Placenta growth factor (PlGF), a novel inducer of plasminogen activator inhibitor-1 (PAI-1) in sickle cell disease (SCD)

Abstract

Sickle cell disease (SCD) is characterized by a prothrombotic state. Plasminogen activator inhibitor-1 (PAI-1) is known to modulate fibrinolysis, lung injury/fibrosis, and angiogenesis. However, its role in SCD is less understood, and the molecular mechanisms underlying increased PAI-1 are unknown. Herein, we show a novel link between PAI-1 and sickle erythropoiesis. Plasma PAI-1 levels were high in SCD patients at steady state and in two humanized sickle mouse models, with increased PAI-1 immunolabeling in sickle mouse lung, bronchial epithelial cells, alveolar macrophages, and pulmonary microvascular endothelial cells. Placenta growth factor (PlGF), released at high levels by sickle erythroblasts, induced PAI-1 expression in primary human pulmonary microvascular endothelial cells and monocytes through activation of c-Jun N-terminal kinase (JNK), NADPH oxidase, and hypoxia-inducible factor-1alpha (HIF-1alpha). Analysis of the human PAI-1 promoter revealed this induction was mediated by hypoxia-response element (HRE)-1, HRE-2, and distal activator protein (AP-1) sites. We also identify the involvement of c-Jun, c-Jun/c-Fos, and JunD, but not JunB, in binding with AP-1 sites of the PAI-1 promoter upon PlGF induction. Consistent with these findings, levels of PAI-1 were low in PlGF knock-out mice and sickle-PlGF knock-out mice; overexpression of PlGF in normal mice increased circulating PAI-1. In conclusion, we identify a novel mechanism of PAI-1 elevation in SCD.

FIGURE 1.

PAI-1 expression in mice and human subjects. A and B, plasma levels of PAI-1 in mice as measured by ELISA. Berk-hemi, hemizygous Berkeley mice; Berk-SS, Berkeley SS mice. C, plasma PlGF levels in mice by ELISA. The respective strain is indicated at the bottom of the figure. D, immunohistochemistry for PAI-1 expression in C57BL/6 (normal) and Berkeley SS mouse lungs (shown in brown; nuclei are stained red). The top panel shows: (i) antibody control and PAI-1 expression in bronchial epithelial cells of normal and (ii) Berkeley SS mice; the bottom panel shows corresponding staining for PAI-1 in their lung parenchyma. Arrows indicate alveolar macrophages, and arrowheads indicate bronchial epithelial layer. E, the levels of PAI-1 were quantified in BAL of indicated strains of mice by ELISA. F, qRT-PCR analysis of PAI-1 mRNA in MNC from SCD subjects (n = 9) and normal controls (n = 9). RQ, relative quantification. G and H, HPMVEC were treated with human recombinant PlGF (250 ng/ml) for the indicated time periods. G, total RNA was subjected to RPA for the expression of indicated genes. H, the culture supernatants were assayed for PAI-1 by ELISA. RPA data are representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Where indicated, the vertical lines show repositioned gel lanes. *, p < 0.05, **, p < 0.01, ***, p < 0.001. ns, not significant.

FIGURE 2.

Cellular signaling pathway of PlGF-induced PAI-1 expression. A and B, HPMVEC were pretreated for 30 min with pharmacological inhibitors of PI3K, LY294002 (10 μm); Erk, PD98059 (10 μm); P38α, SB203580 (1 μm); JNK, SP600125 (0.1 μm); NADPH oxidase, DPI (10 μm); HIF-1α, ascorbate (25 μm), and NF-κB, sulfasalazine (2 mm) followed by treatment with PlGF for 6 h. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C and D, HPMVEC were transfected with indicated siRNA or scrambled (sc) siRNA constructs followed by PlGF treatment for 6 h. A and C, RPA analysis of the indicated genes. Where indicated, the vertical lines show repositioned gel lanes. Data are representative of three independent experiments. B and D, PAI-1 release by ELISA. The data are representative of three independent experiments. ***, p < 0.001. ns, not significant. E and F, HPMVEC were treated with PlGF either in the presence or in the absence of pharmacological inhibitors for 30 min. E, the cytosolic extracts were prepared and subjected to Western blotting for phospho-JNK, and the same blot was reprobed for total JNK. Ab, antibody. F, the nuclear extracts were analyzed for phospho-c-Jun, and the same blot was reprobed for total c-Jun and c-Fos. Data are representative of three independent experiments.

FIGURE 3.

Identification of functional cis-acting elements in PlGF-induced PAI-1 promoter activation. A, schematics of PAI-1 promoter (−806/+19 bp, relative to transcription start site), indicating the location of HRE-1 to -5, proximal and distal AP-1, C/EBP, and Egr-1 binding sites. Luc, luciferase. B, HPMVEC were co-transfected with either WT or individual HRE mutant (mut) constructs of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. C, illustrations of an overlapping region (−158/−147 bp) containing the HRE-1 and AP-1 binding sites in the PAI-1 promoter. The mutated oligonucleotides corresponding to HRE-1, AP-1, and the overlapping region of HRE-1 and AP-1 are underlined. D, HPMVEC were co-transfected with either WT or HRE-1 mutant or AP-1 mutant or HRE-1/AP-1 mutant of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. The luciferase and β-galactosidase activities were estimated. E, nuclear extracts from untreated and PlGF-treated HPMVEC (10 μg) were incubated either with a biotinylated double-stranded oligonucleotide probe corresponding to an overlapping region containing HRE-1 and AP-1 sites or with a probe containing mutations in an overlapping four shared nucleotides or mutation only in the AP-1 site. Data are representative of three independent experiments. ns, not significant.

FIGURE 4.

Role of individual components of AP-1 in PlGF-induced PAI-1 promoter activity. A, HPMVEC were co-transfected with expression plasmid for either c-Fos or c-Jun or both c-Fos + c-Jun or JunB or JunD along with the WT PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. Luciferase and β-galactosidase activities were estimated. PlGF promotes HIF-1α and AP-1 binding to PAI-1 promoter in vivo. ns, not significant. B and C, HPMVEC were pretreated with pharmacological inhibitors of either JNK (SP600125) or HIF-1α (ascorbate) prior to PlGF stimulation for 4 h. The soluble chromatin was isolated and immunoprecipitated with indicated antibody or control rabbit IgG. DNA was amplified with the primers (−366 to −82 bp) corresponding to HRE-1 and -2 and a distal AP-1 site (B) and the primers (−114 to +98 bp) corresponding to a proximal AP-1 binding site (C). The primer sequences are listed in Table 1. The input DNA panel represents the amplification of samples before immunoprecipitation. Data are representative of three independent experiments.

FIGURE 5.

Role of PlGF in PAI-1 expression in sickle mice. Berkeley SS mice (Berk-SS) were bred with PlGF^−/− mice to obtain Berkeley SS/PlGF^−/− mice. The plasma levels of PAI-1 are measured by ELISA in the indicated strains of mice (n = 6).