Angelia D. Lockett

Vascular remodeling leading to occlusion of the pulmonary artery and increased pulmonary vascular resistance is the central feature of Pulmonary Arterial Hypertension (PAH) which leads to death due to right heart failure. The expression of Nicotinamide phosphoribosyltransferase (Nampt) is increased in the lungs and isolated PAECs of PAH patients. Inhibition of NAMPT is protective in preclinical models of pulmonary hypertension. NAMPT regulates multiple mitochondrial processes that control cell proliferation and survival. Using rodent models of PH, we demonstrated that the mitochondrial unfolded protein response (UPR ^mt ) promotes vascular remodeling. Hence we, hypothesized that NAMPT activates the UPR ^mt to promote abnormal pulmonary arterial endothelial cell proliferation. Analysis of PAECs isolated from PAH patients show increased expression of the UPR ^mt pathway mediators, ATF-5, mtHSP70 and ClpP. Human PAEC cell lines were exposed to recombinant Nampt or transduced with lentiviral-Nampt. We observed increased phosphorylation and activation of eIF2α which permits preferential translation of the ATF-5 transcription factor. ATF-5 expression and nuclear localization was increased. Further, we observed increased expression of ATF-5 target genes, mtHSP70, HSP60, ClpP and LonP1 as well as increased PAEC proliferation. Blocking mtHSP70 function reversed the Nampt induced increase in proliferation. Neither UPR ^mt activation nor proliferation was increased in the presence of enzymatically inactive-Nampt. Our observations demonstrate that Nampt can promote PAEC proliferation by activating the UPR ^mt and uncovers novel potential targets to address vascular remodeling in PAH.

Proliferation and vasoconstriction of the intimal smooth muscle layer of the pulmonary artery are pathogenic characteristics of pulmonary arterial hypertension (PAH). Altered mitochondrial function, i.e. glycolysis, ROS generation and fission, are known potentiators of vascular remodeling. However, most current therapeutic interventions fail to effectively address the proliferation of the pulmonary artery smooth muscle cells (PASMCSs) lining the pulmonary vasculature and highlight the importance of identifying novel pathways to target for intervention. The Sphk1/S1P/S1P2 signaling axis is upregulated in PAH patients and is known to induce PASMC proliferation during hypoxia-mediated pulmonary hypertension (HPH). Interestingly, Sphk1 modulates mitochondrial function in that it regulates dynamics, cell growth and survival and in C. elegans , it induces activation of the unfolded protein response (UPR ^mt ). We aimed to investigate if the Sphk1/S1P/S1P2 signaling axis promotes vascular remodeling in PAH via activation of the UPR ^mt . PASMCs isolated from IPAH patients were subjected to RNAseq analysis. The effect of Sphk1 or S1P was interrogated in hPASMC cell lines and the HPH model was used to assess the effect of the UPR ^mt on PAH pathogenesis. RNAseq analysis revealed that pathways involved in mitochondrial respiration were among the top 20 most significantly regulated pathways. Furthermore, ATF-5, the transcription factor that mediates the UPR ^mt was significantly upregulated. In hPASMCs, Sphk1/S1P lead to decreased respiration, increased glycolysis, fission, ROS and UPR ^mt activation. Pharmacological inhibition of the UPR ^mt mediator, mtHSP70, mitigated the Sphk1 induced increase in hPASMC proliferation. Furthermore, mtHSP70 inhibition was protective in hypoxia-mediated pulmonary hypertension (HPH) as we observed a decrease in right ventricular systolic pressure, right ventricular hypertrophy and vascular remodeling. These data suggest that the UPR ^mt promotes vascular remodeling in PAH and may present a novel pathway to target for pharmaceutical intervention.

Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1^f/f TaglnCre⁺) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1^f/f TaglnCre⁺ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by (mal)adaptive remodeling of the pulmonary vasculature, which is associated with inflammation, fibrosis, thrombosis, and neovascularization. Vascular remodeling in PAH is associated with cellular metabolic and inflammatory reprogramming that induce profound endothelial and smooth muscle cell phenotypic changes. Multiple signaling pathways and regulatory loops act on metabolic and inflammatory mediators which influence cellular behavior and trigger pulmonary vascular remodeling in vivo. This review discusses the role of bioenergetic and inflammatory impairments in PAH development.

The aberrant proliferation of pulmonary artery smooth muscle (PASMCs) cells is a defining characteristic of pulmonary arterial hypertension (PAH) and leads to increased vascular resistance, elevated pulmonary pressure, and right heart failure. The sphingosine kinase 1 (SPHK1)/sphingosine-1 phosphate/sphingosine-1 phosphate receptor 2 pathway promotes vascular remodeling and induces PAH. The aim of this study was to identify genes and cellular processes that are modulated by over-expression of SPHK1 in human PASMCs (hPASMCs). RNA was purified and submitted for RNA sequencing to identify differentially expressed genes. Using a corrected p-value threshold of <0.05, there were 294 genes significantly up-regulated while 179 were significantly down-regulated. Predicted effects of these differentially expressed genes were evaluated using the freeware tool Enrichr to assess general gene set over-representation (enrichment) and ingenuity pathway analysis (IPA™) for upstream regulator predictions. We found a strong change in genes that regulated the cellular immune response. IL6, STAT1, and PARP9 were elevated in response to SPHK1 over-expression in hPASMCs. The gene set enrichment mapped to a few immune-modulatory signaling networks, including IFNG. Furthermore, PARP9 and STAT1 protein were elevated in primary hPASMCs isolated from PAH patients. In conclusion, these data suggest a role of Sphk1 regulates pulmonary vascular immune response in PAH.

Protective levels of Alpha-1 antitrypsin (A1AT) are achieved in the lung through the uptake of the pulmonary vasculature of hepatocyte-secreted A1AT. The anti-inflammatory, anti-apoptotic, and anti-protease properties of A1AT are critical toward maintaining the function of pulmonary endothelial and epithelial cells and for the structural integrity of the pulmonary interstitium. To perform these functions A1AT must cross the pulmonary-endothelial barrier. Using transwell inserts, we have demonstrated that the endocytosis of A1AT at the apical surface of endothelial cells, followed by the transcytosis and secretion at the basolateral surface, is a mechanism through which A1AT is transported into the lung epithelium and interstitium.

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30–50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.