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. 2011 Jul-Sep;1(3):405–418. doi: 10.4103/2045-8932.87310

Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells

Weijuan Yao 1,†, Wenbo Mu 2,†, Amy Zeifman 3, Michelle Lofti 4, Carmelle V Remillard 1, Ayako Makino 1,3, David L Perkins 1, Joe G Garcia 3,5, Jason X J Yuan 1,3,5,6, Wei Zhang 7,8,✉
PMCID: PMC3224433  PMID: 22140631

Abstract

Fenfluramine is prescribed either alone or in combination with phentermine as part of Fen-Phen, an anti-obesity medication. Fenfluramine was withdrawn from the US market in 1997 due to reports of heart valvular disease, pulmonary arterial hypertension, and cardiac fibrosis. Particularly, idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension (PPH), was found to be associated with the use of Fen-Phen, fenfluramine, and fenfluramine derivatives. The underlying mechanism of fenfluramine-associated pulmonary hypertension is still largely unknown. We reasoned that investigating drug-induced gene dysregulation would enhance our understanding of the fenfluramine-associated pathogenic mechanism of IPAH. Whole-genome gene expression profiles in fenfluramine-treated human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells (isolated from normal subjects) were compared with baseline expression in untreated cells. Fenfluramine treatment caused dysregulation in a substantial number of genes involved in a variety of pathways and biological processes. In addition to several common pathways and biological processes such as “MAPK signaling pathway,” “inflammation response,” and “calcium signaling pathway” shared between both cell types, pathways and biological processes such as “blood circulation,” “muscle system process,” and “immune response” were enriched among the dysregulated genes in PASMC. Pathways and biological processes such as those related to cell cycle, however, were enriched among the dysregulated genes in PAEC, indicating that fenfluramine could affect unique pathways (or differentially) in different types of pulmonary artery cells. While awaiting validation in a larger cohort, these results strongly suggested that fenfluramine could induce significant dysregulation of genes in multiple biological processes and pathways critical for normal pulmonary vascular functions and structure. The transcriptional and posttranscriptional changes in these genes may, therefore, contribute to the pathogenesis of fenfluramine-associated IPAH.

Keywords: anorexigen, gene expression profile, lysosome, mitochondria, pulmonary hypertension

INTRODUCTION

Fenfluramine (Fen, 3-trifluoromethyl-N-ethylamphetamine), a drug in the class of anorectics (appetite suppressants), has been prescribed either alone or in combination with phentermine (Phen) as part of Fen-Phen, an anti-obesity medication. The drug was withdrawn from the U.S. market in 1997 due to reports of valvular heart disease and pulmonary hypertension, including a condition known as cardiac fibrosis. Though the magnitude and prevalence of their deleterious cardiopulmonary effects remain undetermined, the links between these anorectics and valvular heart disease and pulmonary hypertension are clearly established.[1] For example, idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension (PPH), was found to be associated with the use of Fen-Phen, fenfluramine, and fenfluramine derivatives.[2–6] Fatal cases of pulmonary hypertension and valvular heart disease have been reported to be associated with even short-term fenfluramine usage.[2,7] Furthermore, based on a study of more than 5,700 former fenfluramine users, damage to the heart valve (e.g., regurgitant valvulopathy) continued long after discontinuing use of the medication.[8] Previous studies suggested that nitric oxide deficiency could predispose affected individuals to develop anorexigen-associated pulmonary hypertension.[9] In addition, Fen and its active metabolite norfenfluramine were found to, indirectly, activate mitogenic serotonin 2B (5-HT2B) receptors,[10,11] thus potentially leading to the valvular abnormalities (e.g., abnormal valve cell division) found in patients taking fenfluramine.[12] Particularly, in cells expressing recombinant 5-HT2B receptors, norfenfluramine potently stimulates the hydrolysis of inositol phosphates, increases intracellular Ca2+, and activates the mitogen-activated protein kinase (MAPK) cascade, the latter of which has been linked to mitogenic actions of the 5-HT2B receptors.[13]

