Idiopathic pulmonary arterial hypertension (PAH) is an unrelentingly progressive disease characterized by proliferative, enlarged, and vacuolated endothelial and smooth muscle cells resistant to apoptosis. Singularly missing from discussions of the pathogenesis of PAH is consideration of subcellular membrane- and protein/receptor-trafficking mechanisms between the plasma membrane and the cell interior. We will test the hypothesis that the entire basket of alterations in vasorelevant proteins (including increases/decreases in levels of various receptors, signaling pathway molecules and eNOS) in this disease is largely due to mislocalization of these proteins to the wrong subcellular compartments due to defects in intracellular trafficking at the level of the Golgi apparatus (""""""""Golgi blockade hypothesis""""""""). We implicate both new players (respective Golgi tethers, SNAREs and 1-SNAP and NSF) and old players (mislocalized eNOS and reduced subcellular NO, mutant BMPRII species) in the subcellular mechanisms that """"""""cause"""""""" PAH.
Aim I will deal with high-resolution 3-D imaging studies of the alterations of the Golgi apparatus morphology, enlargement and fragmentation under various PAH-mimetic conditions [NO scavenging, hypoxia and monocrotaline pyrolle (MCTP)] in cultures of primary HPAECs and HPASMCs with a focus on giantin, GM130, p115, GS28 and 1-SNAP.
Aim II will deal with the consequences of these changes in terms of the subcellular mislocalization of eNOS. The studies will be carried out in (1) cultures of HPAECs exposed to NO-scavenging, hypoxia or MCTP, and (2) in cells in formalin-fixed sections of vascular lesions in lungs derived from the MCT-treated rat, human PAH and the SHIV-nef-infected macaque with pulmonary vasculopathies.
Aim III will test the hypothesis that PAH-disease-associated BMPRII mutant species can mislocalize eNOS and trafficking-mediator proteins in HPAECs and HPSMCs causing a dominant-negative effect on intracellular trafficking. Taken together, the proposed studies represent the next step in testing the Golgi blockade hypothesis in PAH. Upon completion of this project we will have elucidated changes in Golgi apparatus morphology in high-resolution 3-D in both cell culture and in cells in tissue sections in this disease, obtained evidence for subcellular mislocalization of eNOS and 1-SNAP, and provide a novel mechanism for how mutations in BMPRII might disrupt intracellular trafficking culminating in the cellular phenotype observed in pulmonary vascular cells in PAH lesions.

Public Health Relevance

Fifteen thousand patients die of pulmonary arterial hypertension (PAH) every year in the U.S. It is not clearly understood what causes PAH. Although mutations in the BMPR2 gene have been implicated, the mechanisms have not been elucidated despite 10 years'of investigations. We propose a novel way of looking at the pathogenesis of this disease in terms of a global disruption of intracellular trafficking in pulmonary arterial endothelial and smooth muscle cells which result in the incorrect placement of vasorelevant proteins on the cell surface and in locations within the cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31HL107013-02
Application #
8230450
Study Section
Special Emphasis Panel (ZRG1-F10A-S (20))
Program Officer
Colombini-Hatch, Sandra
Project Start
2011-02-07
Project End
2012-05-31
Budget Start
2012-02-07
Budget End
2012-05-31
Support Year
2
Fiscal Year
2012
Total Cost
$10,988
Indirect Cost
Name
New York Medical College
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
041907486
City
Valhalla
State
NY
Country
United States
Zip Code
10595
Lee, Jason E; Yang, Yang-Ming; Yuan, Huijuan et al. (2013) Definitive evidence using enucleated cytoplasts for a nongenomic basis for the cystic change in endoplasmic reticulum structure caused by STAT5a/b siRNAs. Am J Physiol Cell Physiol 304:C312-23
Khan, Rasel; Lee, Jason E; Yang, Yang-Ming et al. (2013) Live-cell imaging of the association of STAT6-GFP with mitochondria. PLoS One 8:e55426
Lee, Jason E; Yuan, Huijuan; Liang, Feng-Xia et al. (2013) Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells. Nitric Oxide 33:64-73
Lee, Jason E; Yang, Yang-Ming; Liang, Feng-Xia et al. (2012) Nongenomic STAT5-dependent effects on Golgi apparatus and endoplasmic reticulum structure and function. Am J Physiol Cell Physiol 302:C804-20