary Fig. 7a, b). In accordance with aforementioned results that NPRC deletion rescued the downregulated expression of eNOS in vivo and vitro, eNOS expression was reduced in HAECs after oxLDL treatment which was further declined in NPRC overexpressed HAECs (Fig. 7f, g). The decreased eNOS expression resulted in elevated oxLDL and ROS levels in the aortic tissues in NPRC- overexpressed mice relative to control mice (Fig. 7h–j). Similarly, ROS level was increased in NPRC-overexpressed HAECs assayed by DHE staining (Supplementary Fig. 7c, d). These results suggested that NPRC overexpression aggravated oxidative stress in vivo and vitro induced by pro-atherogenic stimulation.

NPRC overexpression promotes pro-inflammatory cytokine expression, macrophage migration and phagocytosis in vitro

The expression level of pro-inflammatory cytokines and adhesive molecules including TNFα, MCP1, IL-6, ICAM1, and VCAM1, was upregulated time-dependently in HAECs after oxLDL stimulation, while this trend was markedly enhanced by NPRC overexpression in HAECs (Fig. 8a, b). In addition, Raw 264.7 cells exhibited more extensive migration stimulated by the medium of NPRC-overexpressed HAECs than by the medium of control HAECs (Fig. 8c, d). Macrophages stimulated by the medium from NPRC-overexpressed HAECs showed more phagocytosis of lipids than those stimulated by the medium from control HAECs (Fig. 8e, f). These results demonstrated that NPRC overexpression promoted pro-inflammatory cytokine expression in HAECs, which in turn increased macrophage migration and phagocytosis.

Overexpression of NPRC increased inflammation and apoptosis and aggravated migration and phagocytosis of macrophages. a Representative Western blot images of ICAM1, VCAM1, TNFα, MCP1, and IL-6 expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs. b Quantification of ICAM1, VCAM1, TNFα, MCP1, and IL-6 expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs (n = 5 per group). c Representative images of Raw 264.7 migration in oxLDL-stimulated oe-NC and oe-NPRC HAECs (scale bar = 100 μm). d Quantification of crystal violet positive cells in Raw 264.7 stimulated by medium from si-NC and si-NPRC HAECs (n = 5 per group). e Representative images of phagocytosis of Raw 264.7 from si-NC and oe-NPRC HAECs after oxLDL stimulation (scale bar = 100 μm). f Quantification of relative positive staining area of Oil Red O in (e) (n = 5 per group). g Representative images of TUNEL assay in oxLDL-stimulated oe-NC and oe-NPRC HAECs (scale bar = 100 μm). h Quantification of TUNEL-positive cells in oxLDL-stimulated oe-NC and si-NPRC HAECs (n = 5 per group). i Representative Western blot images of p-AKT1, AKT1, Cleaved-caspase 3, Caspase 3, Cleaved-caspase 7, and Caspase 7 expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs. j Quantification of p-AKT1/AKT1, Cleaved-caspase 3/Caspase 3 and Cleaved-caspase 7/Caspase 7 expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs (n = 5 per group). Normal distributions were tested by Shapiro–Wilk method. Unpaired two-tailed Student’s t tests were applied in (d), (f), (h). Two-way ANOVA was used in (b) and (j)

NPRC overexpression aggregates apoptosis of HAECs via inhibiting AKT1 phosphorylation in vitro

Compared with the empty vector group, overexpression of NPRC in HAECs increased cell apoptosis induced by oxLDL (Fig. 8g, h). In addition, the expression levels of several apoptosis-related proteins, such as cleaved-caspase3 and cleaved-caspase7, were dramatically increased in NPRC-overexpressed HAECs versus empty vector-transfected HAECs (Fig. 8i, j). The phosphorylation level of AKT1, which was declined in control HAECs after oxLDL stimulation, was further decreased in NPRC-overexpressed HAECs receiving the same oxLDL treatment (Fig. 8i, j). Taken together, these results demonstrated that NPRC overexpression promoted endothelial cell apoptosis induced by oxLDL via downregulating phosphorylation of AKT1.

