A Metabolic Basis for Endothelial-to-Mesenchymal Transition

•Induction of EndoMT triggers a reduction in FAO•FAO is required to maintain endothelial acetyl-CoA levels•FAO modulates in vitro and in vivo EndoMT Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions. Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions. EndoMT is a specific form of epithelial-to-mesenchymal transition (EMT) characterized by the loss of endothelial features and the acquisition of mesenchymal, fibroblast, or stem-cell-like characteristics (Kovacic et al., 2012Kovacic J.C. Mercader N. Torres M. Boehm M. Fuster V. Epithelial-to-mesenchymal and endothelial-to-mesenchymal transition: From cardiovascular development to disease.Circulation. 2012; 125: 1795-1808Crossref PubMed Scopus (291) Google Scholar, Sanchez-Duffhues et al., 2016Sanchez-Duffhues G. Orlova V. Ten Dijke P. In brief: Endothelial-to-mesenchymal transition.J. Pathol. 2016; 238: 378-380Crossref PubMed Scopus (42) Google Scholar). This process was initially described in the context of endocardial differentiation and it is now apparent that EndoMT contributes to the formation of the atrioventricular cushion, septa, and valves during normal cardiac development (Eisenberg and Markwald, 1995Eisenberg L.M. Markwald R.R. Molecular regulation of atrioventricular valvuloseptal morphogenesis.Circ. Res. 1995; 77: 1-6Crossref PubMed Scopus (533) Google Scholar). While linked to physiological cardiac development, abnormal EndoMT has also been implicated in a growing number of pathological conditions. These include pulmonary hypertension (Stenmark et al., 2016Stenmark K.R. Frid M. Perros F. Endothelial-to-mesenchymal transition: An evolving paradigm and a promising therapeutic target in PAH.Circulation. 2016; 133: 1734-1737Crossref PubMed Scopus (64) Google Scholar), vein graft failure (Cooley et al., 2014Cooley B.C. Nevado J. Mellad J. Yang D. Hilaire C.S. Negro A. Fang F. Chen G. San H. Walts A.D. et al.TGF-β signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.Sci. Transl. Med. 2014; 6: 227ra34Crossref PubMed Scopus (269) Google Scholar), metastatic spread of tumors (Magrini et al., 2014Magrini E. Villa A. Angiolini F. Doni A. Mazzarol G. Rudini N. Maddaluno L. Komuta M. Topal B. Prenen H. et al.Endothelial deficiency of L1 reduces tumor angiogenesis and promotes vessel normalization.J. Clin. Invest. 2014; 124: 4335-4350Crossref PubMed Scopus (46) Google Scholar, Potenta et al., 2008Potenta S. Zeisberg E. Kalluri R. The role of endothelial-to-mesenchymal transition in cancer progression.Br. J. Cancer. 2008; 99: 1375-1379Crossref PubMed Scopus (405) Google Scholar), atherosclerosis (Chen et al., 2015Chen P.Y. Qin L. Baeyens N. Li G. Afolabi T. Budatha M. Tellides G. Schwartz M.A. Simons M. Endothelial-to-mesenchymal transition drives atherosclerosis progression.J. Clin. Invest. 2015; 125: 4514-4528Crossref PubMed Scopus (295) Google Scholar, Evrard et al., 2016Evrard S.M. Lecce L. Michelis K.C. Nomura-Kitabayashi A. Pandey G. Purushothaman K.R. d’Escamard V. Li J.R. Hadri L. Fujitani K. et al.Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability.Nat. Commun. 2016; 7: 11853Crossref PubMed Scopus (283) Google Scholar), and fibrosis in key organs such as the heart and kidney (Piera-Velazquez et al., 2011Piera-Velazquez S. Li Z. Jimenez S.A. Role of endothelial-mesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders.Am. J. Pathol. 2011; 179: 1074-1080Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, Zeisberg et al., 2007Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. et al.Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1621) Google Scholar, Zeisberg et al., 2008Zeisberg E.M. Potenta S.E. Sugimoto H. Zeisberg M. Kalluri R. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.J. Am. Soc. Nephrol. 