The underlying cellular and molecular mechanism of fenfluramine-associated pulmonary hypertension is still largely unknown. Quantitative gene expression is an important intermediate phenotype that situates in the middle of DNA sequence variation, environmental influences (e.g., exposure to drugs) and other cellular/whole-body phenotypes/traits[14–17] including the susceptibility to complex diseases (e.g., IPAH). For example, a previous genomic study of the expression profiles in peripheral blood mononuclear cells from patients with pulmonary arterial hypertension and normal individuals demonstrated a significant number of dysregulated genes between the patient cohort and normal individuals, as well as between patients with IPAH and secondary pulmonary hypertension.[18] Distinct gene signatures derived from lung tissues in IPAH and secondary pulmonary hypertension patients were also identified based on genome-wide expression profiling.[19] Therefore, we reasoned that a comprehensive examination of fenfluramine-induced gene dysregulation (upregulation or downregulation of mRNAs after treatment with fenfluramine) in relevant normal lung tissues, i.e., pulmonary artery smooth muscle cells (PASMC) and pulmonary artery endothelial cells (PAEC), could potentially help shed light on the molecular pathogenesis of fenfluramine-associated pulmonary hypertension.

In order to examine the fenfluramine-associated gene dysregulation, we profiled transcriptional (mRNA) expression using a whole-genome cDNA array (covering 41,000 unique human transcripts with public domain annotations) in human PASMC and PAEC samples derived from normal individuals. We compared gene expression profiles in the PASMC and PAEC samples after treatment with fenfluramine and their baseline expression profiles. Known pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG),[20,21] Gene Ontology (GO)[22] categories (i.e., biological processes, molecular function, cellular components), and gene networks were further evaluated among the dysregulated genes between cells treated and untreated with fenfluramine. Our findings suggest that fenfluramine may contribute to the pathogenesis of IPAH through causing differential expression of genes in certain pathways and biological processes that are critical to the normal functions of pulmonary arteries.

MATERIALS AND METHODS

Cell culture, drug treatment and cell morphological experiments

Normal human PASMC and PAEC (3 samples for each cell type) were purchased from Lonza (Walkersville, Md.) and maintained in cell growth medium supplemented with 10% fetal bovine serum (FBS) and growth factors. Two days before the treatment, 5 × 105 cells were seeded in a 10-cm plate. The cell growth medium was replaced by the medium with 200 μM fenfluramine based on a previous publication,[23] and culturing was continued for 72 hrs. For cell morphological experiments, the cells were stained with the membrane-permeable nucleic acid stain, 4’, 6’-diamidino-2-phenylindole (DAPI, 5 μM). The blue fluorescence emitted at 461 nm was used to visualize the cell nuclei. The smooth muscle α-actin antibody was used to evaluate expression of α-actin in DAPI-stained cells.

Mitochondrial and lysosome imaging

Normal human PASMC and PAEC samples were cultured in medium supplemented with 10% FBS and growth factors. The cover slips with cells were mounted on glass slides and phase contrast images of cells were taken using an Olympus microscopy system. For mitochondrial staining, the media in 6-well plate were removed and pre-warmed medium containing MitoTracker Green FM (100 nM, Invitrogen, Carlsbad, Calif.) was added. The cells were incubated in the incubator for 15 min. The staining solution was removed and cells were rinsed with pre-warmed medium, and cells were visualized with a 100× objective on a fluorescent microscope (Nikon, Japan) coupled to the Solamere Imaging System. For lysosome staining, the media in 6-well plate were removed and a staining solution containing LysoTracker Red (50 nM, Invitrogen, Carlsbad, Calif.) was used. The cells were incubated in the incubator for 30 min. before the staining solution was removed. The cells were then rinsed with pre-warmed medium and observed with a 20× objective on an Olympus microscope. Fluorescent images and phase contrast images, which were taken in the same field, were overlaid using the software included in the Olympus fluorescent imaging system.

