Mitochondrial Regulation of the Hypoxia-Inducible Factor in the Development of Pulmonary Hypertension

Round 1

Reviewer 1 Report

The manuscript written by Zeidan et al., titled Mitochondrial Regulation of the Hypoxia-Inducible Factor in the Development of Pulmonary Hypertension, is a great review.

The authors provide to describe the involvement of the mitochondria in the regulation of HIF that developed during Pulmonary Hypertension.
As described by the authors, Reactive Oxygen Species (ROS) play a crux role in the HIF stabilization and signaling during hypoxia.

However, as suggested by the title, because the mitochondrial role is significant in this pathway, I think that Chapter 4 of the manuscript could be deepened to clarify and emphasize the potentiality of using components that exploit mitochondrial ROS as a target in the treatment of pathology.

Author Response

We thank the reviewer for their time and effort in reading this article. Your valuable comments helped us improve the final manuscript.

We responded to the comment below:

as suggested by the title, because the mitochondrial role is significant in this pathway, I think that Chapter 4 of the manuscript could be deepened to clarify and emphasize the potentiality of using components that exploit mitochondrial ROS as a target in the treatment of pathology

Additional studies have been included (yellow highlights) in Chapter 4, as per your recommendations; the text now reads:

Many investigated compounds have now been utilized in the preclinical stage to assess their ability to target the HIF pathway therapeutically and determine hypoxic responses in the lung [22]. Most of these compounds showed promising results in reversing or inhibiting the incidence of PH in experimental models (Table 1). Multiple studies have specifically targeted PHD2, HIF1α, or HIF2α as components of the HIF pathway, with pharmacological compounds in models of PH. These inhibitors, given by various routes of administration, were found to render and reverse PH in different rodent models of PH (hypoxia, MCT, and SuHx) [23]. For example, camptothecin and topotecan deactivate HIF1 at the level of mRNA expression, while celastramycin, 2-methoxyestradiol, and digoxin decrease protein synthesis [10].

In contrast, the YC-1 molecule targets protein accumulation and transcriptional regulation of the HIF axis. Moreover, apigenin and mAb AA98 have altered specific signaling molecules in HIF1 pathway regulation. Additionally, the link between HIF1α, NF-kB, and phosphatidylinositol 3-kinase (PI3K) signaling in PASMCs [24] and other vascular networks continues to gain interest [25]. A previous study demonstrated that caffeic acid phenethyl ester (CAPE), known to inhibit NF-kB, significantly suppressed the AKT/ERK/HIF1α signaling axis in MCT- or chronic hypoxia-induced animal models of PH. This study showed that the CAPE inhibited vascular remodeling by inhibiting HIF1α-induced activation of AKT and ERK signaling. Suppression of those signaling pathways by CAPE inhibited vascular cell proliferation and enhanced the apoptosis of PASMCs [20].

 Other potential therapeutics include C76, which suppresses HIF-2α at the level of mRNA, heterodimerization, and DNA binding [26]. Notably, C76 showed significant anti-remodeling effects in different animal models of PH [10,23], which manifested as attenuation of vascular muscularization, right-sided hypertrophy, and PAPs in hypoxia-exposed animals, suggesting that HIF2α inhibition could provide a promising therapeutic approach to attenuate hypoxia-induced PH [27-29]. Thus, therapeutic targeting of this route has sparked an increased interest. Iron supplementation is another way to target the HIF system, as PHD activity is iron-dependent. Indeed, iron infusions reduced the rise in pulmonary arterial pressures in response to acute and chronic hypoxia [30,31].

Furthermore, considering the vital role of ROS in the PH pathogenesis, inhibition of mtROS release by the mitochondria-targeted antioxidant MitoQ [32] or via genetic approaches, e.g., AOX overexpression [33] or Cox4i2 disruption [21] showed inhibition of acute hypoxic pulmonary vasoconstriction but not chronic hypoxia-induced PH. Moreover, incubation with an antioxidant inhibited PH development when administered prior to the hypoxic exposure but not simultaneously [34].

