Human Pulmonary Arterial Cells Research Articles

The diverging roles of insulin-like growth factor binding proteins in pulmonary arterial hypertension

Pulmonary hypertension (PH) is a progressive, severe and to date not curable disease of the pulmonary vasculature. Alterations of the insulin-like growth factor 1 (IGF-1) system are known to play a role in vascular pathologies and IGF-binding proteins (IGFBPs) are important regulators of the bioavailability and function of IGFs. In this study, we show that circulating plasma levels of IGFBP-1, IGFBP-2 and IGFBP-3 are increased in idiopathic pulmonary arterial hypertension (IPAH) patients compared to healthy individuals. These binding proteins inhibit the IGF-1 induced IGF-1 receptor (IGF1R) phosphorylation and exhibit diverging effects on the IGF-1 induced signaling pathways in human pulmonary arterial cells (i.e. healthy as well as IPAH-hPASMCs, and healthy hPAECs). Furthermore, IGFBPs are differentially expressed in an experimental mouse model of PH. In hypoxic mouse lungs, IGFBP-1 mRNA expression is decreased whereas the mRNA for IGFBP-2 is increased. In contrast to IGFBP-1, IGFBP-2 shows vaso-constrictive properties in the murine pulmonary vasculature. Our analyses show that IGFBP-1 and IGFBP-2 exhibit diverging effects on IGF-1 signaling and display a unique IGF1R-independent kinase activation pattern in human pulmonary arterial smooth muscle cells (hPASMCs), which represent a major contributor of PAH pathobiology. Furthermore, we could show that IGFBP-2, in contrast to IGFBP-1, induces epidermal growth factor receptor (EGFR) signaling, Stat-3 activation and expression of Stat-3 target genes. Based on our results, we conclude that the IGFBP family, especially IGFBP-1, IGFBP-2 and IGFBP-3, are deregulated in PAH, that they affect IGF signaling and thereby regulate the cellular phenotype in PH.

Abstract 13336: Rasa3 Affects Key Proliferation and Angiogenic Pathways in and Around Pulmonary Artery Endothelial Cells

Introduction: Despite improvements in quality of life and functional status, pulmonary hypertension (PH) remains a highly morbid disease with poor life expectancy with no targeted treatments available. RASA3 is a ubiquitously expressed GTPase activating protein that is integral to angiogenesis and barrier function in endothelial cells (EC). In this study, we sought to explore the differential expression of RASA3 in response to PH-relevant stimuli and downstream markers of EC health when RASA3 expression is lost. Methods: Human pulmonary artery cells (PAECs) from Lonza were grown in culture and treated with 1% hypoxia, increasing concentrations of TNF-α for 24 hours or IL-1β for 48 hours and then harvested. Cells were transfected with siRNA directed against RASA3 or non-targeting control and harvested to evaluate downstream markers of EC health, or plated on Matrigel to evaluate angiogenic capabilities. Non-contact co-culture experiments were performed using human pulmonary artery smooth muscle cells (PASMC) with PAECs that were transfected with siRNA against RASA3 or non-targeting control. Results: We found that when untreated PAECs were exposed to 1% hypoxia for up to 96 hours, RASA3 expression was significantly decreased (p=0.046). When treated with increasing concentrations of TNF-α for 24 hours, RASA3 expression decreased (p=0.0002). When PAECs were treated with IL-1β 10ng/mL for 48 hours, RASA3 expression significantly increased (p=0.001). Silenced RASA3 PAECs had significantly less PCNA (p=0.0474), a trend towards increased HIF-2α expression (p=0.08) and increased VEGFR2 expression (p=0.0878). Cells that had silenced RASA3 expression when plated on Matrigel were significantly less able to form rings compared to non-targeting control (p=0.0152). PASMCs that had been co-cultured with silenced RASA3 had significantly increased PCNA compared to PASMCs co-cultured with non-targeting control PAECs (p=0.0467). Conclusions: RASA3 is essential to PAEC homeostasis, and loss of RASA3 affects endothelial cell health as well as affects its local environment. Further studies are ongoing to determine how loss of RASA3 contributes to the development of PH.

