Dysregulated metabolism, endothelial cell dysfunction and apoptosis, vascular proliferation and remodeling, and inflammation are the hallmarks of several vascular diseases including coronary, carotid and peripheral vascular disease atherosclerotic plaques and pulmonary arterial hypertension (PAH) [1,2]. PAH is a progressive vaso-occlusive disease characterized by increased pulmonary arterial pressure, and if left untreated, it carries a high rate of mortality with the most common cause of death being right-sided heart failure [2,3]. Advanced right ventricular (RV) hypertrophy, dilatation and dysfunction also influence the left ventricle and can lead to left ventricular (LV) dysfunction through RV–LV interactions. On the other hand, pulmonary hypertension is a frequent complication of multiple common left heart diseases (LHDs) that adversely affects the life-style quality and outcome. In addition, RV dysfunction is common in left heart failure with preserved ejection fraction and is associated with unfavorable outcome implying that RV dysfunction might be a plausible therapeutic target in both right and left heart failure .
In the recent years, the ‘degenerative paradigm’ has emerged as a rationale explaining the pathogenesis of PAH and is supported by some evidence [2,4]. It adopts the notion that, initially, endothelial cell dysfunction and undergoes apoptosis which can directly contribute to the dropout of fragile precapillary arterioles leading to microvascular rarefaction and eventually increasing the pulmonary vascular resistance causing PAH [2,4]. Hence, this paradigm implies that a regenerative therapeutic strategy would be quite reasonably adopted in order to restore and promote the lung microvasculature [2,4]. Here is where peroxisome proliferator-activated receptor gamma (PPARγ) activation comes as a viable option. PPARγ is a nuclear receptor that interacts with retinoid X receptor (RXR) and other cofactors to regulate transcription of target genes, thus regulating adipogenesis, metabolism, cellular differentiation and development, inflammation, and tumorigenesis [5,6]. PPARγ is ubiquitously expressed in normal human tissues including normal human pulmonary tissues and is significantly reduced in the plexiform lesions in patients with PAH [2,7].
In this article, we will briefly highlight the potential capacity and role of PPARγ in DNA damage response and repair, thus maintaining endothelial cell DNA stability and integrity, in an attempt to extend and add to the conventional knowledge about PPARγ and its metabolic, antiproliferative and anti-inflammatory effects, in order to build a rationale premise for the future classification and use of its agonists, especially pioglitazone (a thiazolidinedione-class agonist), for regenerative purposes.
DNA damage in pulmonary artery endothelial cell as an example of endothelial cells
Pulmonary artery endothelial cell (PAEC) relies on cellular respiration and is therefore, susceptible to small changes in oxygen concentration . PAH-PAEC, however, exhibits increased glycolysis even at normoxia, leading to enhanced reactive oxygen species (ROS) production and increased oxidative stress . This, together with impaired handling of the oxidative stress secondary to decreased superoxide dismutases expression , contributes, in part, to the pathogenesis of PAH by causing DNA damage . Indeed, genomic instability and an increased propensity for apoptosis are key features of PAECs from PAH patients [11–14]. The mitochondria are deficient in several protective mechanisms; therefore, the mitochondrial DNA is more susceptible to oxidative damage than nuclear DNA . Recently, De Silva et al.  have provided evidence that endothelial PPARγ has an essential role in protecting against endothelial cell dysfunction and oxidative stress during senescence.
The DNA damage response and its relation to peroxisome proliferator-activated receptor gamma
The DNA damage response (DDR) is the linchpin through which the cell senses DNA damage and transduces this signal to modulate numerous pathways initiating several actions including DNA repair. An example of such response is the rapid activation of ATM protein kinase which in turn phosphorylates target proteins resulting in checkpoint activation, apoptosis and DNA repair . The Mre11/Rad50/Nbs1 (MRN) complex [16,17], as well as the ubiquitin ligase UBR5 , are required for ATM signaling and function which is necessary for DNA repair. A recent study by Li et al. , using affinity purification mass spectrometry (AP-MS) in transiently transfected 293T cells, verified PPARγ interactions, and their specificity, with the MRN complex, UBR5 and p53 independent of RXR, hence, implicating a potential role for PPARγ in the DDR pathway which is independent of its transcription function. In the same study, PPARγ depletion led to decreased activation of ATM and its targets in response to hydroxyurea- and doxorubicin-induced DNA damage . Hydroxyurea-induced genotoxicity closely resembles that induced by chronic replication stress in PAECs from PAH patients [12,20]. PPARγ depletion also suppressed UBR5 E3 ubiquitin ligase activity leading to decreased UBR5-mediated ATMIN ubiquitination which was accompanied by an increase in ATMIN protein levels both at baseline and after hydroxyurea-induced injury . ATMIN is a UBR5 substrate which modulates ATM activity and when ubiquitinated, and it releases ATM allowing its phosphorylation and therefore, activation . Li et al.  also found ATMIN mRNA levels not to be significantly altered; thus, indicating that loss of PPARγ can alter cellular protein degradation and that this is not related to PPARγ transcription function . These results show the capacity of PPARγ in promoting ATM signaling and, hence, the necessity of PPARγ in initiating the DDR.