RNA isolation and microarray hybridization

Total RNAs from treated and untreated PASMC and PAEC samples were isolated using standard molecular biology protocols. High-quality RNA samples with no signs of DNA contamination and RNA degradation were hybridized on the Agilent Whole Human Genome 4΄44K Gene Expression Two-color arrays (Agilent Technologies, Santa Clara, Calif.), which contain 41,000 unique human transcripts (targeting 19,596 Entrez Gene RNAs) supported by public sources including RefSeq,[24] Golden Path Ensembl UniGene Human Genome (Build 33) and GenBank (http://www.ncbi.nlm.nih.gov/genbank/) databases, according to the manufacturer's recommended protocol at the UCSD Microarray Core Facility. Two PAEC samples did not pass the quality control of array hybridization. Therefore, in total, 4 samples (3 PASMC and 1 PAEC samples) were included in further analyses.

Microarray data preprocessing

The raw expression data were normalized and summarized with the robust multi-array average (RMA)[25] algorithm using GeneSpring GX v10 (Agilent Technologies, Santa Clara, Calif.). Since the ratio of the 2-color channels is most informative when the intensities are well over background for both the cy3 and cy5 channels, we removed those probesets if both the cy3 and cy5 channels had intensities in the lower quartile across all of the 4 samples. However, a gene could be interesting from a biological point of view, even if it has a meaningless ratio (e.g., a gene expressed in only 1 channel). Therefore, we included those genes with intensities above the cutoff (i.e., lower quartile) in either cy3 or cy5 channel. Only transcripts with unique, unambiguous gene annotations according to the manufacturer's information (retrieved from the Agilent eArray website at http://earray.chem.agilent.com/earray/) were analyzed. In total, ~18,000 gene-level transcripts were included in the final analysis set.

Identification of dysregulated genes

Differentially expressed genes between fenfluramine-treated samples and untreated controls were identified based on a series of fold-changes (e.g., 1.2, 1.5 and 2.0) in the 3 PASMC samples. Due to the exploratory nature of this study and the small sample size, our choices of statistical approaches were limited, so any genes meeting a cutoff (e.g., fold-change>1.5) in at least 2 PASMC samples were considered differentially expressed. For the 1 PAEC sample, a single relatively stringent cutoff (i.e., fold-change>2) was used to control false positives. The expression patterns (i.e., upregulation or downregulation of genes) were also compared between the two cell types.

Western blot validation of microarray data

Three dysregulated genes that met fold-change>1.5: MMP1 (encoding metallopepetidase 1) for PAEC, CYCS (encoding cytochrome C, somatic) and VIM (encoding vimentin) for PASMC were selected for Western blot validation. Cells were washed with ice-cold PBS, suspended in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 μg/ml phenylmethylsulfonyl fluoride, phosphatase inhibitors, and protease inhibitors), and incubated for 30 min. on ice. The cell lysates were then sonicated and centrifuged at 12,000 rpm for 10 min., and the supernatant was collected. Protein concentrations were determined by DC™ Protein Assay (Bio-Rad Laboratories, Hercules, Calif.) using BSA as a standard. Samples were applied on SDS-PAGE (4–20%), and proteins were transferred onto nitrocellulose membranes by electroblot. Membranes were blocked in 5% nonfat milk and incubated overnight at 4°C with primary antibodies and then with secondary antibodies. Blots were developed with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, Ill.).

Gene ontology and pathway analyses

We further searched the KEGG[20,21] and GO[22] databases for any enriched physiological pathways or biological processes among the dysregulated genes relative to the final analysis set using the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/).[26,27] For pathway and GO analyses, genes dysregulated in at least one PASMC or PAEC were included. Significantly enriched pathways or biological processes were determined based on an adjusted P-value after the Benjamini-Horchberg (BH) procedure[28] (e.g., adjusted P-value<0.05) and the size of the gene sets (e.g., a minimum size of 5 or 10 hits). To obtain robust evaluation of enrichment patterns, we focused on those enriched pathways or biological processes across different cutoffs of differential expression.

