This study offers five new discoveries that clarify the timing, reversibility, and cellular origins/mechanisms of the glycolytic shift that has been recognized in human and experimental PAH. First, we clarify the temporal relationship between increased lung FDG-PET and onset of PAH, showing that both occur virtually simultaneously (1–2 wk after MCT; ). Second, we provide the first evidence that PET imaging of increased pulmonary FDG uptake is sufficiently sensitive to allow both early detection of PAH and monitoring of disease regression in response to DCA or direct inhibition of platelet-derived growth factor signaling with imatinib. The fall in lung FDG-PET signal is associated with decreased muscularization of the pulmonary arteries ( and ). Third, we used laser capture microscopy and immunohistochemistry to demonstrate that increased pulmonary FDG uptake reflects increased Glut1 expression in both pulmonary artery endothelial and smooth muscle cells in vivo
( and ). A similar observation was made in the SU5416/hypoxia model (). Furthermore, analysis of inflammatory cells collected by bronchoalveolar lavage shows no glycolytic shift in these cells and pulmonary macrophage depletion does not decrease pulmonary FDG uptake, suggesting that pulmonary FDG uptake is independent of inflammation (Figure E2). Fourth, we used the Seahorse extracellular flux analyzer to validate that the increased FDG seen in vivo
was actually the result of increased glycolysis, which persisted in cultured MCT PASMCs (). Moreover, PDH activity was reduced in PASMCs from MCT animals and PDH activation with DCA decreases lactate formation (), confirming that PDH is not fully active in MCT PASMCs. Finally, we show that activation of HIF-1α occurs in MCT PAH (), which is associated with increased vascular cell proliferation, consistent with prior findings in chronic hypoxic pulmonary hypertension (31
) and PAH in fawn hooded rats (9
) and humans (9
). We also observed a significant up-regulation of the HIF-1α−inducible PDK3 isoform in small pulmonary arteries and in cultured PASMCs from MCT animals, suggesting a molecular connection between HIF-1α activation and increased glycolysis.
These discoveries suggest that monitoring lung FDG uptake allows noninvasive assessment of the status of the pulmonary vasculature in PAH and reflects the metabolic, proliferative diathesis of endothelial and smooth muscle cells. Increased lung FDG uptake has been noted in a small cohort of patients with PAH, and at least some of the signal originates from endothelial cells (11
). However, it remained unclear whether the FDG-PET signal is also derived from PASMCs, airway cells, and/or inflammatory cells. The rat monocrotaline model is characterized by the rapid development of pulmonary hypertension within 3–4 weeks. Using Doppler of the pulmonary artery, the first signs of increased pulmonary artery pressures (shortening of the PAAT [28
]) can be identified after 2 weeks. FDG imaging demonstrates a parallel rise in FDG uptake and pulmonary artery pressure (evident from shortening PAAT). The ability to detect these changes early in the development of PAH suggests FDG-PET has the potential as a noninvasive test for early diagnosis and monitoring of PAH (), as previously suggested (11
Because there is a clear correlation between the expression level of Glut1 and FDG signal intensity in tumors (32
), we were interested in studying this transporter in lung sections to determine which cell type is responsible for the increased FDG uptake that we observed. Using immunohistochemistry, we confirmed that Glut1 is up-regulated both in endothelial cells (as has been described previously in patients with PAH [11
]) and smooth muscle cells (). There is a clear correlation between the degree of muscularization and Glut1 staining intensity, suggesting that the more remodeled blood vessels are responsible for the observed increased FDG uptake (). This finding also indicates the important contribution of PASMCs to the FDG-PET signal in this model of PAH. To prove the general applicability of our findings, we also investigated the SU5416/hypoxia model, which is characterized by endothelial proliferation in addition to PASMC proliferation (27
). Laser capture microscopy shows induction of Glut1 in the small pulmonary arteries, and the pulmonary FDG uptake was increased (). In combination, both the MCT and SU5426/hypoxia models share a glycolytic shift in the pulmonary blood vessels that can be detected by PET imaging.
Interestingly, using a Seahorse extracellular flux analyzer, we confirmed that PASMCs isolated from MCT animals maintain their glycolytic/lactate-producing phenotype in vitro
(). Previous publications describe that endothelial cells of patients with pulmonary arterial hypertension also maintain this glycolytic phenotype in vitro
). Expression levels of Glut1 and hexokinase-1 are up-regulated in early-passage PASMCs derived from MCT animals, which allows these cells to take up and retain a larger amount of glucose (), indicating a glycolytic shift. Indeed, PDH activity, which regulates uptake of pyruvate into the Krebs cycle, is decreased in MCT PASMCs, whereas activation of PDH with DCA leads to increased oxygen consumption and decreased lactate production ().
To establish the basis for this increased glycolysis, we measured HIF-1α activation in PASMCs derived from control and MCT animals, because this transcription factor is known to be pathologically activated in PAH and because HIF-1α regulates the expression of Glut1. We observed that a higher percentage of PASMCs from MCT animals stain positive for HIF-1α (), in agreement with findings in PASMCs from human patients with PAH and spontaneously hypertensive fawn hooded rats (9
). Remarkably, this nuclear localization and activation of HIF-1α persist in culture where the Po2
is less than 100 mm Hg. Analysis of lung sections reveals that MCT animals have a larger number of HIF-1α–positive nuclei in vivo
(). Because Glut1 expression is regulated by HIF-1α (33
), the increased level of Glut1 is likely explained by the larger number of PASMCs containing activated HIF-1α. Costaining for PCNA reveals that HIF-1α–positive cells are more likely to be proliferating (), consistent with the emerging hypothesis that glycolysis is permissive of proliferation (see
review in Reference 34
). Two PDK isoforms, PDK1 and PDK3, have been shown to be up-regulated by HIF-1α in lymphoma cells and stromal/cancer cell lines, respectively (15
). In our experiments, we observed significant up-regulation of PDK3 in small pulmonary arteries and in cultured PASMCs from MCT animals, suggesting that HIF-1α mediates the glycolytic shift in PASMCs by activating PDK3 ().
Because this study focused on the diagnostic utility and cellular origins of the lung FDG-PET signals in PAH, we did not explore the mechanism of HIF-1α activation in this study. However, prior work from our group demonstrates that HIF-1α activation can result from changes in redox state acquired through epigenetic silencing of superoxide dismutase-2 (17
). In addition, deficiency of p53 can alter HIF-1α activation and pulmonary arterial remodeling in hypoxic pulmonary hypertension (35
). Interestingly, activation of HIF-1α promotes proliferation in PASMCs from fawn hooded rats, a rat strain that spontaneously develops pulmonary hypertension (17
), and in the lungs of patients with PAH (9
). The importance of glycolytic energy metabolism as a permissive factor for PASMC proliferation was demonstrated in mice lacking malonyl-CoA decarboxylase (13
We demonstrate that the development of pulmonary hypertension is associated with increased reliance of pulmonary vascular cells (endothelium and PASMCs) on glycolytic energy metabolism, which can be imaged in vivo
by PET-FDG imaging. These findings are consistent with assessments of the origins and significance of the FDG-PET signal in cancer as reflecting the activity of hyperproliferative, glycolytic cells (22
). PET imaging can detect early changes in the vasculature and holds promise for better characterizing therapeutic efficacy at the cellular level.