The peroxisome proliferator-activated receptor gamma-UBR5-ATMIN axis is conserved in endothelial cells
This PPARγ-DDR was found to be conserved in endothelial cells. In PAECs, PPARγ depletion was not found to influence the magnitude of DNA damage after hydroxyurea exposure, but rather the capacity of DNA repair was reduced as evidenced by unresolved damage foci in PPARγ-depleted PAECs in comparison to the control cells . Depleting ATMIN in addition to PPARγ led to resolution of DNA damage foci during recovery confirming the role ATMIN plays in the PPARγ-mediated DNA repair function . This highlights that the PPARγ-UBR5-ATMIN axis is necessary for endothelial cell DNA repair and homeostasis. The common shared response of 293T cells and primary endothelial cells supports the argument that alterations in the PPARγ-UBR5-ATMIN axis could possibly occur in multiple cell types expressing PPARγ, including the myocardium; hence, would be operating in a wide range of disease mechanisms . An evidence supporting this notion is the fact that PPARγ noncanonical agonist ligand activation sensitizes lung cancer cells to platinum-based chemotherapeutics by activating the DDR pathway .
The peroxisome proliferator-activated receptor gamma-DNA damage response function is perturbed in pulmonary arterial hypertension-pulmonary artery endothelial cells
As expected, due to impaired PPARγ function and genomic instability caused by persistent DNA damage  in PAH, PAH-PAECs exhibited increased markers of DNA damage (γH2AX foci and extended comet tails), and reduced phosphorylated-ATM foci upon hydroxyurea treatment when compared to control-PAECs, and thus, confirming the perturbed PPARγ-DDR function in PAH and, hence, loss of pulmonary vascular homeostasis . Interestingly, PAH-PAECs also showed reduced PPARγ–UBR5 interactions in comparison to control-PAECs despite the similar levels of PPARγ in both . Li et al. suggested that this would be due to some sort of posttranslational modifications (PTMs) which confers structural changes and can therefore alter protein–protein interactions [19,22].
The molecular interaction of peroxisome proliferator-activated receptor gamma with Mre11/Rad50/Nbs1
Li et al. demonstrated, using tandem affinity purification and cross-linking-mass spectrometry (XL-MS), that PPARγ binds to MRN via Nbs1 and that three PPARγ peptides crosslinked to NBS1. They found, with reference to the crystal structure of PPARγ , two of the three peptides to be located in the PPARγ DNA-binding domain and the third in the ligand-binding domain (LBD) . To determine if this Nbs1 binding can interfere with PPARγ transcription function or not, size-exclusion chromatography was carried out revealing that Nbs1 and RXR resided in two separate high and low molecular weight PPARγ pools, respectively. This implies the mutually exclusive nature of PPARγ interaction with either Nbs1 or RXR, and hence, Nbs1 binding can interfere with PPARγ transcription factor function . Because the LBD of PPARγ is involved, this also raises the question of whether the canonical agonist ligand activation of PPARγ, using pioglitazone, can enhance the DDR response or not.
Is it safe to use pioglitazone?