Gene network analysis

We used the the Cytoscape (http://www.cytoscape.org/)[29] plug-in from the Reactome (http://www.reactome.org/)[30,31] to find gene networks among the fenfluramine-induced dysregulated genes. This plug-in accesses the Reactome Functional Interaction (FI) network, a highly reliable, manually curated pathway-based protein functional interaction (e.g., activation, inhibition) network covering close to 50% of human proteins.[30,31] We also evaluated the relative importance of the interacting genes based on betweenness centrality, a measure of a node's (i.e., a gene's) centrality in a network.

RESULTS

Cell and mitochondrial morphologies after the treatment of fenfluramine

Cell morphologies of untreated controls and fenfluramine-treated PASMC and PAEC were evaluated. In untreated PASMC and PAEC, the phase contrast images showed that the cells appeared to be flat and have a smooth surface in the cytosplasm, and no intracellular organelle structure could be seen in untreated cells (Fig. 1 left panels). Treatment of the cells with 200 μM of fenfluramine for 72 hrs. seemed to cause significant morphological changes. In fenfluramine-treated cells, the surface membrane of the cytoplasm became rough, while perinuclear organelles became strikingly swollen (Fig. 1, right panels). In addition, we observed significant changes in the mitochondrial morphology in PASMC treated with fenfluramine (Fig. 2). In untreated cells, photomicrographs showed typical images of mitochondria; the mitochondrial structure looked intact and distributed throughout the entire cytoplasm (Fig. 2 left panels). After treatment with fenfluramine, however, the mitochondrial structure was severely damaged; the volume and the tubular frequency were significantly decreased (Fig. 2 right panels). These data imply that fenfluramine treatment may induce mitochondrial fragmentation in PASMC.

Figure 1.

Figure 1

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Morphological changes in human PASMC and PAEC treated with fenfluramine. Phase contrast images of untreated PASMC (upper left panels) and PAEC (lower left panels), and fenfluramine-treated PASMC (upper right panels) and PAEC (lower right panels). The small images depict an enlarged area of a single cell from each of the multi-cell images for PASMC and PAEC.

Figure 2.

Figure 2

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Mitochondrial morphological changes in human PASMC treated with fenfluramine. Photomicrographs show images of mitochondria in untreated PASMC (left panels) and PASMC treated with 200 μM of fenfluramine for 72 hrs. The enlarged images at the bottom show the perinuclear area of an untreated PASMC (left) and a fenfluramine-treated PASMC.

Furthermore, we observed the changes in lysosomes in PAEC treated with fenfluramine. We stained the untreated and fenfluramine-treated PAEC with LysoTracker Red to label the lysosomes. In untreated cells, the fluorescence intensity from lysosomes was relatively low; however, in fenfluramine-treated cells, the fluorescence intensity of lysosomes was significantly increased (Fig. 3). By overlaying the phase contrast images with the LysoTracker Red-stained images, it seemed that the swollen perinuclear organelles in fenfluramine-treated PAEC were lysosomes.

Figure 3.

Figure 3

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The effect of fenfluramine on the lysosomes in human PAEC. Cells were treated with 200 mM fenfluramine for 72 hrs. and the lysosomes in the cells were stained with LysoTracker Red. The phase contrast images (upper panels) of the cells were overlaid with the fluorescent images of the lysosomes (in red, middle panels) to show the location of lysosomes in untreated and fenfluramine-treated PAEC (bottom panels). The LysoTracker Red-staining is mainly localized in the perinuclear area in fenfluramine-treated cells.

