Dysfunctional intracellular trafficking in the pathobiology of pulmonary arterial hypertension

Abstract

Discussions of the initiation of pulmonary arterial hypertension (PAH) in man and in experimental models have centered around intimal and medial proliferation in medium-sized pulmonary arteries. The histologic events are thought to include disordered proliferation of enlarged, vacuolated endothelial cells, neo-muscularization of the affected blood vessels, and vascular pruning. The discovery of the association of familial and sporadic PAH with mutations in BMPR2 has generated intense interest in cytokine receptor trafficking and function in the endothelial cell and how this might be disrupted to yield an enlarged proliferative cell phenotype. Nevertheless, considerations of the subcellular machinery of membrane trafficking in the endothelial cell and consequences of the disruption of this outward and inward membrane trafficking are largely absent from discussions of the pathobiology of PAH. Long-standing electron microscopy data in the PAH field has demonstrated marked disruptions of intracellular membrane trafficking in human and experimental PAH. Further, a role of the membrane-trafficking regulator Nef in simian HIV-induced PAH in macaques and in HIV-induced PAH in man is now evident. Additionally, monocrotaline and hypoxia are known to disrupt the function of Golgi tethers, SNAREs, SNAPs, and N-ethylmaleimide-sensitive factor ("the Golgi blockade hypothesis"). These results, along with recent reports demonstrating the trapping of PAH-associated human BMPR2 mutants in the Golgi, highlight the implications of disrupted intracellular membrane trafficking in the pathobiology of PAH. The purpose of this review is to present a brief overview of the molecular basis of intracellular trafficking and relate these considerations to the pathobiology of PAH.

Figure 1.

Tethers, SNAREs, SNAPs, and NSF in vesicular trafficking. (A) The SNARE cycle in membrane fusion. Initial interaction between a vesicle and its target membrane is mediated by cognate tethers. Membrane fusion is then mediated by the formation of a quaternary-α-helical trans-SNARE complex consisting of one v- (or R-) SNARE on the vesicle and two or three t- (or Q-) SNAREs on the target membrane. After membrane fusion, the cis-SNARE complex is disassembled by the ATPase NSF which is recruited to the cis-SNARE complex from the cytosol by α-SNAP. (B) Classification of SNAREs based on location (on a vesicle [v-] or a target [t-] membrane) or functional amino-acid residue (arginine [R-] or glutamine [Q-]) in their respective SNARE-motifs.

Figure 2.

Dysfunctional intracellular trafficking in the pathobiology of PAH. (A) Productive transcriptional signaling from the plasma membrane to the nucleus along the BMP/Smad 1/5, TGFβ/Smad2, and IL-6/PY-STAT-3 signaling pathways is membrane-associated. IL-6/STAT3 and ERK1/2 signaling is inversely related to loss of caveolar/raft cav-1. (B) Golgi blockade mechanisms in PAH. MCTP and hypoxia lead to a trapping of vesicle tethers, SNAREs, and SNAPs in the Golgi of affected pulmonary arterial endothelial cells. This leads to a block in anterograde trafficking of vasorelevant cargo proteins such as cav-1 and eNOS and reduced caveolar NO production. The intracellularly sequestered eNOS produces NO, which may potentially S-nitrosylate cysteine-rich proteins like NSF, further inhibiting trafficking. Golgi-trapped dominant-negative BMPR2 mutants may also potentially block trafficking of cargo proteins to the plasma membrane.

Figure 3.

Dysfunctional Golgi tethers and SNAREs after hypoxia and monocrotaline. (A and B) Bovine PAEC in culture exposed to hypoxia (1.5% vol/vol) or MCTP (∼ 50 μM equivalent) for 4 d were immunostained for different Golgi tethers (GM130, p115, giantin, golgin 84) and SNAREs (GS28, syntaxin 6, Vti1a, SNAP23). Scale bars = 25 μM in A and 50 μM in B. Adapted from Refs. and with permission. (C) Live-cell caveolar imaging of NO using DAF-2DA in PAEC after 4 d of exposure to hypoxia or MCTP as in A and B. Arrowheads point to caveolar NO. Scale bar = 25 μM. Adapted from Ref. with permission. (D) Enhancement of Golgi tethers (arrowheads) in PAEC in rat lung 4 wk after MCT administration. Scale bars = 20 μM in a and 20 μM in b. Adapted from Ref. 35 with permission.