MicroRNA-21 integrates pathogenic signaling to control pulmonary hypertension: results of a network bioinformatics approach

Abstract

Background: Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA molecules are potential candidates for coordinated regulation of these disease stimuli.

Methods and results: Using a network biology approach, we identify microRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying microRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho-kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haploinsufficiency of BMP receptor type 2. To validate these predictions, we have found that hypoxia and BMP receptor type 2 signaling independently upregulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 downregulates BMP receptor type 2 expression. Furthermore, miR-21 directly represses RhoB expression and Rho-kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is upregulated in pulmonary tissue from several rodent models of PH and in humans with PH. On induction of disease in miR-21-null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH.

Conclusions: A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying microRNA in PH.

Figure 1

A network biology approach identifies PH-modifying miRNA. (A) The PH-network displays substantial functional interconnections. Left graph: The mean LCC size derived from 100,000 randomly chosen modules of 115 genes from the consolidated interactome (4.5 ± 2.5, mean ± standard deviation) is significantly smaller than the LCC of the PH-network (82 nodes). The maximum LCC size (max size) from randomly selected gene modules is 31. (** signifies p<<10⁻⁵). Right graph: The mean number of direct interconnections (edges) within 100,000 randomly chosen modules of 115 genes from the consolidated interactome (9.4 ± 5.6, mean ± standard deviation) is significantly smaller than the number of edges in the PH-network (255 edges). The maximum number of edges (max edges) within randomly selected gene modules is 53. (** signifies p<<10⁻⁵). (B) MiRNA that associate with the PH-network (29 miRNA groups) target a subset of pathways related to hypoxia, inflammation, and/or TGF-β. (C) A subset of miRNA previously associated with hypoxia, inflammation and TGF-β is predicted to target the PH-network. MiRNA groups predicted by enrichment analysis (Table 1) are bolded. (D) Predicted target network of 7 miRNA groups identified both by enrichment analysis and literature review reveals genes that may represent points of coordinated miRNA regulation in PH. Circles: predicted gene targets. Triangles: miRNA. Blue lines: predicted associations of miRNA and targets. Dotted gray lines: gene interactions documented in the consolidated interactome. Circle size is proportional to the number of miRNA groups (among these 7) predicted to target that particular gene.

Figure 2

MiR-21 is up-regulated by hypoxia- and BMPRII-dependent signaling and reciprocally down-regulates BMPRII expression. (A) As measured by RT-PCR, hypoxic HPAECs display increased levels of mature miR-21. (B) Knockdown of AKT2 (si AKT2) negligibly affects miR-21 expression during normoxia but partially inhibits the specific up-regulation of miR-21 during hypoxia. (* signifies p≤0.05 compared with si Control samples at 21% O₂; Ω signifies p≤0.05 compared with si Control samples at 0.2% O₂). (C) Exposure to BMP9 increases mature miR-21 expression in HPAECs; BMP9-dependent up-regulation of miR-21 is abrogated by knockdown of BMPR2 or SMAD5 but not SMAD4. (* signifies p≤0.05, NS signifies p>0.05 as compared with vehicle control; Ω signifies p>0.05 as compared with BMP9 + si Control; # signifies p≤0.05 as compared with vehicle + si Control). (D) Lungs of BMPR2 (+/−) mice display reduced expression of miR-21 compared to wildtype littermates (WT). (E) (Left blots) As measured by immunoblot densitometry, BMPRII expression is decreased in HPAECs previously transfected with miR-21 oligonucleotide mimics. (Right blots) BMPRII expression is increased in hypoxic HPAECs after inhibition of miR-21 (AS-21). In (A–D), control miR-21 expression is assigned a fold change of 1, to which other conditions are compared. In (E), immunoblots are representative of experiments performed at least in triplicate; gel densitometry is normalized to actin levels and compared as arbitrary units. In all panels, error bars reflect SEM; (*) denotes p≤ 0.05 (N≥3), NS signifies p>0.05 (N≥3). For all experiments, N≥3.