Genes dysregulated in fenfluramine-treated pulmonary artery cells

In total, 17,877 gene-level transcripts met our criteria to be included in the analysis set. These transcripts had expression intensities above the lower quartile in at least 1 channel, as well as unique, unambiguous gene annotations (excluding putative genes encoding hypothetical proteins) according to the manufacturer's information. Figure 4 shows a general characterization of the dysregulated genes in fenfluramine-treated PASMC and PAEC samples using different fold-change cutoffs. At fold-change>1.5, 881 genes were found to be dysregulated (497 upregulated and 384 downregulated) in the fenfluramine-treated PASMC samples; and 2,534 genes were found to be differentially expressed in the fenfluramine-treated PAEC sample (1,226 upregulated and 1,308 downregulated) (Fig. 4a). In contrast, at fold-change>2.0, there were 122 (74 upregulated and 48 downregulated) and 633 (285 upregulated and 348 downregulated) genes found to be dysregulated in the fenfluramine-treated PASMC and PAEC, respectively (Fig. 4a). Notably, there were more dyregulated genes with the same direction (e.g., upregulated in both cell types) in both PASMC and PAEC than those with different direction (e.g., upregulated in PASMC, but downregulated in PAEC) (Fig. 4b). At fold-change>2.0, there were 39 dysregulated genes (32 upregulated and 7 downregulated) in both PASMC and PAEC samples (Table 1). Figure 4c shows a heatmap of the 384 differential genes (i.e., dysregulated in at least 1 cell type) in both PASMC and PAEC. Particularly, the PASMC samples were clustered together, showing a similar dysregulation pattern relative to PAEC.

Figure 4.

Figure 4

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A general characterization of the genes dysregulated in PASMC and PAEC after fenfluramine treatment. (a) Numbers of genes that are affected by fenfluramine in PASMC and PAEC. The dysregulated genes are classified into five categories: expression level changed (fold-change>1.5) - “total changed,” upregulated - “all upregulated,” upregulated greater than 2-fold - “upregulated (>2),” downregulated - “all downregulated,” and downregulated greater than 2-fold - “downregulated (>2)” in PASMC (left) and PAEC (right). (b) Numbers of genes that are upregulated in both PASMC and PAEC, upregulated in PASMC but downregulated in PAEC, downregulated in PASMC but upregulated in PAEC, or downregulated in both PASMC and PAEC at different fold-changes (<1.5- fold, 1.5- to 2-fold, or >2-fold). (c) A heatmap of the 384 dysregulated genes (fold-change>2.0) in PASMC or PAEC.

Table 1.

Genes dysregulated in both PASMC and PAEC samples (fold-change>2.0)

graphic file with name PC-1-405-g005.jpg

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Western blot validation of microarray data

Three dysregulated genes (fold-change>1.5), MMP1, CYCS and VIM, after fenfluramine treatment were selected for Western blot validation in the same PASMC and PAEC samples. The gene dysregulation patterns for these 3 genes were recaptured by the Western blot experiments (Fig. 5a and b). Particularly, the mRNA expression of MMP1 in PAEC after fenfluramine treatment (our microarray data) was upregulated by approximately 200% (Fig. 5c), while the protein expression level was upregulated by 50% (Fig. 5b). The fenfluramine-mediated mRNA expression upregulation of CYCS and VIM, determined by the microarray data (Fig. 5c), was also consistent with the fenfluramine-mediated protein expression upregulation determined by Western blot analysis (Fig. 5b).

Figure 5.

Figure 5

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Western blot validation of microarray data. Validation of microarray expression data was performed using Western blot in PASMC and PEAC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. (a and b) Western blot analysis on matrix metallopeptidase 1 (MMP1) in untreated and fenfluramine-treated PAEC, and on somatic cytochrome c (CYCS) and vimentin in untreated and fenfluramine-treated PASMC. The data shown in B are mean±SE; *P<0.05 vs. control (Cont). (c) The fold-changes of mRNA expression levels of MMP1, CYCS and vimentin in untreated and fenfluramine-treated cells as indicated by the microarray data.

