Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype

Abstract

The transforming growth factor-beta superfamily member bone morphogenetic protein-2 (BMP-2) is up-regulated in atherosclerotic arteries; however, its effects on the endothelium are not well characterized. Using microdissected coronary arterial endothelial cells (CAECs) and cultured primary CAECs, we demonstrated endothelial mRNA expression of BMP-2 and BMP-4. The proinflammatory cytokine tumor necrosis factor-alpha and H2O2 significantly increased endothelial expression of BMP-2 but not BMP-4. In organ culture, BMP-2 substantially decreased relaxation of rat carotid arteries to acetylcholine and increased production of reactive oxygen species, events inhibited by pharmacologically blocking protein kinase C (PKC) or NAD(P)H oxidase. BMP-2 activated nuclear factor-kappaB in CAECs, and BMP-2 and BMP-4 substantially increased adhesion of monocytic THP-1 cells, which was reduced by pharmacologically inhibiting p42/44 MAP kinase pathway (also by siRNA down-regulating ERK-1/2) or PKC. Incubation of rat carotid arteries with BMP-2 ex vivo also increased adhesion of mononuclear cells to the endothelium, requiring p42/44 MAP kinase and PKC. Western blotting showed that in CAECs and carotid arteries BMP-2 elicited phosphorylation of p42/44 MAP kinase, which was reduced by blocking MAP kinase kinase and PKC. Collectively, expression of BMP-2 is regulated by proinflammatory stimuli, and increased levels of BMP-2 induce endothelial dysfunction, oxidative stress, and endothelial activation. Thus, the proinflammatory effects of BMP-2 may play a role in vascular pathophysiology.

Figure 1

A and B: Expression of BMP-2 in endothelial and smooth muscle cells microdissected from coronary arteries (A) and in cultured primary rat CAECs and VSMCs (B). Analysis of mRNA expression was performed by real-time QRT-PCR. GAPDH was used for normalization. Data are mean ± SEM (n = 3 to 5 for each group). *P < 0.05. C: Fluorescent photomicrographs showing that immunolabeling for BMP-2 (green) is present in endothelial cells (double arrows) and medial smooth muscle cells (bold arrow, smooth muscle cells show red immunofluorescent labeling for α-smooth muscle actin). D: VISTA plot showing the percentage of conservation between the mRNAs transcribed from the rat BMP-2 and BMP-4 genes. E and F: Expression of BMP-4 in endothelial and smooth muscle cells microdissected from coronary arteries (E) and in cultured CAECs and VSMCs (F). Data are mean ± SEM (n = 3 to 5 for each group). *P < 0.05.

Figure 2

Effect of TNF-α (1 or 10 ng/ml, for 24 hours) on the expression of BMP-2 mRNA (A, C: QRT-PCR) and protein (B, D: Western blotting) in cultured primary rat CAECs and VSMCs. Data are mean ± SEM (n = 4 to 8 for each group). *P < 0.05 versus untreated control. E: Expression of BMP-2 mRNA in endothelial and smooth muscle cells microdissected from control and TNF-α-treated (10 ng/ml, for 24 hours) cultured rat aortas. Data are mean ± SEM. *P < 0.05 versus untreated control. F: Reporter gene assay showing the effects of TNF-α (10 ng/ml) on NF-κΒ reporter activity in CAECs and VSMCs. Cells were transiently co-transfected with NF-κΒ-driven firefly luciferase and CMV-driven Renilla luciferase constructs followed by TNF-α stimulation. Cells were then lysed and subjected to luciferase activity assay. After normalization relative luciferase activity was obtained from seven independent transfections. In control experiments PDTC was used to inhibit NF-κB activity. Data are mean ± SEM. *P < 0.05 versus control. G: Effect of TNF-α (1 or 10 ng/ml, for 24 hours) on the expression of BMP-4 mRNA (QRT-PCR) in CAECs and VSMCs (H). Data are mean ± SEM. *P < 0.05 versus untreated control.

Figure 3

Effect of H₂O₂ on BMP-2 (A) and BMP-4 (B) mRNA expression in CAECs (QRT-PCR). Data are mean ± SEM (n = 5 for each group). *P < 0.05 versus untreated control.