Figure 3

MiR-21 inhibits RhoB expression to suppress Rho kinase activity in HPAECs. (A–B) As measured by immunoblot densitometry, RhoB expression is increased in hypoxic HPAECs after inhibition of miR-21 (A) and is decreased in hypoxic HPAECs transfected with miR-21 mimic (B). (C) Rho kinase-dependent phosphorylation of threonine-853 (MP853) in myosin phosphatase (MP) is increased in hypoxic HPAECs after inhibition of miR-21. (D) NOS3 expression is decreased in hypoxic HPAECs after inhibition of miR-21. (E) Intensity of staining with phalloidin-FITC (green) to detect F-actin formation is increased in hypoxic HPAECs after inhibition of miR-21. Furthermore, as demonstrated by more fibers traversing nuclei, increased complexity of F-actin network formation is evident in AS-21-transfected cells (asterisk) as compared with AS-Cont-transfected cells (double asterisk). In (A–D), error bars reflect SEM; (*) denotes p≤ 0.05 (N≥3), NS signifies p>0.05 (N≥3). Immunoblots and micrographic images are representative of experiments performed at least in triplicate; gel densitometry is normalized to actin levels and compared as arbitrary units. For all experiments, N≥3.

Figure 4

MiR-21 expression is increased in in vivo models of PH. (A) MiR-21 expression is increased in mouse lung over-expressing IL-6 (N=5 mice per group). (B) RT-PCR reveals that miR-21 is up-regulated in the lungs of VHL-null mice as compared with wildtype littermates (N=6 mice per group). (C) MiR-21 is up-regulated in the lungs of mice exposed to a combination of SU5416 and 10% O₂ for 1–3 weeks compared to time 0 (N=3 mice per group). (D) MiR-21 steadily increases over time in lungs of rats after 1–4 weeks post-monocrotaline exposure (N=3 rats per treatment group) compared to time 0. Control miR-21 expression is assigned a fold change of 1, to which other conditions are compared. In all panels, error bars reflect SEM; (*) denotes p≤ 0.05 (N≥3), NS signifies p>0.05 (N≥3).

Figure 5

MiR-21 expression is increased in remodeled pulmonary vessels in animal models of PH and human PAH patients. (A–B) In situ staining and quantitation reveal increased expression of miR-21 in <100 µm pulmonary vessels of VHL-null mice and IL-6 transgenic mice as compared with wildtype control mice (N=3 mice per group) (A) and in <100 µm pulmonary vessels of monocrotaline-exposed rats as compared with vehicle (N= 3 rats per group) (B). (C) Increased expression of miR-21 in <200 µm pulmonary vessels from lungs of 3 human patients with PAH (Patients A, B, C) as compared with 3 non-diseased human lung specimens (Patients 1, 2, 3). In all panels, error bars reflect SEM; (*) denotes p≤ 0.05 (N≥3), NS signifies p>0.05 (N≥3).

Figure 6

miR-21-null (−/−) mice display exaggerated manifestations of PH when exposed to SU5416 and chronic hypoxia. (A–C) After exposure to SU5416 and 10% O₂, miR-21-null mice display increased RVSP (A), increased relative right ventricular mass (RV/LV+S) (B), and increased pulmonary vascular remodeling (C). In (C), remodeling is evident in miR-21-null mice (right panels, arrows), with increased medial thickening and cellularity, as compared with pulmonary vessels (left panels, arrowheads) in wildtype littermates (α-smooth muscle actin stain). A significant increase in medial wall thickness relative to vessel diameter in <100 µm pulmonary vessels (right graph) is evident in miR-21-null mice. (N=8 mice per treatment group in A–C). (D) Immunohistochemistry (arrows) and quantitation (graph) reveal increased expression of RhoB in <100 µm pulmonary vessels of treated miR-21-null (−/−) mice (N=5 mice per group). (E) Rho kinase-dependent expression of phosphorylated myosin phosphatase [p-MP (T853), arrows, left panels] is increased in <100 µm pulmonary vessels of treated miR-21-null mice. Stain for total myosin phosphatase in consecutive sections is shown (MP, arrows, right panels) (N=5 mice per group). (F) Lung tissue from treated miR-21-null mice display increased transcript levels of endothelin-1. Wildtype expression is assigned a fold change of 1, to which expression in miR-21-null mice is compared. (N=4 mice per group). In appropriate panels, error bars reflect SEM; (*) denotes p≤ 0.05 (N≥3), NS signifies p>0.05 (N≥3).

Figure 7

MiR-21 integrates multiple pathogenic signals to regulate pulmonary hypertension. A molecular model is presented whereby hypoxia, inflammation, and BMP-dependent signaling up-regulate miR-21 in the pulmonary vasculature. In response, miR-21 represses Rho kinase activation and, perhaps, other pathways to modulate the development of PH in vivo.