2008; 19: 2282-2287Crossref PubMed Scopus (680) Google Scholar). Although transforming growth factor β (TGF-β) signaling is a potent inducer of EndoMT (Sanchez-Duffhues et al., 2016Sanchez-Duffhues G. Orlova V. Ten Dijke P. In brief: Endothelial-to-mesenchymal transition.J. Pathol. 2016; 238: 378-380Crossref PubMed Scopus (42) Google Scholar), the intracellular metabolic mediators regulating this process within endothelial cells are incompletely characterized. Although proliferating endothelial cells rely primarily on glycolysis (De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar), a recent study demonstrated that this cell type also requires fatty acid oxidation (FAO) (Schoors et al., 2015Schoors S. Bruning U. Missiaen R. Queiroz K.C. Borgers G. Elia I. Zecchin A. Cantelmo A.R. Christen S. Goveia J. et al.Fatty acid carbon is essential for dNTP synthesis in endothelial cells.Nature. 2015; 520: 192-197Crossref PubMed Scopus (367) Google Scholar). Indeed, the mitochondrial-dependent, β-oxidation of long-chain fatty acids (LCFAs) is central to energy homeostasis in a wide array of cells (Houten et al., 2016Houten S.M. Violante S. Ventura F.V. Wanders R.J. The biochemistry and physiology of mitochondrial fatty acid β-oxidation and its genetic disorders.Annu. Rev. Physiol. 2016; 78: 23-44Crossref PubMed Scopus (304) Google Scholar). For successful transport from the cytosol into the mitochondrial matrix, LCFAs must undergo two successive enzymatic reactions, a sequence of events known as the carnitine shuttle. These reactions involve CPT1, located on the outer mitochondrial membrane, followed by CPT2, located on the inner mitochondrial membrane (Bonnefont et al., 2004Bonnefont J.P. Djouadi F. Prip-Buus C. Gobin S. Munnich A. Bastin J. Carnitine palmitoyltransferases 1 and 2: Biochemical, molecular and medical aspects.Mol. Aspects Med. 2004; 25: 495-520Crossref PubMed Scopus (432) Google Scholar). As such, the sequential activity of both CPT1 and CPT2 are required to import and hence, metabolize LCFA. Here, we demonstrate a novel role for endothelial FAO in restraining EndoMT. In particular, we show that TGF-β signaling-induced EndoMT is accompanied by an inhibition of FAO. Furthermore, FAO inhibition potentiates EndoMT through regulation of intracellular acetyl-CoA levels and SMAD7 signaling. We further show that genetic disruption of Cpt2 modulates in vivo EndoMT. Together, these results establish endothelial FAO as an important regulator of the EndoMT process. Based on a previous strategy (Rieder et al., 2011Rieder F. Kessler S.P. West G.A. Bhilocha S. de la Motte C. Sadler T.M. Gopalan B. Stylianou E. Fiocchi C. Inflammation-induced endothelial-to-mesenchymal transition: A novel mechanism of intestinal fibrosis.Am. J. Pathol. 2011; 179: 2660-2673Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), we found that primary cultures of human pulmonary microvascular endothelial cells (HPMVECs) could be stimulated to undergo EndoMT by treating these cells with a cytokine combination of TGF-β1 and interleukin-1β (IL-1β). These cytokine-treated endothelial cells underwent a clear morphological transition adopting a more fibroblast or mesenchymal appearance (Figure 1A). Coincident with this morphological switch, cytokine treatment of endothelial cells induced a host of mesenchymal markers, as well as a simultaneous decrease in endothelial markers (Figures 1B, S1A, and S1B). To investigate potential novel mediators of EndoMT, we performed a metabolomics profile using this in vitro system. This analysis revealed that EndoMT was accompanied by a rise in certain short chain acylcarnitines and a fall in glycolytic and tricarboxylic acid (TCA) cycle-linked organic acid metabolites (Figure 1C). This suggested that EndoMT might potentially