Project 4 Update

The overall goal of our proposed studies is to understand the mechanisms for the increased production of HA by PASMCs through the study of the effects of NO on the abnormal HA matrix in IPAH. The innovative approach in this proposal is enhanced exponentially by sharing several common themes with other components in the Program Project.

AIM 1: Our initial assessment of HA in IPAH showed increased levels in the lung and plasma of patients (AJP-Lung 2008). We determined that PAECs do not produce HA spontaneously or after stimulation with hypoxia. We evaluated epigenetic abnormalities in HAS2 and were not able to find differences in histone deacetylation of HAS2 and HYALl2 between PH and control PASMCs cells. We have focused on post-translational modifications (i.e., O-GlcNAc) and the hexosamine biosynthetic pathway (HBP) as facilitators of HA production and sustainability in IPAH. Our characterization and mechanistic insight into the HBP and its effects on the O-GlcNAc transferase (OGT) in IPAH was recently accepted for publication (Barnes J, Tian L, Heresi G, Carol Farver, Kewal Asosingh, Suzy Comhair, Kulwant Aulak, Dweik RA. O-GlcNAc Transferase Directs Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension. Circulation, 2015, in press NIHMSID: 661905). We believe that exploring the HBP in IPAH is the source of HA production since cytosolic UDP-GlcNAc is a substrate for O-GlcNAcylation and HA biosynthesis. Also, O-GlcNAc can regulate HAS enzymes. We are further exploring the relationship between HA, HASs, HYAL, and the HBP in IPAH patient lung tissue and isolated pulmonary arterial cells.

AIM 2: Our current model suggests that HA is crucial for smooth proliferation and inflammatory cell binding. Our exploration of the HBP has shown that manipulation of this pathway regulates HA production in IPAH. We currently believe that the HBP contributes to smooth muscle cell proliferation (Barnes et al, Circulation 2015) and changes in NO production in IPAH. We are currently working on the relationship between glucose dysregulation and HBP activation. Our hypothesis is that glucose metabolic flux into the HBP causes increased eNOS O-GlcNAc modification and regulates eNOS phosphorylation and activity resulting in decreased NO production. We evaluated the eNOS O-GlcNAc modification and determined that it is elevated 2-fold in IPAH PAECs compared to control PAECs. We have validated the modification of eNOS by demonstrating that recombinant O-GlcNAc transferase can modify eNOS in a bacterial co-expression system. Our preliminary data suggest that the HBP flux can facilitate increased O-GlcNAc modification of eNOS. In line with this, we analyzed the phosphorylation of eNOS (at ser-1177) and determined that this site has a reduction in phosphorylation (and increased O-GlcNAc) in IPAH compared to control.

AIM 3: We recently published a report on the modification of HA by heavy chains in human lungs of IPAH patients (Lauer and Aytekin et al. JBC 2014).

Trainee Activity, Project 4

The trainees within this project have been engaged in the Glycosciences Skills Development and Resource Cores of the PEG. They have had the opportunity to attend National and International meetings to showcase their work funded by the PEG projects. Our trainees have been awarded poster and platform talks at society meetings outside the fields of glycobiology, which is a great tool to educate and advance other science realms about glycobiology and its importance in disease. For some of these conferences, travel awards or abstract scholarships were awarded due to the impact of the research within the field. The respective meetings including: The Society for Glycobiology, American Thoracic Society, the Pulmonary Hypertension Summit, and the Respiratory Institute Research Day. With this PEG support, our trainees have been able to meet and interact with world-renown specialists in the fields of HA and glycobiology as well as the pulmonary vascular diseases. In addition to conferences, the Glycoscience Skills Core has supported and provided training platform for the funding of an F32 Postdoctoral Fellowship to one of our trainees Dr. Jarrod Barnes, PhD (F32HL120629).

Publications (complete list below) and National Meeting Presentation of Posters and Talks.

