Linking Vascular Remodeling and Inflammation in Pulmonary Arterial Hypertension: Is There a Common Root Cause?

One of the fundamental questions about pulmonary hypertension is why small artery remodeling and perivascular inflammation are seen together in obstructive lung lesions, which are the hallmark of this disease. Simply put, does inflammation stimulate vascular pathology or does vascular pathology cause secondary inflammation? Even more intriguing is the idea that defects in a single transcription factor or related transcription factors could simultaneously cause both small vessel occlusive remodeling and perivascular inflammation. This has been one of the “holy grail” issues for scientists who study pulmonary arterial hypertension. Recent evidence suggests that there are families of transcription factors, related by sequence homology, that regulate gene clusters whose protein products are involved in angiogenesis, cellular homeostasis, cell junction integrity, and inflammation. Of these, the E-26 transformation-specific (ETS) proteins, a group of 28 related transcription factors, are emerging as master regulators of proliferation, apoptosis, inflammation, and fibrosis (1). ETS transcription factors share a highly conserved 85 amino acid DNA binding domain (ETS domain) that binds to a DNA core consensus motif, 59GGA(A/T)39. These factors can act as transcriptional activators, repressors, or both depending on the cellular context. The ETS-related gene (ERG) and Friend leukemia integration-1 (FLI-1) transcription factors are the most closely related by protein structure of all the ETS factors (2). ERG expression is restricted to endothelial cells, megakaryocyteerythroid progenitor cells, T and B early lineage-committed cells, and myocardium (3), whereas FLI-1 expression is restricted to endothelial cells, hematopoietic progenitors and mature derivative cells (megakaryocytes, monocytes, erythroblasts, and natural killer cells), areas of the developing brain, and dermal fibroblasts (4). Interestingly, pathologic expression of ERG and FLI1 due to chromosomal rearrangement resulting in the juxtaposition of strong tissue-specific promoters adjacent to these genes has been found in human prostate cancer (for ERG [5]) and Ewing’s sarcoma (for FLI1 or ERG [6]), indicating their role as protooncogenes in tissues where these genes are otherwise not expressed in the adult. Several studies employing knockout mice have shed light on the functional roles of the Erg and Fli-1 transcription factors. Homozygous knockout of Erg resulted in a spectrum of lethality (E8.5 to 3 months after birth) from cardiac defects, vascular permeability, and hemorrhage in utero (7). Homozygous knockout of Fli1 causes embryonic lethality (E11.0–E12.5) with bleeding from the dorsal aorta, neural tube, and brain secondary to loss of brain epithelial integrity, as well as defects in hematopoietic colony-forming cells (8). Interestingly, recent breakthroughs in pulmonary vasculopathy have been made using Erg null, Fli1:Klf5 double heterozygote, and endothelial-specific Fli:Tie2-Cre knockout mice. Lathen and colleagues found that although most Erg mice died in utero, a small proportion survived after birth and spontaneously developed pulmonary venoocclusive disease, leading to lethality at 4–12 wk of age (9). In contrast, Noda and colleagues showed that doubly heterozygous Fli1:Klf5 (a member of the Kruppel-like factor family) mice spontaneously developed clinical characteristics of systemic sclerosis (SSc) and SSc-induced pulmonary arterial hypertension (10). They also found that Fli1:Klf5 mice manifested dermal fibrosis, increased B cell accumulation and collagen deposition in the lung, and nonspecific interstitial pneumonia (the most common pattern of SSc lung disease), leading to obliteration of precapillary arterioles at 4 months of age from a-smooth muscle actin (SMA)–positive cells, simulating the pulmonary arterial hypertensive phenotype. These doubly heterozygous mice have become the principal animal model for the study of SSc vasculopathy. In addition, endothelial-specific Fli1 knockout (Fli1:Tie2-Cre) mice have been shown to develop arteriolar stenosis, dilation of capillaries, and increased vascular permeability without concomitant fibrosis in both skin and lung (11). Collectively, these studies led to the concept that the suppression or absence of Erg or Fli1 in the pulmonary endothelium of mice may be associated with different forms of pulmonary occlusive vasculopathy. A large body of literature is now emerging to suggest that ERG and FLI-1 turn on sets of genes that concomitantly control endothelial function, proliferation, homeostasis, cell–cell interaction, and vascular inflammation (see Figure 1). As such, they serve as intriguing “master regulators” of sets of genes whose protein products have been implicated in causing pulmonary arterial hypertension. Notably, ERG has been found in multiple studies to repress the transcription of genes that encode inflammatory mediators (e.g., CXCL8, IL8, and NFkB) while augmenting the transcription of genes that promote endothelial cell–cell-junction tightness (e.g., CDH5, ICAM2, and CLDN5), endothelial angiogenesis (e.g., DLL4, FLK1, and FLT1), and endothelial homeostasis (e.g., ENG, APLNR, and NOS3) (reviewed in Reference 12). In contrast, FLI-1 is emerging as a transcription factor that represses genes that induce fibrosis (COL1A1 and COL1A2) while augmenting the transcription of inflammatory genes (CXCL5 and MCP1) and genes that promote angiogenesis (TIE2, VEGFA, NOTCH1, FLI1, and EGF17) (reviewed in Ref. 13). Thus, although they are similar in structure, distinctly separate roles are being defined for ERG and FLI-1. In their paper in this issue of the Journal, Looney and colleagues (pp. 121–131) examine the inflammatory signature that results from knockdown of ERG and FLI1 in human pulmonary artery endothelial cells in vitro, with the goal of studying whether dual knockdown is associated with the pulmonary hypertensive phenotype (14). They find that simultaneous small-interferingRNA (siRNA) knockdown of ERG and FLI1 in pulmonary artery endothelial cells leads to up-regulation of proinflammatory genes and interferon pathway-related genes at magnitudes exceeding those obtained from knockdown of ERG or FLI1 alone. Microarray