involve a shift in the relative role of fatty acid and carbohydrate metabolism. Interestingly, upon induction of EndoMT, cytokine treatment induced an early decline in the level of CPT1A, the enzyme that plays a rate-limiting and obligate role in FAO (Figures 1D, S1C, and S1D). This decline in CPT1A expression is transient but specific (e.g., does not include CPT2) and precedes the induction of various mesenchymal markers (Figures S1E–S1H). This reduction of CPT1A expression is consistent with an emerging role for TGF-β1 in regulating expression of genes involved in FAO (Kang et al., 2015Kang H.M. Ahn S.H. Choi P. Ko Y.A. Han S.H. Chinga F. Park A.S. Tao J. Sharma K. Pullman J. et al.Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development.Nat. Med. 2015; 21: 37-46Crossref PubMed Scopus (716) Google Scholar). In that regard, our in vitro model of EndoMT was associated with a decrease in proliferator-activated receptor (PPAR)-dependent signaling pathways which in turn, appears to regulate CPT1A expression (Figures S1I–S1N). The fall in CPT1A expression appears to have functional consequences. For instance, untreated endothelial cells could respond to an exogenous palmitate challenge with an increase in their oxygen consumption rate (OCR). In contrast, following induction of EndoMT, or after treatment with the CPT1 inhibitor etomoxir, this metabolic response to fatty acids was absent (Figure 1E). Moreover, a direct assessment of palmitate oxidation revealed a marked decline in endothelial FAO following TGF-β stimulation (Figure 1F). To better understand the importance of this EndoMT-induced decline in FAO, we expressed CPT1A using a heterologous promoter in endothelial cells. Surprisingly, constitutive CPT1A expression inhibited the induction of EndoMT mesenchymal markers following TGF-β1/IL-1β treatment, as well as inhibiting the cytokine-induced decline in oxygen consumption (Figures 2A, 2B, and S2A). We next used small hairpin RNAs (shRNAs) to stably knock down CPT1A (CPT1AKD; Figures S2B and S2C). In the absence of exogenous cytokines, endothelial cells with reduced CPT1A expression exhibited a more fibroblast-like morphology (Figure 2C). Moreover, these cells activated the EndoMT program (Figures 2D–2G, S2D, and S2E). Similar results were obtained using another shRNA directed against CPT1A (Figure S2F) or using shRNAs directed against CPT2, a protein that is also required for FAO (Figures S2G and S2H). In addition, while endothelial cells from different vascular beds are known to exhibit markedly different functional characteristics (Nolan et al., 2013Nolan D.J. Ginsberg M. Israely E. Palikuqi B. Poulos M.G. James D. Ding B.S. Schachterle W. Liu Y. Rosenwaks Z. et al.Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.Dev. Cell. 2013; 26: 204-219Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar), we found that aortic and umbilical vein derived endothelial cells behaved similarly to HPMVECs after FAO inhibition (Figure S2I). While CPT1AKD endothelial cells exhibited the signature of EndoMT in the absence of any exogenous cytokine stimulation, it is important to note that most cells in culture secrete TGF-β and can respond in an autocrine fashion. In this context, we found that reducing CPT1A expression increased phosphorylation of the TGF-β downstream effector SMAD2, suggesting that inhibiting FAO acted to augment endogenous TGF-β signaling (Figure 3A). Moreover, addition of SB431542, a small molecule inhibitor of TGF-β signaling (Laping et al., 2002Laping N.J. Grygielko E. Mathur A. Butter S. Bomberger J. Tweed C. Martin W. Fornwald J. Lehr R. Harling J. et al.Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542.Mol. Pharmacol. 