 

Moreover, we have added a table for preclinical studies that utilized various therapeutic strategies targeting HIF signaling in PH as per recommended by reviewer no. 2

 

Table 1: Preclinical studies that utilized various therapeutic strategies targeting HIF signaling in PH development.

Compound	Experimental Setting	Target	Main findings	Reference
Compound 76 (C76)	In vitro: PASMCs, PAECs, lung samples from iPAH patients In vivo:  MCT and SuHx models of PAH.	Ý IRP1 to inhibit HIF2a signaling.	ß RVSP ß RVH ß RVR	[9,10]
3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1)	In vitro: hPASMCs exposed to hypoxia In vivo:  chronic hypoxia(28 day) PH mouse model	Ý sGC signaling to inhibit HIF-1α expression.	ß  PVR ß RVH	[11]
Topotecan (TPT)	In vitro: hPASMCs exposed to hypoxia In vivo:  chronic hypoxia(28 day) PH rat model PH	ß HIF-1α protein accumulation.  ß HIF-1α target genes expression.	ß PASMCs proliferation. ß PVR	[12]
Celastramycin	In vitro: PASMCs from iPAH patients.	ß HIF-1α mRNA & protein levels.	ß  PASMCs proliferation. ß markers of oxidative stress and inflammation.	[13]
2-methoxyestradiol (2-ME2)	In vitro: hPASMCs exposed to hypoxia In vivo:  chronic hypoxia (28 day) PH rat model PH	Ý MnSOD activity ß ROS production  ß HIF-1a protein expression.	ß PASMCs proliferation. ß RVSP ß RVH ß PVR	[14,15]
Apigenin	In vivo:  chronic hypoxia (28 day) PH rat model PH	ß Akt signaling ß  HIF-1α expression.	ß RVH ß PVR	[16]
Digoxin	In vivo:  chronic hypoxia (28 day) PH mouse model PH	ß HIF-1α transcription and protein synthesis.	ß RVP ß RVR	[17]
Anti-CD146 mAb AA98	In vivo:  chronic hypoxia (28 day) PH mouse model PH, and MCT mouse model	ß CD146 dimerization  & ß HIF-1α hypoxic response.	Ý cardiac function ß PH development	[18]
PHD2 activator (R59949)	In vivo:  chronic hypoxia (28 day) PH mouse model PH	Ý PHD2 & ß HIF-1α ß levels	ß PVR	[19]
Caffeic acid phenethyl ester (CAPE)	In vivo: MCT mouse model	ß AKT/ERK activation & HIF-1α expression	ß proliferation & apoptosis resistance. ß RVSP ß PVR	[20]
Abbreviations; AKT, phosphorylated protein kinase B; IRP1, iron-regulatory protein ; MCT, monocrotaline; PASMCS, pulmonary artery smooth muscle cells; PAECs, pulmonary artery endothelial cells;PVR, pulmonary vascular remodeling; RVH, right ventricular hypertrophy; RVP, right ventricular pressure; RVR, right-ventricular remodelling; RVSP, right ventricular systolic pressure, SU/HX; sugen/hypoxia.

 

 

Thank you

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The review focuses on a very important topic on the mitochondrial regulation of hypoxia-inducible factor in the development of pulmonary hypertension (PH). Given the emerging evidence on the important role of HIF signaling in the pathogenesis of PH, this review is very timely and will be a great addition to the PH literature. The authors presented a general discussion of HIF signaling in PH, mitochondrial ROS regulation of HIF signaling, and preclinical studies on targeting HIF signaling in PH. Please see the comments noted below and the revised manuscript may be accepted for a publication in JCM if the comments are addressed.

Major comments

1.      The objective of the review was not stated in the introduction. In the introduction before moving onto the sub section 1.1, a brief statement on authors main impetus to write this review and how it informs the field (specifically pertaining to mitochondrial regulation HIF signaling in PH) can be added.