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells.

Simple SummaryThe Jagged family of ligands are aberrantly expressed during multiple myeloma progression and contributes to activate Notch signaling both in myeloma cells and in the nearby bone marrow cell populations activating several pro-tumor effects. This work elucidates, in vitro, in vivo as well as in patients’ bone marrow biopsies, different mechanisms by which tumor cell-derived Jagged1 and 2 contribute to myeloma-associated angiogenesis. These include the ability to induce myeloma and bone marrow stromal cell secretion of VEGF along with a direct activation of the pro-angiogenic Notch signaling pathway in endothelial cells. This research provides a rational for a Jagged-directed therapy in multiple myeloma.Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1–2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines’ ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients’ BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands.

Factors Associated with Heritable Pulmonary Arterial Hypertension Exert Convergent Actions on the miR-130/301-Vascular Matrix Feedback Loop.

Pulmonary arterial hypertension (PAH) is characterized by occlusion of lung arterioles, leading to marked increases in pulmonary vascular resistance. Although heritable forms of PAH are known to be driven by genetic mutations that share some commonality of function, the extent to which these effectors converge to regulate shared processes in this disease is unknown. We have causally connected extracellular matrix (ECM) remodeling and mechanotransduction to the miR-130/301 family in a feedback loop that drives vascular activation and downstream PAH. However, the molecular interconnections between factors genetically associated with PAH and this mechano-driven feedback loop remain undefined. We performed systematic manipulation of matrix stiffness, the miR-130/301 family, and factors genetically associated with PAH in primary human pulmonary arterial cells and assessed downstream and reciprocal consequences on their expression. We found that a network of factors linked to heritable PAH converges upon the matrix stiffening-miR-130/301-PPARγ-LRP8 axis in order to remodel the ECM. Furthermore, we leveraged a computational network biology approach to predict a number of additional molecular circuits functionally linking this axis to the ECM. These results demonstrate that multiple genes associated with heritable PAH converge to control the miR-130/301 circuit, triggering a self-amplifying feedback process central to pulmonary vascular stiffening and disease.

Effect of endogenous sulfur dioxide on the apoptosis induced by cobalt chloride in the human pulmonary arterial endothelial cells

Objective To explore the effect of endogenous sulfur dioxide (SO2) on the apoptosis induced by cobalt chloride (CoCl2) in the human pulmonary arterial endothelial cells (HPAECs). Methods CoCl2 was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1), and the key enzyme generating endogenous SO2 were over-expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups: vehicle group, vehicle+ CoCl2 group, AAT1 group and AAT1+ CoCl2 group.The expressions of AAT1, B-cell lymphoma-2 (bcl-2), bcl-associated X protein (bax), Caspase-3 and activated Caspase-3 (cleaved Caspase-3) in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Results There were significant differences in the endogenous SO2 content, the expre-ssions of AAT1 and bcl-2, and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F=147.364, 23.738, 6.521, 64.884, all P 0.05). Compared with vehicle group, AAT activity [(0.96±0.24) Carmen′s unit/μg vs.(2.21±0.60) Carmen′s unit/μg], endogenous SO2 content (40.71±7.72 vs.105.60±16.20) and bcl-2 expression (0.59±0.19 vs.1.02±0.20) in the HPAECs of vehicle+ CoCl2 group were significantly decreased, while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56±0.25 vs.0.95±0.13) were significantly increased (all P 0.05). The SO2 content (351.50±42.43 vs.105.60±16.20) and AAT1 expression (1.22±0.33 vs.0.53±0.11) in the HPAECs of AAT1 group were higher than those of vehicle group (all P 0.05). Moreover, no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51±0.17 vs.0.50±0.11) between the two AAT1-overexpressed groups (all P>0.05). Conclusion Endo-genous SO2 inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2. Key words: Sulfur dioxide; Aspartate aminotransferase 1; Cobalt chloride; Human pulmonary arterial endothelial cells; Apoptosis