Enriched pathways and GO biological processes among the fenfluramine-induced dysregulated genes

We evaluated the enrichment of KEGG[20,21] pathways and GO[22] biological processes among the genes dysregulated in PASMC and PAEC samples after fenfuramine treatment. Figure 6compares the top 15 enriched pathways and biological processes (enrichment significant at adjusted P-value<0.001 after the BH procedure;[28] a minimum gene set size of 5 hits) between different cutoffs for differential expression (i.e., fold-change>1.5 and 2.0), as well as between both cell types. A significant number of enriched KEGG[20,21] pathways (Table 2) and GO[22] biological processes (Table 3) were identified using both fold-changes of 1.5 and 2.0 for differential expression. Notably, 14 KEGG[20,21] pathways (e.g., “MAPK signaling pathway,” “calcium signaling pathway,” “cell adhesion molecules”) (Table 2) and 7 GO[22] biological processes (e.g., “inflammatory response,” “response to organic substance,” “regulation of cell proliferation”) (Table 3) were enriched among the dysregulated genes in both PASMC and PAEC. Furthermore, some pathways and biological processes were also found to be enriched specifically among the dysregulated genes in either PASMC or PAEC. For example, the KEGG[20,21] pathways: “dilated cardiomyopathy” and “complement and coagulation cascades” (Table 2), as well as the GO[22] biological processes: “regulation of blood pressure” and “ muscle system process” (Table 3), were enriched among the dysregulated genes in PASMC. In contrast, the KEGG[20,21] pathways: “steroid biosynthesis” and “cell cycle” (Table 2), as well as the GO[22] biological process “cell proliferation” and “cell division” (Table 3), were enriched among the dysregulated genes in PAEC.

Figure 6.

Figure 6

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Enriched pathways and biological processes among the dysregulated genes. Top 15 enriched KEGG pathways and GO biological processes for the genes that are changed by fenfluramine in PASMC (a and c) and PAEC (b and d) at fold-change greater than 1.5 (a and b) or 2.0 (c and d).

Table 2.

Enriched KEGG pathways (FDR<0.001) among the dysregulated genes after fenfluramine treatment

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Table 3.

Enriched GO biological processes (FDR<0.001) among the dysregulated genes after fenfluramine treatment

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Gene network analysis

Some of the dysregulated genes in PASMC or PAEC after fenfluramine treatment were found to be connected with each other through certain FIs (e.g., activation, inhibition). Specifically, the Reactome[30,31] FI database was queried to identify FI relationships among the dysregulated genes after fenfluramine treatment in either PASMC (Fig. 7a) or PAEC (Fig. 8a). We also evaluated the relative importance of the genes in the identified FI networks based on betweenness centrality, a measure in graph theory for a node's (i.e., a gene's) centrality in a particular network (Fig. 7b for PASMC and Fig. 8b) for PAEC). Particularly, encoding bone morphogenetic protein receptor, type IB (BMPR1B) was located in the hub based on betweenness centrality in the networks comprised of the dysregulated genes in PASMC, while encoding repulsive guidance molecule A (RGMA) was found to be the most important gene based on betweenness centrality in the networks comprised of the dysregulated genes in PAEC (Figs. 7 and 8).

Figure 7.

Figure 7

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Gene networks comprised of the dysregulated genes in PASMC after fenfluramine treatment. A substantial proportion of the dysregulated genes after fenfluramine treatment (fold-change>2.0) are connected with each other through functional interactions. (a) A gene network comprised of the dysregulated genes in PASMC. Red: upregulated genes; Blue: downregulated genes. (b) A gene network comprised of the dysregulated genes in PASMC showing betweenness centrality, a measure for the relative importance of genes in a network. Green: low value; Red: high value; Orange/ Yellow: intermediate value. (c) Bone morphogenetic protein receptor, type IB (BMPR1B) is the hub based on betweenness centrality. AMHR2: Anti-Mullerian hormone receptor, type II; GDF10: Growth differentiation factor 10; STK35: Serine/threonine kinase 35; TLL2: Tolloid-like 2.

Figure 8.