2.      As noted by the authors, while some studies show that mitochondrial ROS (mtROS) regulates HIF signaling in PH, others show no role for mtROS, please provide your insight on this dichotomy-whether a pulmonary vascular cell specific HIF signaling accounts for this (vascular smooth cells vs endothelial cells).

3.      Given the objective of the review, please consider focusing on mitochondrial regulation of HIF in PH (section 3) and condense discussions on HIF and vascular signaling (sections 1.1 and 2).

For example, discussion on some of the important studies on mitochondrial regulation of HF mediated PH were omitted.

Please cite and discuss studies:

a.       Marsboom et al. Circ Res. DOI: 10.1161/CIRCRESAHA.111.263848,

b.      Chen et al., Circulation DOI: 10.1161/CIRCULATIONAHA.117.031258,

c.       Bonnet et al., DOI: 10.1161/CIRCULATIONAHA.105.609008

d.      Waypa et al., DOI: 10.1165/rcmb.2013-0191OC  

e.       Fijalkowska et al., DOI: 10.2353/ajpath.2010.090832   

4.      Section 1.1 titles HIF regulation in hypoxia induced PH. However, the discussion on HIF signalling in PH is very limited. This section instead can be put under section titled ‘HIF signaling in hypoxia’ then transition into section 2 to introduce HIF signaling in PH.  

5.      Line 399: mitochondrial specific antioxidants (mitoQ) was assessed in chronic hypoxia mouse model of PH (DOI: 10.1183/13993003.01024-2017) cited elsewhere in the manuscript. Please include and discuss in the section 3.

6.      In section 4, please consider adding a table listing all the preclinical studies that utilized various therapeutic strategies targeting HIF signaling in PH.

Minor comments

1.      Lines 36-40: References appears to be not related, especially reference Reference#5. Please cite standard guidelines on the classification of pulmonary hypertension.

2.      Lines 58-60: Reference# 20 is unrelated to the statement, please revise.

3.      Line 98-99: Please provide a reference for “repress mitochondrial respiration”

4.      Lines 155-157: Please provide a reference.

5.      Line 163: “inhibition of BMP signaling” appears unrelated to this reference cited. Please revise.

6.      Lines 180-182: Cited study (Reference# 6) shows the data on endothelial colony forming cells not pulmonary artery endothelial cells. Please replace the citations with original studies that supports the statement.

7.      Line 185: Cited references# 51, 52 do not include HIF signaling in pulmonary artery endothelial cells rather focuses on HIF singling in cancer. Please revise the references to support the statement.

8.      Line 190: Cited reference# 53 focuses on HIF regulation of Arginase II solely in pulmonary endothelial cells but not in mice as stated by the authors. Please revise the citation to support the statement.

9.      Please provide expansion of abbreviations in Figure 2 in the legend.

10.  Please double check the phrases noted below for the accuracy

a.       Line 250: the word “upstream ROS precursor”

b.      Line 273 “which produces hyperpolarization of the mitochondrial membrane”

c.       Line 370: “shows its value”

11.  Line 393: Cited reference #94 not related specifically to PH rather COPD, please revise.

Author Response

Response to Reviewer 1 Comments

 

The authors wish to thank the reviewer for reading the manuscript, for valuable comments, and for insightful input.

We have addressed the comments below point-by-point:

Major comment;

Point 1: The objective of the review was not stated in the introduction. In the introduction before moving onto the sub section 1.1, a brief statement on the authors main impetus to write this review and how it informs the field (specifically pertaining to mitochondrial regulation HIF signaling in PH) can be added.

Response 1: The following paragraph was added to the end of the Introduction (yellow highlights in the manuscript).

                          “Although evidence implicates mitochondrial reactive oxygen species (ROS) in the development of hypoxia-induced PH, possibly via stabilization of the hypoxia-inducible factor-1 (HIF-1), the direct role of ROS in the development of PH, hence the therapeutic potential of antioxidant treatment is controversial. Thus, this review focuses on mitochondrial regulation of HIF1a signaling in chronic hypoxia-induced PH.”