NPRC overexpression increases inflammation via upregulating NF-κB pathway

In the present study, NPRC-knockout in endothelial cells and NPRC-knockdown in HAECs markedly reduced the phosphorylation level of P65 and IKKα/β in vivo and in vitro, respectively. In addition, the phosphorylation level of P65 and IKKα/β was significantly increased in NPRC-overexpressed HAECs compared with that in control HAECs after oxLDL stimulation (Fig. 9a, b). Furthermore, p-P65 protein translocated into nucleus more significantly in NPRC-overexpressed HAECs than in control HAECs (Fig. 9c, d). These results suggested that overexpression of NPRC in HAECs aggravated inflammation via upregulating pro-inflammatory NF-κB signaling pathway.

Activation of PKA pathway upregulated eNOS and p-AKT1, and inhibited p-p65 expression. a Representative Western blot images of p-Iκκα/β, Iκκα, Iκκβ, p-P65, and P65 expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs. b Quantification of p-Iκκα/β, Iκκα, Iκκβ, p-P65, and P65 expression in oxLDL-stimulated oe-NC and oe-NPRC HAECs (n = 5 per group). c Representative immunofluorescence images of p-P65 in oe-NC and oe-NPRC HAECs stimulated by oxLDL (scale bar = 50 μm). d Quantification of p-P65 immunofluorescent intensity in nuclei of oe-NC and oe-NPRC HAECs stimulated by oxLDL (n = 5 per group). e Representative Western blot images of p-PKA-substrate expression in HAECs treated by forskolin and H89. f Quantification of p-PKA-substrate expression in HAECs treated by forskolin and H89 (n = 5 per group). g Representative Western blot images of eNOS expression in HAECs treated by forskolin and H89. h Quantification of eNOS expression in HAECs treated by forskolin and H89 (n = 5 per group). i Representative Western blot images of p-P65, P65, p-AKT1, and AKT1 expression in HAECs treated by forskolin and H89. j Quantification of p-P65/P65 expression in HAECs treated by forskolin and H89 (n = 5 per group). k Representative images of TUNEL assay in HAECs pretreated by forskolin and H89, and then stimulated by oxLDL (scale bar = 100 μm). l Quantification of TUNEL-positive cells in HAECs pretreated by forskolin and H89, and then stimulated by oxLDL (n = 5 per group). Normal distributions were tested by Shapiro–Wilk method. Two-way ANOVA was applied in (b). Unpaired two-tailed Student’s t tests were applied in (d). One-way ANOVAs were used in (f), (h), (j), and (l)

Activation of PKA signaling pathway inhibits pro-inflammatory cytokine expression and endothelial apoptosis in vitro

To understand why loss of NPRC increased the expression of eNOS in the aortic tissues and HAECs, we used forskolin, an agonist of PKA, and H89, an antagonist of PKA, to treat HAECs and found that expression of p-PKA substrates was markedly increased in HAECs treated by forskolin, which was reversed by H89 treatment in HAECs (Fig. 9e, f). Moreover, expression of eNOS was substantially enhanced in HAECs stimulated by forskolin for 24 h, which was offset by H89 treatment in HAECs (Fig. 9g, h). We then used forskolin and H89 to explore the relation between PKA pathway activation and NF-κB signaling in HAECs, and found that the phosphorylation level of P65 was significantly decreased after stimulation of forskolin but remarkably increased by H89 treatment in HAECs (Fig. 9i, j). Finally, as PKA/p-AKT1 pathway is reportedly to play a major role in modulating endothelial cell apoptosis, we further explored the relation between PKA/p-AKT1 pathway and HAECs apoptosis by using forskolin and H89. We found that treatment with forskolin increased, whereas treatment with H89 dramatically decreased, the phosphorylation level of AKT1 in HAECs (Fig. 9i, j). To verify this result, we applied forskolin and H89 to HAECs after oxLDL stimulation for 24 h, and the result showed that the number of apoptotic cells detected by TUNEL was decreased when pretreated HAECs with forskolin, but markedly increased when pretreated by H89 (Fig. 9k, l). Taken together, these data demonstrated that activation of PKA signaling pathway inhibited NF-κB signaling and endothelial apoptosis.