2002; 62: 58-64Crossref PubMed Scopus (532) Google Scholar), abrogated FAO-dependent SMAD2 activation, as well as the ability of CPT1A inhibition to activate the EndoMT program (Figures 3A, S3A, and S3B). We next asked how a decrease in endothelial FAO could potentiate TGF-β signaling and thereby modulate the threshold for EndoMT. We thought it possible that stress signaling initiated by altered energetics might play a role; however, we saw no evidence that AMP-activated protein kinase (AMPK) was activated or that there were appreciable alterations in malonyl-CoA levels (Figures S3C–S3E). To further pursue this question, we took note of the increasing evidence suggesting that altering acetyl-CoA levels has broad effects on growth, gene expression, protein function, and cell fate (Cai et al., 2011Cai L. Sutter B.M. Li B. Tu B.P. Acetyl-CoA induces cell growth and proliferation by promoting the acetylation of histones at growth genes.Mol. Cell. 2011; 42: 426-437Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, Lee et al., 2014Lee J.V. Carrer A. Shah S. Snyder N.W. Wei S. Venneti S. Worth A.J. Yuan Z.F. Lim H.W. Liu S. et al.Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation.Cell Metab. 2014; 20: 306-319Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, Wellen et al., 2009Wellen K.E. Hatzivassiliou G. Sachdeva U.M. Bui T.V. Cross J.R. Thompson C.B. ATP-citrate lyase links cellular metabolism to histone acetylation.Science. 2009; 324: 1076-1080Crossref PubMed Scopus (1461) Google Scholar). Moreover, FAO has, in some cases, been shown to be necessary to maintain cellular acetyl-CoA levels (Pougovkina et al., 2014Pougovkina O. te Brinke H. Ofman R. van Cruchten A.G. Kulik W. Wanders R.J. Houten S.M. de Boer V.C. Mitochondrial protein acetylation is driven by acetyl-CoA from fatty acid oxidation.Hum. Mol. Genet. 2014; 23: 3513-3522Crossref PubMed Scopus (100) Google Scholar). Consistent with this, cytokine induction of EndoMT resulted in a fall in acetyl-CoA levels (Figure 3B). To further assess whether this fall in acetyl-CoA levels acts as a trigger for EndoMT, we took advantage of recent observations that supplementing the culture media with acetate can directly increase acetyl-CoA levels through the action of acetyl-CoA synthetase (ACSS2) (Figures 3C and S3F) (Balmer et al., 2016Balmer M.L. Ma E.H. Bantug G.R. Grählert J. Pfister S. Glatter T. Jauch A. Dimeloe S. Slack E. Dehio P. et al.Memory CD8(+) t cells require increased concentrations of acetate induced by stress for optimal function.Immunity. 2016; 44: 1312-1324Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, Moussaieff et al., 2015Moussaieff A. Rouleau M. Kitsberg D. Cohen M. Levy G. Barasch D. Nemirovski A. Shen-Orr S. Laevsky I. Amit M. et al.Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells.Cell Metab. 2015; 21: 392-402Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, Schoors et al., 2015Schoors S. Bruning U. Missiaen R. Queiroz K.C. Borgers G. Elia I. Zecchin A. Cantelmo A.R. Christen S. Goveia J. et al.Fatty acid carbon is essential for dNTP synthesis in endothelial cells.Nature. 2015; 520: 192-197Crossref PubMed Scopus (367) Google Scholar, Schug et al., 2015Schug Z.T. Peck B. Jones D.T. Zhang Q. Grosskurth S. Alam I.S. Goodwin L.M. Smethurst E. Mason S. Blyth K. et al.Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.Cancer Cell. 2015; 27: 57-71Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). We reasoned that if a decline in acetyl-CoA was critical, acetate treatment might prevent cells from undergoing EndoMT. Consistent with this notion, we observed that acetate supplementation inhibited cytokine-stimulated SMAD2 activation (Figure 3D). Acetate treatment also effectively inhibited the EndoMT program (Figures 3E and 3F). We next asked whether a reciprocal reduction in acetyl-CoA levels could stimulate EndoMT. To lower acetyl-CoA