 

Point 2: As noted by the authors, while some studies show that mitochondrial ROS (mtROS) regulates HIF signaling in PH, others show no role for mtROS, please provide your insight on this dichotomy-whether a pulmonary vascular cell specific HIF signaling accounts for this (vascular smooth cells vs endothelial cells). 

Response 2: As per the reviewer’s recommendation, we have modified the discussion in section 3. “Mitochondrial regulation of HIF in hypoxia-induced PH”. We highlighted the changes in yellow and added the following paragraph to the end of the text (Section 3) (green highlights in the manuscript).:

Together, these findings demonstrated that, under certain conditions, HIF1a stabilization was independent of distal mtROS inhibition. This contradiction with other research results could be related to changes in the concentration of oxygen used in different experiments, the duration of hypoxia, or the cell type-specific processes of HIF1a stabilization. Therefore, the triggering factors and mechanisms of HIF1a signaling pathways still need further investigation to understand the differential modulation of HIF1a stabilization via mtROS during chronic hypoxia-induced PH—such valuable research will allow the discovery of novel therapeutic approaches.

 

Point 3: Given the objective of the review, please consider focusing on mitochondrial regulation of HIF in PH (section 3) and condense discussions on HIF and vascular signaling (sections 1.1 and 2).

Response 3: We sincerely thank the reviewer for their recommendation of the seminal articles that were missed while writing the first version of the manuscript. We have made some changes to the text as per your recommendations. We reduced sections 1.2 & 2 and discussed more studies in section 3. The following sections were added to the text (yellow highlights in the manuscript).

Mitochondria are essential as signaling organelles for initiating and spreading several homeostatic mechanisms. A critical aspect of mitochondria regulation comprises a dynamic network that undergoes constant merging (fusion) and fragmenting (fission). The GTPases mitofusin-1 and mitofusin-2 control the merging process. In contrast, dynamin-related protein-1 (DRP1) and fission-1 regulate mitochondrial fragmentation [1]. Interestingly, PASMCs isolated from PAH patients showed excessive mitochondrial fragmentation, primarily due to activated DRP1, which translocates to the mitochondria, multimerizes, and induces fission [2]. The activity of DRP1 is controlled by two mitochondrial dynamic proteins: MiD49 and MiD51. Epigenetic activation of MiDs enhances mitotic fission, promoting pathologic cellular proliferation and resistance to apoptosis. Other studies reported elevated expression of MiD49 and MiD51 in PASMCs from human PAH patients or rodent models of PAH induced by monocrotaline (MCT) or Sugen/hypoxia [3]. Moreover, stabilization of HIF1α by CoCl₂ in lung sections or normal PASMC resulted in DRP1-mediated mitochondrial fission. On the other hand, inhibiting HIF1α decreased DRP1 activation, mitochondrial fragmentation, and PASMC proliferation [2]. These findings proved that HIF1α activation mediates mitochondrial fragmentation resulting in enhanced cell proliferation.

……………………………….. Besides, a previous study showed that a genetic abnormality on chromosome-1 resulted in decreased mitochondrial ROS production and consequent activation of HIF1α under normoxic conditions in a rat model of PH [4]. This abnormality inhibited the expression of the oxygen-sensitive, voltage-gated Kv channels (e.g., Kv1.5). Such a change increases membrane depolarization and elevates cytosolic K⁺ and Ca²⁺, favoring a proliferative, apoptosis-resistant PASMC phenotype in PAH [4]. These findings showed that mitochondrial abnormalities and reduced ROS production are upstream of normoxic HIF1a activation, irrespective of PO₂, creating a pseudo-hypoxic environment analogous to that observed in idiopathic human PAH and the pathophysiological changes in chronically hypoxic rats [4].