Figure 8

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Gene networks comprised of the dysregulated genes in PAEC after fenfluramine treatment. A substantial proportion of the dysregulated genes after fenfluramine treatment (fold-change>2.0) can be connected with each other through functional interactions. (a) A gene network comprised of the dysregulated genes in PAEC. Red: upregulated genes; Blue: downregulated genes. (b) A gene network comprised of the dysregulated genes in PAEC showing betweenness centrality, a measure for the relative importance of genes in a network. Green: low value; Red: high value, Orange/Yellow: intermediate value. (c) Repulsive guidance molecule A (RGMA) is the hub based on betweenness centrality. CHRDL1: Chordin-like 1; ZFYVE16: Zinc finger, FYVE domain containing 16.

DISCUSSION

Fenfluramine-associated IPAH is likely a complex phenotype caused by multiple genetic and non-genetic factors, as well as by molecular changes after exposure to the drug. Elucidating the effects of this drug on relevant lung tissues (i.e., PASMC and PAEC) can potentially provide much-needed information on the underlying mechanism of fenfluramine-associated IPAH. In terms of cell and mitochondrial morphologies, there were obvious differences observed in PASMC and PAEC before and after treatment with fenfluramine (Figs. 1–3). These morphological data suggest that fenfluramine treatment not only results in mitochondrial damage (or fragmentation) in PASMC but also causes lysosome swelling in PAEC. However, it is unclear how these effects are related to the development of pulmonary hypertension.

We hypothesized that gene expression dysregulation after fenfluramine treatment in normal pulmonary artery tissues might contribute to the pathogenesis of fenfluramine-associated IPAH. Using a whole-genome cDNA microrray, a comprehensive evaluation of gene expression profiles in PASMC and PAEC samples showed a substantial number of dysregulated genes after fenfluramine treatment (Fig. 4), suggesting a wide range of molecular changes in lung tissues after fenfluramine exposure. For example, 122 and 633 genes were identified to be dysregulated for at least 2-fold in PASMC and PAEC, respectively (Fig. 4).

In addition to a number of genes that were dysregulated in both PASMC and PAEC (i.e., upregulated or downregulated in both cell types) (Table 1), the majority of dysregulated genes after fenfluramine treatment showed changes in either PASMC or PAEC only (Fig. 4b), thus potentially indicating a differential effect of fenfluramine on different cell types. Therefore, the microarray results suggested that with the exception of some commonly dysregulated genes in PASMC and PAEC, fenfluramine exposure could cause unique or differential changes in different cell types. Notably, some commonly and cell type-specifically dysregulated genes have been implicated in either pulmonary hypertension or related biological processes, demonstrating the relevance of these genes with fenfluramine-associated IPAH. For example, CECR1 (encoding cat eye syndrome chromosome region, candidate 1), which was upregulated (fold-change>2.0) in both PASMC and PAEC (Table 1), has been implicated in cat eye syndrome, a rare disease that features abnormal pulmonary venous return, potentially leading to pulmonary hypertension.[32] PPP1R12 (encoding protein phosphatase 1, regulatory [inhibitor] subunit 12B), an upregulated (fold-change>2.0) gene in both PASMC and PAEC after fenfluramine treatment (Table 1), is a myosin phosphatase (also known as MYPT2) that regulates muscle contraction, thus potentially affecting blood pressure.[33] In addition, mutations in BMP family including BMPR1B, an upregulated (fold-change>2.0) gene after fenfluramine treatment in PASMC, have been associated with IPAH.[34,35] In contrast, MYLK (encoding myosin, light chain kinase, transcript variant 1), an upregulated gene (fold-change>2.0) in PAEC, has been implicated in endothelial cell contraction and barrier dysfunction.[36–38]