Effect of emodin on the proliferation and migration of hypoxia pulmonary artery smooth muscle cells

Objective To observe the inhibitory effect of emodin on the proliferation and migration of pulmonary artery smooth muscle cells. Methods Primary cultured human pulmonary arterial smooth muscle cells (HPASMC) were cultured in a routine manner to establish an hypoxic cell model. Emodin at different concentrations (0, 5, 10, 15 and 20 μmol/L) 48 and 72 h later, the proliferation and migration of hypoxic pulmonary artery were detected by methyl thiazol tetrazolium (MTT) assay and Transwell invasion assay. Western blotting assay was used to determine the expression of p38 protein in hypoxia HPASMC induced by emodin. Results Emodin inhibited the proliferation and migration of hypoxic HPASMC in a time-and concentration-dependent manner. When the concentration was 20 μmol/L, the effect was the most significant, and the inhibitory rates at 12, 24, 48 and 72 h were (53.46±1.81)%, (61.32±2.69)%, (76.34±3.05)% and (84.43±3.18)%, respectively. (68.90±8.49), (46.10±5.63), (27.56±5.38) in the Transwell experimental group (5, 10, 15 and 20 μmol/[(12.61±3.68)] compared with the control group [(88.59±8.56)]. Compared with the control group, the experimental group p38 protein expression was reduced (P=0.001). Conclusion Emodin can significantly inhibit the proliferation and migration of human pulmonary artery smooth muscle cells under hypoxic conditions and down-regulate the expression of p38 protein. Key words: Emodin; Pulmonary artery smooth muscle cells; Proliferation; Migration

Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery Cells: Implications in Pulmonary Arterial Hypertension.

Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16α-hydroxyestrone (16αOHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)-induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen-Nox-dependent processes was studied in female Nox1(-/-) and Nox4(-/-) mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16αOHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-α, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16αOHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16αOHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid-related factor 2 activity and expression of nuclear factor erythroid-related factor 2-regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1(-/-) but not Nox4(-/-) mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1(-/-) mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid-related factor 2 whereby 16αOHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH, particularly important in women.

Histone Acetylation Regulates the Cell-Specific and Interferon-γ–Inducible Expression of Extracellular Superoxide Dismutase in Human Pulmonary Arteries

Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme present in the vascular wall, and is responsible for both the protection of vessels from oxidative stress and for the modulation of vascular tone. Concentrations of EC-SOD in human pulmonary arteries are very high relative to other tissues, and the expression of EC-SOD appears highly restricted to smooth muscle. The molecular basis for this smooth muscle-specific expression of EC-SOD is not known. Here we assessed the role of epigenetic factors in regulating the cell-specific and IFN-γ-inducible expression of EC-SOD in human pulmonary artery cells. The analysis of CpG site methylation within the promoter and coding regions of the EC-SOD gene demonstrated higher levels of DNA methylation within the distal promoter region in endothelial cells compared with smooth muscle cells. Exposure of both cell types to DNA demethylation agents reactivated the transcription of EC-SOD in endothelial cells alone. However, exposure to the histone deacetylase inhibitor trichostatin A (TSA) significantly induced EC-SOD gene expression in both endothelial cells and smooth muscle cells. Concentrations of EC-SOD mRNA were also induced up to 45-fold by IFN-γ in smooth muscle cells, but not in endothelial cells. The IFN-γ-dependent expression of EC-SOD was regulated by the Janus tyrosine kinase/signal transducers and activators of transcription proteins signaling pathway. Simultaneous exposure to TSA and IFN-γ produced a synergistic effect on the induction of EC-SOD gene expression, but only in endothelial cells. These findings provide strong evidence that EC-SOD cell-specific and IFN-γ-inducible expression in pulmonary artery cells is regulated, to a major degree, by epigenetic mechanisms that include histone acetylation and DNA methylation.