Previous research showed the importance of sirtuins, a class of NAD-dependent deacetylases that regulate mitochondrial function, oxidative stress, and inflammation especially Sirt3, being essential in the pathogenesis of conditions of oxygen and glucose deprivation through its effect on redox signaling [5]. Interestingly, in a previous study of PH mice model, mice lacking Sirt3 demonstrated amplified left ventricular hypertrophic changes in response to pharmacological signals, an effect attenuated by Sirt3 overexpression [6]. The Sirt3-deficient hearts displayed increased ROS signaling and diminished expression of necessary antioxidant enzymes such as catalase and manganese superoxide dismutase (MnSOD) [6]. The results of other studies, showing increased ROS output and HIF1α stabilization of HIF1 after Sirt3 deletion, support the importance of Sirt3-mediated maintenance of mitochondrial antioxidant levels, particularly MnSOD expression [7,8]. Conversely, in a different study, PASMCs from Sirt3 knockout mice showed unaltered ROS signaling responses in the mitochondria, neither in the matrix nor the other compartments, upon acute hypoxic exposure (1.5% O₂; 30 min) [9]. However, the sustained hypoxic exposure (1.5%O₂; 16 h) following Sirt3 deletion resulted in increased ROS signaling only in the mitochondrial matrix, which was not followed by enhancing HIF1a stabilization. In addition, a 30-day in vivo exposure to chronic hypoxia of Sirt3 knockout mice resulted in the development of PH, as in the WT mice [9]. The discrepancy between such results [6-9] regarding the Sirt3-mediated responses and its regulation of ROS/HIF1a be attributed to the variations in cell type, experimental settings, or the possible genetic modulation, feedback, or compensatory mechanisms in genetically modified settings.

 

Point 4: Section 1.1 titles HIF regulation in hypoxia induced PH. However, the discussion on HIF signalling in PH is very limited. This section instead can be put under section titled ‘HIF signaling in hypoxia’ then transition into section 2 to introduce HIF signaling in PH.  

                                  

Response 4: This was done.

 

Point 5: Line 399: mitochondrial specific antioxidants (mitoQ) was assessed in chronic hypoxia mouse model of PH (DOI: 10.1183/13993003.01024-2017) cited elsewhere in the manuscript. Please include and discuss in the section 3.

 

Response 5:  In line 399, the RISP (DOI:10.3390/biomedicines10050957.) study was discussed and cited in section 3, and also the (mitoQ) Study was discussed and cited in section 3.

 

Point 6: In section 4, please consider adding a table listing all the preclinical studies that utilized various therapeutic strategies targeting HIF signaling in PH.

                         

Response 6: The following table was added. Thank you for this important addition.

Table 1: Preclinical studies that utilized various therapeutic strategies targeting HIF signaling in PH development.

Compound	Experimental Setting	Target	Main findings	Reference
Compound 76 (C76)	In vitro: PASMCs, PAECs, lung samples from iPAH patients In vivo:  MCT and SuHx models of PAH.	Ý IRP1 to inhibit HIF2a signaling.	ß RVSP ß RVH ß RVR	[10,11]
3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1)	In vitro: hPASMCs exposed to hypoxia In vivo:  chronic hypoxia(28 day) PH mouse model	Ý sGC signaling to inhibit HIF-1α expression.	ß  PVR ß RVH	[12]
Topotecan (TPT)	In vitro: hPASMCs exposed to hypoxia In vivo:  chronic hypoxia(28 day) PH rat model PH	ß HIF-1α protein accumulation.  ß HIF-1α target genes expression.	ß PASMCs proliferation. ß PVR	[13]
Celastramycin	In vitro: PASMCs from iPAH patients.	ß HIF-1α mRNA & protein levels.	ß  PASMCs proliferation. ß markers of oxidative stress and inflammation.	[14]
2-methoxyestradiol (2-ME2)	In vitro: hPASMCs exposed to hypoxia In vivo:  chronic hypoxia (28 day) PH rat model PH	Ý MnSOD activity ß ROS production  ß HIF-1a protein expression.	ß PASMCs proliferation. ß RVSP ß RVH ß PVR	[15,16]
Apigenin	In vivo:  chronic hypoxia (28 day) PH rat model PH	ß Akt signaling ß  HIF-1α expression.	ß RVH ß PVR	[17]
Digoxin	In vivo:  chronic hypoxia (28 day) PH mouse model PH	ß HIF-1α transcription and protein synthesis.	ß RVP ß RVR	[18]
Anti-CD146 mAb AA98	In vivo:  chronic hypoxia (28 day) PH mouse model PH, and MCT mouse model	ß CD146 dimerization  & ß HIF-1α hypoxic response.	Ý cardiac function ß PH development	[19]
PHD2 activator (R59949)	In vivo:  chronic hypoxia (28 day) PH mouse model PH	Ý PHD2 & ß HIF-1α ß levels	ß PVR	[20]
Caffeic acid phenethyl ester (CAPE)	In vivo: MCT mouse model	ß AKT/ERK activation & HIF-1α expression	ß proliferation & apoptosis resistance. ß RVSP ß PVR	[21]
Abbreviations; AKT, phosphorylated protein kinase B; IRP1, iron-regulatory protein ; MCT, monocrotaline; PASMCS, pulmonary artery smooth muscle cells; PAECs, pulmonary artery endothelial cells;PVR, pulmonary vascular remodeling; RVH, right ventricular hypertrophy; RVP, right ventricular pressure; RVR, right-ventricular remodelling; RVSP, right ventricular systolic pressure, SU/HX; sugen/hypoxia.

