Proline and glucose metabolic reprogramming supports vascular endothelial and medial biomass in pulmonary arterial hypertension
Bradley M. Wertheim, Rui-Sheng Wang, Christelle Guillermier, Christiane V.R. Hütter, William M. Oldham, Jörg Menche, Matthew L. Steinhauser, Bradley A. Maron
Bradley M. Wertheim, Rui-Sheng Wang, Christelle Guillermier, Christiane V.R. Hütter, William M. Oldham, Jörg Menche, Matthew L. Steinhauser, Bradley A. Maron
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Resource and Technical Advance Cardiology Pulmonology

Proline and glucose metabolic reprogramming supports vascular endothelial and medial biomass in pulmonary arterial hypertension

Abstract

In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-Seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all aa and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest that elevated glucose and proline avidity underlie increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.

Authors

Bradley M. Wertheim, Rui-Sheng Wang, Christelle Guillermier, Christiane V.R. Hütter, William M. Oldham, Jörg Menche, Matthew L. Steinhauser, Bradley A. Maron

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Figure 4

Anabolic convergence of glucose and proline as substrate for biomass in remodeling vessels.

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Anabolic convergence of glucose and proline as substrate for biomass in ...
(A) Top: MIMS quantification of proline utilization correlates with glucose utilization in the endothelial cells of inflammatory PAH vessels, but not control vessels. Bottom: MIMS quantification of proline utilization by medial cells correlates with glucose in both PAH and control vessels. Correlations assessed by linear regression model. (B) High-resolution MIMS imaging demonstrates punctate hotspots of hyperutilization of glucose and proline. Line arrows, hyperintense in both glucose and proline; arrowheads, hyperintense in proline but not glucose. Scale bar: 5 μm. (C) 15N-labeling distributions for glucose hotspots (n = top 40), which are largely above the mean for the vessel wall (inclusive of nonhotspots and hotspots) (red line) and partially overlap with the distribution of top 40 15N-proline hotspots (gray). (D) Complementary analysis to C as the 2H-glucose labeling for the top 15N-proline hotspots partially overlaps with the distribution of top 40 2H-glucose hotspots. In C and D, the mean signal for each vessel wall is provided as a reference (red line). Hotspots were 5 × 5 pixels. (E) TEM adjacent to section imaged with MIMS. TEM image inset demonstrates extracellular matrix with collagen fibers arrayed in both cross section (left) and longitudinally (right). ROI were generated to correspond to regions of ECM on the TEM and the data extracted and expressed as a percent above background (table) indicative of glucose and proline labeling. Scale bars: 500 nm.