We further examined whether these dysregulated genes were enriched in any biological processes or known physiological pathways, which could help elucidate the underlying mechanism of fenfluramine-associated pathogenesis of IPAH. Similar to the comparison of gene dysregulation, a number of common KEGG[20,21] pathways and GO[22] biological processes were identified among the genes dysregulated in PASMC and PAEC samples, respectively, in addition to other cell type-specific pathways and biological processes (Fig. 4, Tables 2 and 3). Notably, the enriched pathways (Table 2) and biological processes (Table 3) were robust across different cutoffs for differential expression (i.e., fold-change>1.5 and 2.0). Particularly, the dysregulated genes in PASMC samples were found to be enriched in a number of GO[22] biological processes including “immune response,” “defense response,” “blood circulation,” and “circulatory system process,” as well as processes related to cell adhesion and cell migration (Table 3). Similarly, a number of KEGG[20,21] pathways were enriched specifically among the dysregulated genes in PASMC, including “dilated cardiomyopathy,” and a number of signaling pathways such as “Toll-like receptor signaling pathway,” and “JAK-STAT signaling pathway” (Table 2). Among the dysregulated genes in PAEC, however, there were enriched pathways and biological processes including “mitotic cell cycle,” “regulation of phosphorylation,” and “TGF-β signaling pathway” (Tables 2 and 3). In addition, there were several pathways and biological processes, including “inflammatory response,” “PPAR signaling pathway,” “MAPK signaling pathway,” and “calcium signaling pathway,” that were enriched among the dysregulated genes in both PASMC and PAEC (Tables 2 and 3). Clearly, some of these enriched pathways and biological processes have been implicated in pulmonary hypertension or related physiological processes. For example, the MAPK signaling pathway has been demonstrated to be activated by norfenfluramine, a derivative of fenfluramine, together with the increase of intracellular Ca2+ and the activation of the hydrolysis of inositol phosphates.[13] Previous studies have also implicated the PAAR signaling pathway in PAH.[39,40] Our pathway analysis results also indicated that genes involved in cell permeability, inflammation and immune response, such as “calcium signaling pathway,” and “cell adhesion,” could play critical roles in fenfluramine-associated pathogenesis.

Furthermore, our gene network analysis demonstrated that a substantial proportion of the dysregulated genes in PASMC and PAEC after fenfluramine treatment could be linked with each other through certain functional interactions (e.g., activation, inhibition) (Figs. 7 and 8), suggesting that fenfluramine exposure may cause changes in a series of interacting proteins. Particularly, BMPR1B (Fig. 7c) and RGMA (Fig. 8c) were among the most important genes in the networks comprised of the dysregulated genes in PASMC and PAEC, respectively, based on betweenness centrality, indicating their potentially critical roles in determining the fenfluramine-associated pathogenic process in these cells. Interestingly, repulsive guidance molecule, a BMP co-receptor has been found to alter utilization of bone morphogenetic protein (BMP) type II receptors,[41,42] which have been implicated in pulmonary hypertension.[43]

CONCLUSIONS

In summary, our findings strongly suggest that fenfluramine exposure could cause a wide range of gene dysregulation. Significantly, our results confirmed that fenfluramine-associated IPAH is likely due to a complex pathogenic process that could be caused by genes involved in a variety of pathways and biological processes, including those related to normal functions of blood vessels (e.g., cell permeability, cell adhesion), inflammation response, immune response, and cell cycle. On the other hand, significant genetic and expression variations exist both within human populations and between normal subjects and patients with cardiopulmonary disease[44–47] that may in turn affect the cellular response to fenfluramine. Future investigations with more samples and populations are necessary to validate our findings. Finally, expanding molecular profiling to include other transcriptional targets such as microRNAs and integrating gene expression with genetic and epigenetic variations in the future could provide a more comprehensive picture of the complex cellular response to fenfluramine exposure as well as the pathogenesis of fenfluramine-associated IPAH or pulmonary arterial hypertension in general.

Footnotes

Source of Support: This work was supported, in part, by grants from the NIH/NHLBI (HL066012 and HL098053 to JX-JY and DK083506 to AM). WZ is supported by the new faculty research start-up from the University of Illinois at Chicago. ML was supported by the NSF ASCEND Program.

Conflict of Interest: None declared.

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