 

Minor comments

Point 1: Lines 36-40: References appears to be not related, especially reference Reference#5. Please cite standard guidelines on the classification of pulmonary hypertension

 Response 1: We removed Reference#5 and cited standard guidelines, reference#4 (doi:10.1183/13993003.01913-2018).

Point 2: Lines 58-60: Reference# 20 is unrelated to the statement, please revise.

Response 2: We removed Reference# 20.

Point 3: Line 98-99: Please provide a reference for “repress mitochondrial respiration”

 Response 3: We cited references #29, 30 (doi:10.1152/ajplung.00331.2017,  doi:10.1016/j.yexcr.2017.03.034).

Point 4: Lines 155-157: Please provide a reference.

Response 4: We removed the mentioned study in order to condense this section, as per your recommendation.

 

Point 5: Line 163: “inhibition of BMP signaling” appears unrelated to this reference cited. Please revise

                  

Response 5: We added the right one, references #42 (doi:10.1038/srep12098.)

Point 6: Lines 180-182: Cited study (Reference# 6) shows the data on endothelial colony forming cells not pulmonary artery endothelial cells. Please replace the citations with original studies that supports the statement.

Response 6: We removed no.6 and cited more related one, references #44-46 (doi:10.1152/ajplung.00538.2017,    doi:10.1172/JCI5732, doi:10.1016/j.ebiom.2018.06.003.)

Point 7: Line 185: Cited references# 51, 52 do not include HIF signaling in pulmonary artery endothelial cells rather focuses on HIF singling in cancer. Please revise the references to support the statement

Response 7: We removed no. 51, 52, and cited more related one, references #47, 48 (doi:10.1016/j.biocel.2014.08.012 , doi:10.14814/phy2.13986.              

Point 8: Line 190: Cited reference# 53 focuses on HIF regulation of Arginase II solely in pulmonary endothelial cells but not in mice as stated by the authors.

Response 8: We removed no. 53, and cited more related one, references #49 (doi:10.1073/pnas.1602978113).

Point 9: Please revise the citation to support the statement.Please provide expansion of abbreviations in Figure 2 in the legend

Response 9:We expanded all the abbreviations in Figure 2

Point 10: Please double check the phrases noted below for the accuracy

1. Line 250: the word “upstream ROS precursor”

Response 10. A: We modified the sentence, we used the “upstream ROS source”

1. Line 273 “which produces hyperpolarization of the mitochondrial membrane”

Response 10. B: We double-checked the cited work, and we quoted the following:

“We found that the mitochondrial complex IV subunit 4 isoform 2 initiates acute HPV by mitochondrial hyperpolarization, which promotes the superoxide release preferentially at complex III of the ETC, previously suggested as an essential step of HPV (8, 13).”

