Endothelial cell PHD2-HIF1α-PFKFB3 contributes to right ventricle vascular adaptation in pulmonary hypertension

Abstract

Humans and animals with pulmonary hypertension (PH) show right ventricular (RV) capillary growth, which positively correlates with overall RV hypertrophy. However, molecular drivers of RV vascular augmentation in PH are unknown. Prolyl hydroxylase (PHD2) is a regulator of hypoxia-inducible factors (HIFs), which transcriptionally activates several proangiogenic genes, including the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We hypothesized that a signaling axis of PHD2-HIF1α-PFKFB3 contributes to adaptive coupling between the RV vasculature and tissue volume to maintain appropriate vascular density in PH. We used design-based stereology to analyze endothelial cell (EC) proliferation and the absolute length of the vascular network in the RV free wall, relative to the tissue volume in mice challenged with hypoxic PH. We observed increased RV EC proliferation starting after 6 h of hypoxia challenge. Using parabiotic mice, we found no evidence for a contribution of circulating EC precursors to the RV vascular network. Mice with transgenic deletion or pharmacological inhibition of PHD2, HIF1α, or PFKFB3 all had evidence of impaired RV vascular adaptation following hypoxia PH challenge. PHD2-HIF1α-PFKFB3 contributes to structural coupling between the RV vascular length and tissue volume in hypoxic mice, consistent with homeostatic mechanisms that maintain appropriate vascular density. Activating this pathway could help augment the RV vasculature and preserve RV substrate delivery in PH, as an approach to promote RV function.

Keywords: pulmonary hypertension; right ventricle; stereology; vascular adaptation.

Figure 1.

Characterization of the time course of RV vascular proliferation. A: quantification of the volume fraction of endothelial cell proliferation and a representative image of PCNA and lectin staining (arrows mark representative proliferating endothelial cells; scale bar: 50 µm; N = 13, 8, 8, 8, and 8 per group, respectively; ANOVA P < 0.0001; post hoc Tukey’s test vs. control group: *P < 0.05, ***P < 0.05). B: correlation between total RV free wall volume and total RV vascular length in normoxic, hypoxic, and combined groups of mice. C: total volume of the RV free wall in control and hypoxic mice (N = 13, 11, 8, 8, and 8 per group, respectively; ANOVA P = 0.03; post-hoc Tukey’s test: *P < 0.05). D: radius of tissue served per vessel in control and hypoxic mice (N = 18, 11, 11, 14, and 8 per group, respectively; ANOVA P = NS). In this experiment, all female mice were used. NS, not significant; RV, right ventricular.

Figure 2.

Parabiosis does not find evidence of circulating endothelial cell contribution to RV vascular homeostasis. A: gating of flow cytometry on RV single-cell digest to identify endothelial cells. B: representative histograms of GFP signal from RV endothelial cells obtained from a Ub^GFP-wildtype parabiotic mouse pair. The red curve is the histogram of GFP signal in RV ECs from the wildtype mouse; the green curve is the histogram of GFP signal in RV ECs from the Ub^GFP mouse. In this experiment, all female mice were used. EC, endothelial cell; GFP, green fluorescent protein; RV, right ventricular; WT, wildtype.

Figure 3.

HIF1α contributes to RV vascular homeostasis. A: correlations between total RV vascular length and RV free wall volume in HIF1α^fl (control) mice and HIF1α^UbcERT2 mice, both exposed to 5 wk hypoxia. B: radius of tissue served per vessel in HIF1α^fl and HIF1α^UbcERT2 mice (N = 10, 9 per group; t test P = NS). C: volume fraction of EC proliferation in HIF1α^fl and HIF1α^UbcERT2 mice (N = 5 per group; t test P = NS). D: echocardiography endpoints in HIF1α^fl and HIF1α^UbcERT2 mice, treated for 30 days hypoxia (N = 8, 6 per group; t test P = NS for all comparisons). In the experiment, in A–C, in the HIF1α^fl group, there were 3 female and 7 male mice, and in the HIF1α^UbcERT2 group, there were 5 female and 4 male mice (18). In D, all mice were male. EC, endothelial cell; HIF1α, hypoxia-inducible factor 1α; HIF1α^UbcERT2, HIF1α^fl/fl;Ubc^CreERT2; NS, not significant; RV, right ventricular.

Figure 4.

PHD2 contributes to RV vascular homeostasis. A: radius of tissue served per vessel in PHD2^Tie2 mice in normoxia and hypoxia (N = 8, 10 per group; t test P = NS). B: volume fraction of EC proliferation in PHD2^Tie2 mice in normoxia and hypoxia (N = 8, 10 per group; t test P = NS). C: correlation between total RV vascular length and RV free wall volume in the normoxic, hypoxic, and combined groups of PHD2^Tie2 mice. In this experiment, in the normoxic group, there were 4 female and 4 male mice, and in the hypoxic group, there were 5 female and 5 male mice. EC, endothelial cell; NS, not significant; PHD2, prolyl hydroxylase; PHD2^Tie2, Egln1^fl/fl;Tie2-Cre; RV, right ventricular.

Figure 5.

PFKFB3 contributes to RV vascular homeostasis. A: correlations between total RV vascular length and RV free wall volume in PFKFB3^VECadERT2 mice treated with vehicle (control) or tamoxifen to induce gene deletion in ECs. B: radius of tissue served per vessel in PFKFB3^VECadERT2 mice treated with vehicle or tamoxifen (N = 13, 15 per group; t test P = NS). C: volume fraction of EC proliferation in PFKFB3^VECadERT2 mice treated with vehicle or tamoxifen (N = 13, 15 per group; t test P = 0.0744). In this experiment, in the vehicle group, there were 7 female and 6 male mice, and in the tamoxifen group, there were 8 female and 7 male mice. EC, endothelial cell; NS, not significant; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; RV, right ventricular; Tam, tamoxifen; Veh, vehicle.

Figure 6.

The PFKFB3 inhibitor 3PO inhibits RV vascular homeostasis. A: correlations between total RV vascular length and RV free wall volume in wildtype mice treated with vehicle (control) or 3PO. B: radius of tissue served per vessel in wildtype mice treated with vehicle or 3PO (N = 6 per group; t test P = NS). C: volume fraction of EC proliferation in wildtype mice treated with vehicle or 3PO (N = 6 per group; t test P = NS). In this experiment, all female mice were used. EC, endothelial cell; NS, not significant; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; RV, right ventricular; Veh, vehicle.

Figure 7.

Graphical summary. A: summary of experimental results. B: PHD2 promotes HIF1α degradation. HIF1α is a transcriptional factor that increases PFKFB3 expression. PFKFB3 promotes EC proliferation and the maintenance of RV vascular homeostasis. C: there is a baseline setpoint of RV vascular density, which is maintained under homeostatic conditions (green zone). Mechanisms that maintain the correct RV vascular density include PHD2-HIF1α-PFKFB3 signaling. There is likely a range in which vascular density can vary without harmful physiological consequences (yellow zones). Outside of this safe range, there will be negative physiological consequences (red zones), resulting from either inadequate delivery of metabolites (radius too large) or an excessive number of vessels (radius too small). EC, endothelial cell; HIF1α, hypoxia-inducible factor 1α; HIF1α^UbcERT2, HIF1α^fl/fl;Ubc^CreERT2; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; PFKFB3^VECadERT2, PFKFB3^fl/fl;Cdh5^CreERT2; PHD2, prolyl hydroxylase; PHD2^Tie2, Egln1^fl/fl;Tie2-Cre; RV, right ventricular.