Figure 4

Relaxations to acetylcholine (A) and the NO donor S-nitrosopenicillamine (B) in ring preparations of rat carotid arteries maintained in vessel culture (for 24 hours) in the absence and presence of recombinant BMP-2 (20 or 200 ng/ml). Data are mean ± SEM (n = 4 to 6 for each group). *P < 0.05. C: Compared to the untreated controls (left), BMP-2 elicited significant increases in endothelial O₂^.− production, as indicated by the intensive red fluorescent staining of the endothelial nuclei by ethidium bromide (right). Green autofluorescence is shown for orientation purposes (L, lumen; A, adventitia). D: Demonstration of increased O₂^.− production in BMP-2-treated cultured carotid arteries using lucigenin chemiluminescence (mean ± SEM, n = 5 for each group; *P < 0.05 versus untreated control). E: BMP-2 induced generation of reactive oxygen species in CAECs (assessed by a homovanillic acid/horseradish peroxidase method in the presence of SOD, to convert O₂^.− to H₂O₂) in the absence and presence of the NAD(P)H oxidase inhibitor DPI and apocynin (APO) or the PKC inhibitor chelerythrine. Results are normalized to the control mean values (n = 5 for each group; *P < 0.05 versus untreated control, ^#P < 0.05 versus BMP-2 treatment, data are mean ± SEM).

Figure 5

Reporter gene assay showing the effects of NAD(P)H oxidase inhibitors (3 × 10⁻⁴ mol/L APO, 10⁻⁵ DPI), SOD (200 U/ml), or catalase (200 U/ml) on BMP-2-induced NF-κΒ reporter activity in CAECs. Concentration-dependent TNF-α-induced NF-κΒ reporter activity is shown for comparison. Endothelial cells were transiently co-transfected with NF-κΒ-driven firefly luciferase and CMV-driven Renilla luciferase constructs followed by BMP-2 or TNF-α stimulation. Cells were then lysed and subjected to luciferase activity assay. After normalization relative luciferase activity was obtained from four to seven independent transfections. Data are mean ± SEM. *P < 0.05 versus control.

Figure 6

A: Results of monocyte adhesion assay (see Materials and Methods). Treatment of primary human CAECs with increasing concentrations of BMP-2 and BMP-4 (2 hours) significantly increased the adhesion of fluorescently labeled PMA-stimulated monocytes. TNF-α (10 ng/ml) was used as positive control. The effects of 10 ng/ml BMP-2 and BMP-4 were also assessed after pretreatment with inhibitors on p42/44 MAP kinase (10 μmol/L PD 98059, 10 μmol/L U0126), p38 MAP kinase [10 μmol/L SB203580, PKC (10 μmol/L chelerythrine (CHEL), 10 μmol/L staurosporine (STAURO)], NAD(P)H oxidase (10 μmol/L DPI, 3 × 10⁻⁴ mol/L APO), and the free radical scavenger SOD plus catalase (200 U/ml). Data are mean ± SEM (n = 8 for each group). *P < 0.05 versus untreated control. ^#P < 0.05 versus BMP treatment. B: Representative fluorescent images showing the effect of BMP-2 and TNF-α treatments (2 hours) on the adhesion of activated monocytes (green fluorescence) to the endothelium of rat aortic segments (en face preparation). C: Summary bar graphs are shown (data are normalized to control mean values). D: Effects of PD 98059, SB203580, and chelerythrine on BMP-2 induced monocyte adhesion to the aortic endothelium. Data are mean ± SEM. *P < 0.05 versus control, ^#P < 0.05 versus BMP-2 treatment. Original magnifications, ×40.

Figure 7

A: Representative Western blot (left) and densitometric data (right) showing the effect of increasing concentrations of anti-p42/p44 siRNAs on the expression of p42/44 MAP kinase in primary human coronary arterial endothelial cells (HCAECs). B: Effect of pretreatment with anti-p42/p44 siRNAs on BMP-2- and BMP-4- (10 ng/ml, for 2 hours) induced adhesion of fluorescently labeled PMA-stimulated monocytes to HCAECs. PD98059 (30 minutes, 10 μmol/L) was used to pharmacologically inhibit MAP kinase activity. TNF-α (10 ng/ml) was used as positive control. Data are mean ± SEM. *P < 0.05 versus control, ^#P < 0.05 versus BMP-2/4 treatment. C–F: Representative Western blots (C, E) and densitometric data (D, F) showing the time course of p42/44 MAP kinase phosphorylation in BMP-2 (10 ng/ml)-treated HCAECs (C, D) and rat carotid arterial segments (E, F). G: Representative Western blot (top) and densitometric data (bottom) showing BMP-2 (10 ng/ml, 10 minutes)-induced phosphorylation of p42/44 MAP kinase in HCAECs pretreated with DPI, chelerythrine, and PD98059.