S154 Is there a role for IL-33 in the pathogenesis of pulmonary arterial hypertension?

Introduction and objectivesIL-33 is a 31KDa cytokine which is a member of the IL-1 family. It resides in the nucleus of endothelial and epithelial cells as a chromatin-associated factor in...

DHEA decreases HIF-1α accumulation under hypoxia in human pulmonary artery cells: Potential role in the treatment of pulmonary arterial hypertension

Previous work showed that dehydroepiandrosterone (DHEA) prevents and reverses chronic hypoxic pulmonary artery hypertension in rat via targeting smooth muscle cells. In our study, DHEA was tested on human pulmonary arterial smooth muscle cells (HPASMC) to identify its mechanism of action under hypoxia in vitro. We show that DHEA decreased HIF-1α accumulation under both “chemical hypoxia” with treatment by the iron chelator deferroxamin and gas hypoxia (1% O 2). The mRNA levels of HIF-1α were unchanged whether or not DHEA was applied under chemical and gas hypoxia, as compared to controls in normoxia, suggesting a post-transcriptional effect of the steroid. Protein levels of prolyl hydroxylases responsible for HIF-1α degradation were not modified by DHEA treatment. In addition, a synthetic derivative of DHEA, 3β-methyl-Δ5-androsten-17-one (which cannot be metabolized), was as active as DHEA on HIF-1α accumulation, as well as testosterone and 17β-estradiol (E 2). In HPASMC cultures under normoxia and both types of hypoxia, DHEA gave rise to Δ5-androstene-3β,17β-diol (ADIOL) and DHEA-sulfate (DHEA-S). Neither testosterone, nor E 2 were found. In addition, ADIOL, DHEA-S, 7α-hydroxy-DHEA and Δ4-androstene-3,17-dione were ineffective on HIF-1α accumulation. The effect of DHEA per se reducing HIF-1α accumulation may be relevant to reduced hypoxia effects in pulmonary arterial hypertension.

Antiproliferative Effects of Phosphodiesterase Type 5 Inhibition in Human Pulmonary Artery Cells

Phosphodiesterase Type 5 (PDE5) inhibition represents a novel strategy for the treatment of pulmonary hypertension. Our aim was to establish the distribution of PDE5 in the pulmonary vasculature and effects of PDE5 inhibition on pulmonary artery smooth muscle cells (PASMCs). PDE5 expression was examined by immunohistochemistry and Western blotting, in both normal and hypertensive lung tissues. DNA synthesis, proliferation, PDE activity, and apoptosis were measured in distal human PASMCs treated with soluble guanylyl cyclase activators (nitric oxide donors and BAY41-2272) and sildenafil. Cells containing PDE5 and alpha-smooth muscle actin occurred throughout the pulmonary vasculature, including obstructive intimal lesions. Three molecular forms of PDE5 were identified and protein expression was greater in hypertensive than control lung tissue. Most cyclic guanosine monophosphate hydrolysis (about 80%) in cultured cells was attributed to PDE5. Sildenafil induced a greater elevation of intracellular cyclic guanosine monophosphate levels compared with nitric oxide donors and BAY41-2272 (about 10-fold versus about 2-fold) and cotreatment had a synergistic effect, increasing cyclic nucleotide levels up to 50-fold. Dual stimulation of soluble guanylyl cyclase and inhibition of PDE5 activities also had significant downstream effects, increasing phosphorylation of vasodilator-stimulated phosphoprotein, reducing DNA synthesis and cell proliferation, and stimulating apoptosis, and these effects were mimicked by cyclic guanosine monophosphate analogs. Phosphodiesterase Type 5 is the main factor regulating cyclic guanosine monophosphate hydrolysis and downstream signaling in human PASMCs. The antiproliferative effects of this signaling pathway may be significant in the chronic treatment of pulmonary hypertension with PDE5 inhibitors such as sildenafil.