Meetings with presentations: American Thoracic Society 2014 (San Diego); PH Summit 2014 (Cleveland); Respiratory Research Day (Cleveland); and Society for Glycobiology 2014 (Hawaii, HI).

AIM 1: We plan to explore the relationship between HA, HASs, HYAL, and the HBP in IPAH patient lung tissue and isolated pulmonary arterial cells and determine the role and progression of these molecules in the disease pathogenesis.

AIM 2: Using Mass Spectrometry, we have located the site at which the eNOS protein is specifically O-GlcNAc modified. Our MS assessment determined that OGT modifies eNOS at ser-615. In addition, we have validated, using control and IPAH PAECs, that eNOS ser-615 has reduced phosphorylation in IPAH compared to control. Combined with these results, we are currently investigating the role of the O-GlcNAc modification of ser-615 and its effect on the reduction of ser-1177 phosphorylation and eNOS function. Clinically, we will continue to correlate the HBP activation state, HA, and NO levels with disease outcomes (functional capacity, disease progression, and mortality) in patients with IPAH as we accumulate more patients.

AIM 3: We will access the progression of HA biosynthesis during the course of the disease. We plan to explore the mechanisms associated with heavy chain transfer and IPAH disease progression.

1) Navaneethan SD, Wehbe E, Heresi GA, Gaur V, Minai OA, Arrigain S, Nally JV Jr, Schold JD, Rahman M, Dweik RA. Presence and Outcomes of Kidney Disease in Patients with Pulmonary Hypertension. Clin J Am Soc Nephrol. 2014 May;9(5):855-63. PMCID: PMC4011456 [Available on 2015-05-07].

2) Hanouneh IA, Zein NN, Cikach F, Dababneh L, Grove D, Alkhouri N, Lopez R, Dweik RA. The breathprints in patients with liver disease identify novel breath biomarkers in alcoholic hepatitis. Clin Gastroenterol Hepatol. 2014 Mar; 12(3):516-23. PMCID: PMC3971429.

3) Dababneh L, Cikach F, Alkukhun L, Dweik RA, Tonelli AR. Sublingual Microcirculation in Pulmonary Arterial Hypertension. Annals of the American Thoracic Society. 2014 May;11(4):504-12. PMCID: PMC4225801 [Available on 2015-05-01].

4) Lauer ME, Aytekin M, Comhair SA, Loftis J, Tian L, Farver CF, Hascall VC, Dweik RA. Modification of hyaluronan by heavy chains of inter-alpha-inhibitor in idiopathic pulmonary arterial hypertension. J Biol Chem. 2014 Mar 7;289(10):6791-8. PMCID: PMC3945340.

5) Patel N, Alkhouri N, Eng K, Cikach F, Mahajan L, Yan C, Grove D, Rome ES, Lopez R, Dweik RA. Metabolomic analysis of breath volatile organic compounds reveals unique breathprints in children with inflammatory bowel disease: a pilot study. Aliment Pharmacol Ther. 2014; 40:498-507. PMCID: PMC4127094 [Available on 2015-09-01].

6) Aytekin M, Tonelli AR, Farver CF, Feldstein AE, Dweik RA. Leptin deficiency recapitulates the histological features of pulmonary arterial hypertension in mice. Int J Clin Exp Pathol. 2014;7:1935-46. PMCID: PMC4069962.

7) Navaneethan U, Parsi MA, Gutierrez NG, Bhatt A, Venkatesh PG, Lourdusamy D, Grove D, Hammel JP, Jang S, Sanaka MR, Stevens T, Vargo JJ, Dweik RA. Volatile organic compounds in bile can diagnose malignant biliary strictures in the setting of pancreatic cancer: a preliminary observation. Gastrointest Endosc. 2014 Dec;80(6):1038-45. NIHMSID: 629941.

8) Barnes J, Dweik RA. Is pulmonary hypertension a metabolic disease? Am J Respir Crit Care Med. 2014; 190(9):973-5. PMCID: PMC4299590 [Available on 2015-11-01].


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