As shown, these novel findings link PPARγ activation to a potential capacity in DNA repair which would prevent the persistent DNA damage seen in PAH. Considering this, together with the other known multifaceted beneficial effects of PPARγ, including the metabolic, the antiproliferative (antifibrotic), anti-inflammatory, proangiogenic, and proapoptotic effects, PPARγ agonists would serve as a perfect therapeutic option to treat PAH and other cardiopulmonary disorders [2,23,24]. Unfortunately, erroneous reports on serious side effects and misinterpretations of early diabetes studies in the past decade raised concerns regarding the well tolerated use of PPARγ agonists (especially the thiazolidinediones-class) leading to abandoning its clinical use [3,24]. It is worth mentioning that pioglitazone possesses a better clinical adverse effect profile when compared to rosiglitazone [25,26]. Furthermore, applying supraphysiological doses of pioglitazone to cultured PAECs from patients with idiopathic PAH, as well as from controls, did not demonstrate any toxicity. Neither it did with neonatal rat cardiomyocytes . In the important randomized controlled Insulin Resistance Intervention After Stroke trial which followed 3876 prediabetic/insulin resistant patients for 4.8 years , pioglitazone therapy lacked significant toxicity in high-risk cardiovascular disease population except for increased incidence of weight gain, edema, and bone fractures. Pioglitazone therapy did not increase the risk of heart failure or any malignancy when compared to placebo-treated patients [25,28,29]. On the contrary, pioglitazone therapy reduced the composite outcome of myocardial infarction, hospitalized heart failure, or stroke, demonstrating a positive net cardiovascular effect [25,28,29]. In addition, pioglitazone achieved a preclinical rigor score of 9 ranking the second highest score among 22 different potential therapies that were reviewed. This scoring assesses the rigor of the available preclinical data that would favor the use of a particular therapy regarding PAH .
Taken together, it is quite obvious that the pathobiology of PAH is very intricate, including several different metabolic, inflammatory, and genetic abnormalities contributing to its development. The currently available medications – so far – are ineffective in preventing disease progression and the need for a lung transplant. A regenerative therapeutic strategy sounds as a plausible option given the context of PAH, and other cardiopulmonary diseases, pathobiology. PPARγ activation, using pioglitazone, presents as a strong potential on that front. It has the advantage of addressing almost every single abnormal pathway contributing to PAH, and it does so simultaneously. Studies also unraveled its DDR-function, and hence, can initiate DNA repair, leading to prevention or reduction of PAH-PAECs DNA damage, therefore, maintaining pulmonary vascular homeostasis. In addition, pioglitazone did not show significant toxicity in high-risk populations. Therefore, we recommend timely conduction of preclinical studies, using models with severe PAH resembling the human disease, and clinical studies to characterize the biological and clinical effects of the pharmacological activation of PPARγ, using pioglitazone, in PAH patients opening the door toward its ‘repurposing’.
We would like to thank Prof. Osama Hussein for his endless support to our Research accessibility team. We want also to thank, Prof. Iman Ehsan for championing our research efforts. Finally, yet importantly, we want to acknowledge the role of our colleague Maria Ashraf, who is involved in the field of 3D organ printing and regenerative engineering.
Conflicts of interest
There are no conflicts of interest.
1. Sakao S, Tatsumi K, Voelkel NF. Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation. Respir Res. 2009; 10:95
2. Afdal P, AbdelMassih AF. Is pulmonary vascular disease reversible with PPAR γ agonists? Microcirculation. 2018; 25:e12444
3. Hansmann G, Calvier L, Risbano MG, Chan SY. Activation of the metabolic master regulator PPARγ: a potential PIOneering therapy for pulmonary arterial hypertension. Am J Respir Cell Mol Biol. 2020; 62:143–156
4. Chaudhary KR, Taha M, Cadete VJ, Godoy RS, Stewart DJ. Proliferative versus degenerative paradigms in pulmonary arterial hypertension: have we put the cart before the horse? Circ Res. 2017; 120:1237–1239
5. Lehrke M, Lazar MA. The many faces of PPARgamma. Cell. 2005; 123:993–999
6. Chandra V, Huang P, Hamuro Y, Raghuram S, Wang Y, Burris TP, Rastinejad F. Structure of the intact PPAR-gamma-RXR- nuclear receptor complex on DNA. Nature. 2008; 456:350–356