Our text now reads:

“Nonetheless, acute hypoxia-induced superoxide generation requires the specific activity of cytochrome c oxidase subunit 4 (Cox4i2); its production hyperpolarizes the mitochondrial membrane. The latter effect enhances the ETC's superoxide output of complex I (or III) [22].”

1. Line 370: “shows its value”

Response 10. C:  We have rephrased this part to read:

                                           Therefore, the triggering factors and mechanisms of HIF1a signaling pathways still need            further investigation to understand the differential modulation of HIF1a stabilization via mtROS during chronic hypoxia-induced PH—such research will allow the discovery of novel therapeutic approaches.

Point 11: Line 393: Cited reference #94 not related specifically to PH rather COPD, please revise.

    Response 11: We removed no. 94 and changed in the text to add more studies as recommended by reviewer 1.

             New studies references 71, 98, 11, and 117.

   

         Thank you.

1. Westermann, B. Mitochondrial fusion and fission in cell life and death. Nature reviews. Molecular cell biology 2010, 11, 872-884, doi:10.1038/nrm3013.
2. Marsboom, G.; Toth, P.T.; Ryan, J.J.; Hong, Z.; Wu, X.; Fang, Y.H.; Thenappan, T.; Piao, L.; Zhang, H.J.; Pogoriler, J.; et al. Dynamin-related protein 1-mediated mitochondrial mitotic fission permits hyperproliferation of vascular smooth muscle cells and offers a novel therapeutic target in pulmonary hypertension. Circ Res 2012, 110, 1484-1497, doi:10.1161/CIRCRESAHA.111.263848.
3. Chen, K.H.; Dasgupta, A.; Lin, J.; Potus, F.; Bonnet, S.; Iremonger, J.; Fu, J.; Mewburn, J.; Wu, D.; Dunham-Snary, K.; et al. Epigenetic Dysregulation of the Dynamin-Related Protein 1 Binding Partners MiD49 and MiD51 Increases Mitotic Mitochondrial Fission and Promotes Pulmonary Arterial Hypertension: Mechanistic and Therapeutic Implications. Circulation 2018, 138, 287-304, doi:10.1161/CIRCULATIONAHA.117.031258.
4. Bonnet, S.; Michelakis, E.D.; Porter, C.J.; Andrade-Navarro, M.A.; Thebaud, B.; Bonnet, S.; Haromy, A.; Harry, G.; Moudgil, R.; McMurtry, M.S.; et al. An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension. Circulation 2006, 113, 2630-2641, doi:10.1161/CIRCULATIONAHA.105.609008.
5. Almalki, W.H.; Alzahrani, A.; Mahmoud El-Daly, M.E.; Fadel Ahmed, A.H.F. The emerging potential of SIRT-3 in oxidative stress-inflammatory axis associated increased neuroinflammatory component for metabolically impaired neural cell. Chemico-biological interactions 2021, 333, 109328, doi:10.1016/j.cbi.2020.109328.
6. Sundaresan, N.R.; Gupta, M.; Kim, G.; Rajamohan, S.B.; Isbatan, A.; Gupta, M.P. Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice. J Clin Invest 2009, 119, 2758-2771, doi:10.1172/JCI39162.
7. Finley, L.W.; Carracedo, A.; Lee, J.; Souza, A.; Egia, A.; Zhang, J.; Teruya-Feldstein, J.; Moreira, P.I.; Cardoso, S.M.; Clish, C.B.; et al. SIRT3 opposes reprogramming of cancer cell metabolism through HIF1alpha destabilization. Cancer cell 2011, 19, 416-428, doi:10.1016/j.ccr.2011.02.014.