levels, we first took advantage of a specific pharmacological inhibitor of ATP citrate lyase (ACLY), the enzyme that generates acetyl-CoA from citrate (Figure 3C) (Hatzivassiliou et al., 2005Hatzivassiliou G. Zhao F. Bauer D.E. Andreadis C. Shaw A.N. Dhanak D. Hingorani S.R. Tuveson D.A. Thompson C.B. ATP citrate lyase inhibition can suppress tumor cell growth.Cancer Cell. 2005; 8: 311-321Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar). Interestingly, treatment of primary human endothelial cells with the ACLY chemical inhibitor SB-204990 acted as a strong inducer of EndoMT (Figure 3G). A similar effect was obtained by stable knockdown of ACLY (Figures S3G–S3I) or by inhibiting the mitochondrial citrate transporter (CTP; Figure S3J). Besides its direct effect of acetyl-CoA levels, ACLY also modulates de novo fatty acid synthesis (Hatzivassiliou et al., 2005Hatzivassiliou G. Zhao F. Bauer D.E. Andreadis C. Shaw A.N. Dhanak D. Hingorani S.R. Tuveson D.A. Thompson C.B. ATP citrate lyase inhibition can suppress tumor cell growth.Cancer Cell. 2005; 8: 311-321Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar). However, we saw no induction of EndoMT following stable knockdown of fatty acid synthase (FASN), suggesting that a decrease in fatty acid synthesis is unlikely to be sufficient to mediate cell-fate changes (Figures S3K and S3L). Together, these results suggest that increasing acetyl-CoA levels via acetate supplementation suppresses TGF-β-induced EndoMT, while reducing acetyl-CoA levels via ACLY or CTP inhibition activates EndoMT. In an attempt to better understand how acetyl-CoA levels might regulate EndoMT, we took advantage of previous observations that SMAD7 acts as a potent inhibitor of TGF-β signaling (Miyazawa and Miyazono, 2017Miyazawa K. Miyazono K. Regulation of TGF-β family signaling by inhibitory Smads.Cold Spring Harb. Perspect. Biol. 2017; 9 (Published online March 1, 2017)https://doi.org/10.1101/cshperspect.a022095Crossref PubMed Scopus (262) Google Scholar) and that the post-translational acetylation of SMAD7 on two lysine residues (K64 and K70) has been demonstrated to increase SMAD7 protein stability (Grönroos et al., 2002Grönroos E. Hellman U. Heldin C.H. Ericsson J. Control of Smad7 stability by competition between acetylation and ubiquitination.Mol. Cell. 2002; 10: 483-493Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). We therefore reasoned that acetyl-CoA and hence FAO might modulate the strength of TGF-β-associated signaling by regulating the acetylation, and, hence, the post-translational stability of SMAD7. Consistent with this notion, we found that, in endothelial cells, increasing or decreasing acetyl-CoA levels by treating with acetate or an ACLY inhibitor resulted in a corresponding increase or decrease in endogenous SMAD7 protein expression (Figure S3M). In accordance with a post-translational mechanism, we observed that acetate treatment increased SMAD7 acetylation and overall protein levels, without altering SMAD7 message levels (Figures S3N and S3O). While the effects of acetyl-CoA manipulation on SMAD7 protein levels were seen with wild-type SMAD7 protein (Figure 3H), these same metabolic manipulations did not affect a SMAD7 construct lacking the two known lysine residues that are the targets for acetylation sites (Figure 3I). Similarly, the ability of acetyl-CoA to modulate downstream SMAD2 signaling appeared to require SMAD7 acetylation (Figures S3P and S3Q). Based on these results, we propose a model in which TGF-β signaling results in a positive feedback loop catalyzed by a fall in FAO, a subsequent decline in acetyl-CoA levels and a reduction in SMAD7 inhibition. Our data suggest this enhancement in TGF-β signaling is necessary to drive endothelial cell-fate changes and thus serve as