7. Ameshima S, Golpon H, Cool CD, Chan D, Vandivier RW, Gardai SJ, et al. Peroxisome proliferator-activated receptor gamma (PPARgamma) expression is decreased in pulmonary hypertension and affects endothelial cell growth. Circ Res. 2003; 92:1162–1169
8. Humbert M, Guignabert C, Bonnet S, Dorfmüller P, Klinger JR, Nicolls MR, et al. Pathology and pathobiology of pulmonary hypertension: state of the art and research perspectives. Eur Respir J. 2019; 53:1801887
9. Diebold I, Hennigs JK, Miyagawa K, Li CG, Nickel NP, Kaschwich M, et al. BMPR2 preserves mitochondrial function and DNA during reoxygenation to promote endothelial cell survival and reverse pulmonary hypertension. Cell Metab. 2015; 21:596–608
10. Archer SL, Marsboom G, Kim GH, Zhang HJ, Toth PT, Svensson EC, et al. Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target. Circulation. 2010; 121:2661–2671
11. Ranchoux B, Meloche J, Paulin R, Boucherat O, Provencher S, Bonnet S. DNA damage and pulmonary hypertension. Int J Mol Sci. 2016; 171990
12. Aldred MA, Comhair SA, Varella-Garcia M, Asosingh K, Xu W, Noon GP, et al. Somatic chromosome abnormalities in the lungs of patients with pulmonary arterial hypertension. Am J Respir Crit Care Med. 2010; 182:1153–1160
13. Meloche J, Pflieger A, Vaillancourt M, Paulin R, Potus F, Zervopoulos S, et al. Role for DNA damage signaling in pulmonary arterial hypertension. Circulation. 2014; 129:786–797
14. Federici C, Drake KM, Rigelsky CM, McNelly LN, Meade SL, Comhair SA, et al. Increased mutagen sensitivity and DNA damage in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2015; 192:219–228
15. De Silva TM, Li Y, Kinzenbaw DA, Sigmund CD, Faraci FM. Endothelial PPARγ (peroxisome proliferator-activated receptor-γ) is essential for preventing endothelial dysfunction with aging. Hypertension. 2018; 72:227–234
16. Lee JH, Paull TT. Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science. 2004; 304:93–96
17. Reinhardt HC, Yaffe MB. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response. Nat Rev Mol Cell Biol. 2013; 14:563–580
18. Zhang T, Cronshaw J, Kanu N, Snijders AP, Behrens A. UBR5-mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function. Proc Natl Acad Sci U S A. 2014; 111:12091–12096
19. Li CG, Mahon C, Sweeney NM, Verschueren E, Kantamani V, Li D, et al. PPARγ interaction with UBR5/ATMIN promotes DNA repair to maintain endothelial homeostasis. Cell Rep. 2019; 26:1333–1343.e7
20. de Jesus Perez VA, Yuan K, Lyuksyutova MA, Dewey F, Orcholski ME, Shuffle EM, et al. Whole-exome sequencing reveals TopBP1 as a novel gene in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2014; 189:1260–1272
21. Khandekar MJ, Banks AS, Laznik-Bogoslavski D, White JP, Choi JH, Kazak L, et al. Noncanonical agonist PPARγ ligands modulate the response to DNA damage and sensitize cancer cells to cytotoxic chemotherapy. Proc Natl Acad Sci U S A. 2018; 115:561–566
22. Choi JH, Choi SS, Kim ES, Jedrychowski MP, Yang YR, Jang HJ, et al. Thrap3 docks on phosphoserine 273 of PPARγ and controls diabetic gene programming. Genes Dev. 2014; 28:2361–2369
23. Hansmann G, Zamanian RT. PPARG activation. 2009; 1:1–6
24. Kökény G, Calvier L, Legchenko E, Chouvarine P, Mózes MM, Hansmann G. PPARγ is a gatekeeper for extracellular matrix and vascular cell homeostasis: beneficial role in pulmonary hypertension and renal/cardiac/pulmonary fibrosis. Curr Opin Nephrol Hypertens. 2020; 29:171–179
25. Kernan WN, Viscoli CM, Furie KL, Young LH, Inzucchi SE, Gorman M, et al.; IRIS Trial Investigators. Pioglitazone after ischemic stroke or transient ischemic attack. N Engl J Med. 2016; 374:1321–1331
26. Lazar MA. Reversing the curse on PPARγ. J Clin Invest. 2018; 128:2202–2204
27. Legchenko E, Chouvarine P, Borchert P, Fernandez-Gonzalez A, Snay E, Meier M, et al. PPARγ agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation. Sci Transl Med. 2018; 10:1–18
28. Young LH, Viscoli CM, Schwartz GG, Inzucchi SE, Curtis JP, Gorman MJ, et al.; IRIS Investigators. Heart failure after ischemic stroke or transient ischemic attack in insulin-resistant patients without diabetes mellitus treated with pioglitazone. Circulation. 2018; 138:1210–1220
29. Spence JD, Viscoli CM, Inzucchi SE, Dearborn-Tomazos J, Ford GA, Gorman M, et al.; IRIS Investigators. Pioglitazone therapy in patients with stroke and prediabetes: a post hoc analysis of the IRIS randomized clinical trial. JAMA Neurol. 2019; 76:526–535
30. Prins KW, Thenappan T, Weir EK, Kalra R, Pritzker M, Archer SL. Repurposing medications for treatment of pulmonary arterial hypertension: what’s old is new again. J Am Heart Assoc. 2019; 8:1–32