Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

Abstract

BACKGROUND: Epigenetic regulation of vascular remodeling in pulmonary hypertension (PH) is poorly understood. Transcription regulating, histone acetylation code alters chromatin accessibility to promote transcriptional activation. Our goal was to identify upstream mechanisms that disrupt epigenetic equilibrium in PH. METHODS: Human pulmonary artery smooth muscle cells (PASMCs), human idiopathic pulmonary arterial hypertension (iPAH):human PASMCs, iPAH lung tissue, failed donor lung tissue, human pulmonary microvascular endothelial cells, iPAH:PASMC and non-iPAH:PASMC RNA-seq databases, NanoString nCounter, and cleavage under targets and release using nuclease were utilized to investigate histone acetylation, hyperacetylation targets, protein and gene expression, sphingolipid activation, cell proliferation, and gene target identification. SPHK2 (sphingosine kinase 2) knockout was compared with control C57BL/6NJ mice after 3 weeks of hypoxia and assessed for indices of PH. RESULTS: We identified that Human PASMCs are vulnerable to the transcription-promoting epigenetic mediator histone acetylation resulting in alterations in transcription machinery and confirmed its pathological existence in PH:PASMC cells. We report that SPHK2 is elevated as much as 20-fold in iPAH lung tissue and is elevated in iPAH:PASMC cells. During PH pathogenesis, nuclear SPHK2 activates nuclear bioactive lipid S1P (sphingosine 1-phosphate) catalyzing enzyme and mediates transcription regulating histone H3K9 acetylation (acetyl histone H3 lysine 9 [Ac-H3K9]) through EMAP (endothelial monocyte activating polypeptide) II. In iPAH lungs, we identified a 4-fold elevation of the reversible epigenetic transcription modulator Ac-H3K9:H3 ratio. Loss of SPHK2 inhibited hypoxic-induced PH and Ac-H3K9 in mice. We discovered that pulmonary vascular endothelial cells are a priming factor of the EMAP II/SPHK2/S1P axis that alters the acetylome with a specificity for PASMC, through hyperacetylation of histone H3K9. Using cleavage under targets and release using nuclease, we further show that EMAP II–mediated SPHK2 has the potential to modify the local transcription machinery of pluripotency factor KLF4 (Krüppel-like factor 4) by hyperacetylating KLF4 Cis-regulatory elements while deletion and targeted inhibition of SPHK2 rescues transcription altering Ac-H3K9. CONCLUSIONS: SPHK2 expression and its activation of the reversible histone H3K9 acetylation in human pulmonary artery smooth muscle cell represent new therapeutic targets that could mitigate PH vascular remodeling.

Emerging Epigenetic Targets and Their Molecular Impact on Vascular Remodeling in Pulmonary Hypertension

“…For example, increased levels of aldehyde dehydrogenase, ALDH1A3 (aldehyde dehydrogenase family 1 member 3) promote acetyl coenzyme A to acetylate histone H3K27, while inducing the highly proliferative and glycolytic pulmonary artery SMC (PASMC) in PH [17]. Histone H3K9 acetylation was significantly increased in the lungs of PH patients and promoted PASMC proliferation via Sphingosine Kinase 2 [18]. CircRNA, Hsa_circ_0001402 acts as an miR-183-5p sponge to inhibit SMC proliferation and migration while activating VSMC autophagy to alleviate neointimal hyperplasia [19].…”

“…Importantly, this process is mediated by epigenetic histone acetylation [30]. Other studies determined that endothelial-secreted factors promote the hyperproliferation of SMCs using epigenetic mechanisms such as histone acetylation and microRNA [18,31]. Future exploration is needed to further determine the epigenetic contribution that cell-cell interaction has to PH vascular remodeling and to assess the reversibility of epigenetic targeting in PH (Figure 1).…”

“…Histone acetylation, as previously established, is one of the major epigenetic alterations that governs angiogenesis in LC. Surprisingly, it is a modification that occurs often in PH as well [18,63,64]. Unfortunately, medications that inhibit HDAC (HDACi) such as Vorinostat (Zolinza), Romidepsin (Istodax), and Belinostat (Beleodaq), already approved by the FDA as safe for use in certain lymphoma cases, lack conclusive data regarding their efficacy in treating solid tumors or whether tumors with specific genetic changes may respond better to these drugs [64][65][66].…”