8. Bell, E.L.; Emerling, B.M.; Ricoult, S.J.; Guarente, L. SirT3 suppresses hypoxia inducible factor 1alpha and tumor growth by inhibiting mitochondrial ROS production. Oncogene 2011, 30, 2986-2996, doi:10.1038/onc.2011.37.
9. Waypa, G.B.; Osborne, S.W.; Marks, J.D.; Berkelhamer, S.K.; Kondapalli, J.; Schumacker, P.T. Sirtuin 3 deficiency does not augment hypoxia-induced pulmonary hypertension. Am J Respir Cell Mol Biol 2013, 49, 885-891, doi:10.1165/rcmb.2013-0191OC.
10. Zimmer, M.; Ebert, B.L.; Neil, C.; Brenner, K.; Papaioannou, I.; Melas, A.; Tolliday, N.; Lamb, J.; Pantopoulos, K.; Golub, T.; et al. Small-molecule inhibitors of HIF-2a translation link its 5'UTR iron-responsive element to oxygen sensing. Molecular cell 2008, 32, 838-848, doi:10.1016/j.molcel.2008.12.004.
11. Dai, Z.; Zhu, M.M.; Peng, Y.; Machireddy, N.; Evans, C.E.; Machado, R.; Zhang, X.; Zhao, Y.Y. Therapeutic Targeting of Vascular Remodeling and Right Heart Failure in Pulmonary Arterial Hypertension with a HIF-2alpha Inhibitor. Am J Respir Crit Care Med 2018, 198, 1423-1434, doi:10.1164/rccm.201710-2079OC.
12. Huh, J.W.; Kim, S.Y.; Lee, J.H.; Lee, Y.S. YC-1 attenuates hypoxia-induced pulmonary arterial hypertension in mice. Pulmonary pharmacology & therapeutics 2011, 24, 638-646, doi:10.1016/j.pupt.2011.09.003.
13. Jiang, Y.; Zhou, Y.; Peng, G.; Liu, N.; Tian, H.; Pan, D.; Liu, L.; Yang, X.; Li, C.; Li, W.; et al. Topotecan prevents hypoxia-induced pulmonary arterial hypertension and inhibits hypoxia-inducible factor-1alpha and TRPC channels. The international journal of biochemistry & cell biology 2018, 104, 161-170, doi:10.1016/j.biocel.2018.09.010.
14. Kurosawa, R.; Satoh, K.; Kikuchi, N.; Kikuchi, H.; Saigusa, D.; Al-Mamun, M.E.; Siddique, M.A.H.; Omura, J.; Satoh, T.; Sunamura, S.; et al. Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension. Circ Res 2019, 125, 309-327, doi:10.1161/CIRCRESAHA.119.315229.
15. Docherty, C.K.; Nilsen, M.; MacLean, M.R. Influence of 2-Methoxyestradiol and Sex on Hypoxia-Induced Pulmonary Hypertension and Hypoxia-Inducible Factor-1-alpha. Journal of the American Heart Association 2019, 8, e011628, doi:10.1161/JAHA.118.011628.
16. Wang, L.; Zheng, Q.; Yuan, Y.; Li, Y.; Gong, X. Effects of 17beta-estradiol and 2-methoxyestradiol on the oxidative stress-hypoxia inducible factor-1 pathway in hypoxic pulmonary hypertensive rats. Experimental and therapeutic medicine 2017, 13, 2537-2543, doi:10.3892/etm.2017.4243.
17. He, Y.; Fang, X.; Shi, J.; Li, X.; Xie, M.; Liu, X. Apigenin attenuates pulmonary hypertension by inducing mitochondria-dependent apoptosis of PASMCs via inhibiting the hypoxia inducible factor 1alpha-KV1.5 channel pathway. Chemico-biological interactions 2020, 317, 108942, doi:10.1016/j.cbi.2020.108942.
18. Abud, E.M.; Maylor, J.; Undem, C.; Punjabi, A.; Zaiman, A.L.; Myers, A.C.; Sylvester, J.T.; Semenza, G.L.; Shimoda, L.A. Digoxin inhibits development of hypoxic pulmonary hypertension in mice. Proc Natl Acad Sci U S A 2012, 109, 1239-1244, doi:10.1073/pnas.1120385109.
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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

My comments have been addressed.