metabolic underpinning for EndoMT susceptibility (Figure S3R). To further pursue the physiological implications of these observations, we bred the previously described floxed Cpt2 mice with transgenic VE-cadherin Cre+ mice (Alva et al., 2006Alva J.A. Zovein A.C. Monvoisin A. Murphy T. Salazar A. Harvey N.L. Carmeliet P. Iruela-Arispe M.L. VE-cadherin-Cre-recombinase transgenic mouse: A tool for lineage analysis and gene deletion in endothelial cells.Dev. Dyn. 2006; 235: 759-767Crossref PubMed Scopus (328) Google Scholar, Lee et al., 2015Lee J. Ellis J.M. Wolfgang M.J. Adipose fatty acid oxidation is required for thermogenesis and potentiates oxidative stress-induced inflammation.Cell Rep. 2015; 10: 266-279Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) to generate endothelial-specific Cpt2 knockout (Cpt2E-KO) mice (Figures S4A and S4B). As expected, expression of CPT2 was markedly reduced in endothelial cells isolated from Cpt2E-KO animals (Figures 4A and S4C). Moreover, although wild-type (WT) endothelial cells could readily metabolize a fatty acid substrate (palmitate) as evidenced by a marked increase in OCR, this metabolic response was absent in endothelial cells derived from Cpt2E-KO mice (Figure 4B). The inability to metabolize LCFA did not, however, affect the apparent growth rate of CPT2-deficient endothelial cells (Figure S4D). This may reflect previous observations that under resting conditions, endothelial cells mostly rely on glycolysis for their energetic needs (De Bock et al., 2013De Bock K. Georgiadou M. Schoors S. Kuchnio A. Wong B.W. Cantelmo A.R. Quaegebeur A. Ghesquière B. Cauwenberghs S. Eelen G. et al.Role of PFKFB3-driven glycolysis in vessel sprouting.Cell. 2013; 154: 651-663Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar). In addition, as we had observed following CPT1A silencing (Figure 3A), CPT2-knockout endothelial cells also had a constitutive increase in TGF-β-associated SMAD signaling that was inhibited by SB431542 treatment (Figure S4E). Moreover, as we noted following TGF-β stimulation, CPT2-knockout endothelial cells had reduced acetyl CoA levels (Figure 4C). Moreover, acetate treatment could restore acetyl-CoA levels in CPT2-knockout endothelial cells (Figure 4C) and could correspondingly inhibit SMAD2 activation in these cells (Figure 4D). We next asked whether endothelial FAO modulates EndoMT in an in vivo context. During development, endocardial cells activated by TGF-β and other ligands are stimulated to undergo EndoMT, triggering their migration into the endocardial cushion and eventual forming the heart valve mesenchyme (Eisenberg and Markwald, 1995Eisenberg L.M. Markwald R.R. Molecular regulation of atrioventricular valvuloseptal morphogenesis.Circ. Res. 1995; 77: 1-6Crossref PubMed Scopus (533) Google Scholar, von Gise and Pu, 2012von Gise A. Pu W.T. Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease.Circ. Res. 2012; 110: 1628-1645Crossref PubMed Scopus (283) Google Scholar). This process is particularly important for formation of the atrioventricular valves (mitral and tricuspid), while the semilunar valves (aortic and pulmonary) rely less on EndoMT and more on cells derived from the neural crest and the second heart field (von Gise and Pu, 2012von Gise A. Pu W.T. Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease.Circ. Res. 2012; 110: 1628-1645Crossref PubMed Scopus (283) Google Scholar). Consistent with a role for FAO in modulating the threshold for EndoMT, morphometric analysis of the mitral valve of Cpt2E-KO mice demonstrated that endothelial Cpt2 deletion resulted in marked thickening of the mitral valve (Figures 4E and 4F). Analysis of the aortic valves of Cpt2E-KO mice revealed a more modest effect of Cpt2 deletion, consistent with the increased importance of EndoMT in atrioventricular valve formation (Figures S4F and S4G). To further establish that the mitral valve thickening was a consequence of increased EndoMT, we performed lineage tracing analysis using a double-fluorescent reporter mouse (R26-mTmG) (Muzumdar et al., 2007Muzumdar M.D. Tasic B. Miyamichi K. Li L. Luo L. A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2258) Google Scholar). Consistent with previous reports (Alva et al., 2006Alva J.A. Zovein A.C. Monvoisin A. Murphy T. Salazar A. Harvey N.L. Carmeliet P. Iruela-Arispe M.L. VE-cadherin-Cre-recombinase transgenic mouse: A tool for lineage analysis and gene deletion in endothelial cells.Dev. Dyn. 2006; 235: 759-767Crossref PubMed Scopus (328) Google Scholar), Cpt2E-WT;mTmG mouse embryos exhibited GFP-positive endothelial cells lining the outer surface of the valve, as well as GFP-positive cells located in the valve interior (Figure 4G). The latter cell type is consistent with cells that have undergone EndoMT during development. Subsequent quantification revealed a significant increase in GFP-positive interstitial cells in Cpt2-deficient mouse embryos, consistent with a role for endothelial FAO in restraining the magnitude of physiological EndoMT during embryogenesis (Figure 4H). These differences persisted in adult Cpt2-deficient mice (Figures S4H–S4J). We next sought to ascertain whether endothelial FAO might play a role in restraining EndoMT in the adult animals. We first sought evidence of spontaneous EndoMT using our dual fluorescent reporter to identify vascular endothelial cells (GFP-positive) that simultaneously stained positive for the mesenchymal marker vimentin. Compared to control animals, we observed increased co-localization of GFP and vimentin in the endothelial cells of Cpt2E-KO mice (Figures 4I and 4J). To begin to physiologically assess the functional consequences of such changes, we took advantage of previous observations that, following EndoMT, endothelial cells can lose their junctional properties and therefore have impaired barrier function (Good et al., 2015Good R.B. Gilbane A.J. Trinder S.L. Denton C.P. Coghlan G. Abraham D.J. Holmes A.M. Endothelial to mesenchymal transition contributes to endothelial dysfunction in pulmonary arterial hypertension.Am. J. Pathol. 2015; 185: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Consistent with this concept, our analysis using Evans Blue infusions revealed a significant increase in vascular permeability in the kidney, spleen, and lung of the Cpt2E-KO mice (Figure 4K). There is increasing evidence that EndoMT might contribute to a wide range of pathological conditions including atherosclerosis, pulmonary hypertension, cardiac valve disease, and organ fibrosis (Kovacic et al., 2012Kovacic J.C. Mercader N. Torres M. Boehm M. Fuster V. Epithelial-to-mesenchymal and endothelial-to-mesenchymal transition: From cardiovascular development to disease.Circulation. 2012; 125: 1795-1808Crossref PubMed Scopus (291) Google Scholar, Sanchez-Duffhues et al., 2016Sanchez-Duffhues G. Orlova V. Ten Dijke P. In brief: Endothelial-to-mesenchymal transition.J. Pathol. 2016; 238: 378-380Crossref PubMed Scopus (42) Google Scholar, Yang et al., 2017Yang B. Zhou W. Jiao J. Nielsen J.B. Mathis M.R. Heydarpour M. Lettre G. Folkersen L. Prakash S. Schurmann C. et al.Protein-altering and regulatory genetic variants near GATA4 implicated in bicuspid aortic valve.Nat. Commun. 2017; 8: 15481Crossref PubMed Scopus (61) Google Scholar). The link to fibrosis is particularly intriguing since a previous report has established that kidney fibrosis and subsequent chronic kidney disease (CKD) in both mouse models and human patients is associated with a decrease in FAO (Kang et al., 2015Kang H.M. Ahn S.H. Choi P. Ko Y.A. Han S.H. Chinga F. Park A.S. Tao J. Sha