No abstract

Persistent accumulation of monocytes/macrophages in the pulmonary artery adventitial/perivascular areas of animals and humans with pulmonary hypertension has been documented. The cellular mechanisms contributing to chronic inflammatory responses remain unclear. We hypothesized that perivascular inflammation is perpetuated by activated adventitial fibroblasts, which, through sustained production of pro-inflammatory cytokines/chemokines and adhesion molecules, induce accumulation, retention, and activation of monocytes/macrophages. We further hypothesized that this pro-inflammatory phenotype is the result of abnormal activity of histone-modifying enzymes, specifically, class I histone deacetylases (HDACs). Methods and Results Pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (termed PH-Fibs) expressed a constitutive and persistent pro-inflammatory phenotype defined by high expression of IL-1β, IL-6, CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES), CCR7, CXCR4, GM-CSF, CD40, CD40L, VCAM-1. The pro-inflammatory phenotype of PH-Fibs was associated with epigenetic alterations as evidenced by increased activity of HDACs, and the findings that class I HDAC inhibitors markedly decreased cytokine/chemokine mRNA expression levels in these cells. PH-Fibs induced increased adhesion of THP-1 monocytes, and produced soluble factors that induced increased migration of THP-1 and murine bone marrow-derived macrophages (BMDMs), as well as activated monocytes/macrophages to express pro-inflammatory cytokines and pro-fibrogenic mediators (TIMP1 and COL1) at the transcriptional level. Class I HDAC inhibitors markedly reduced the ability of PH-Fibs to induce monocyte/migration and pro-inflammatory activation. Conclusions The emergence of a distinct adventitial fibroblast population with an epigenetically-altered pro-inflammatory phenotype capable of recruiting, retaining and activating monocytes/macrophages characterizes pulmonary hypertension-associated vascular remodeling, and thus could contribute significantly to chronic inflammatory processes in the pulmonary artery wall.

The NAD(P)H:quinone oxidoreductase 1 (NQO1)*2 polymorphism is characterized by a single proline-to-serine amino acid substitution. Cell lines and tissues from organisms genotyped as homozygous for the NQO1*2 polymorphism are deficient in NQO1 activity. In studies with cells homozygous for the wild-type allele and cells homozygous for the mutant NQO1*2 allele, no difference in the half-life of NQO1 mRNA transcripts was observed. Similarly, in vitro transcription/translation studies showed that both wild-type and mutant NQO1 coding regions were transcribed and translated into full-length protein with equal efficiency. Protein turnover studies in NQO1 wild-type and mutant cell lines demonstrated that the half-life of wild-type NQO1 was greater than 18 h, whereas the half-life of mutant NQO1 was 1.2 h. Incubation of NQO1 mutant cell lines with proteasome inhibitors increased the amount of immunoreactive NQO1 protein, suggesting that mutant protein may be degraded via the proteasome pathway. Additional studies were performed using purified recombinant NQO1 wild-type and mutant proteins incubated in a rabbit reticulocyte lysate system. In these studies, no degradation of wild-type NQO1 protein was observed; however, mutant NQO1 protein was completely degraded in 2 h. Degradation of mutant NQO1 was inhibited by proteasome inhibitors and was ATP-dependent. Mutant NQO1 incubated in rabbit reticulocyte lysate with MG132 resulted in the accumulation of proteins with increased molecular masses that were immunoreactive for both NQO1 and ubiquitin. These data suggest that wild-type NQO1 persists in cells whereas mutant NQO1 is rapidly degraded via ubiquitination and proteasome degradation.

Objective: This study was performed to assess the utility of selective small-molecule inhibitors of class I HDACs in a preclinical model of pulmonary hypertension. Methods and Results:Rats were exposed to hypobaric hypoxia for 3 weeks in the absence or presence of a benzamide HDAC inhibitor, MGCD0103, which selectively inhibits class I HDACs 1, 2, and 3. The compound reduced pulmonary arterial pressure more dramatically than tadalafil, a standard-of-care therapy for human pulmonary hypertension that functions as a vasodilator. MGCD0103 improved pulmonary artery acceleration time and reduced systolic notching of the pulmonary artery flow envelope, which suggests a positive impact of the HDAC inhibitor on pulmonary vascular remodeling and stiffening. Similar results were obtained with an independent class I HDAC-selective inhibitor, MS-275. Reduced pulmonary arterial pressure in MGCD0103-treated animals was associated with blunted pulmonary arterial wall thickening because of suppression of smooth muscle cell proliferation. Right ventricular function was maintained in MGCD0103-treated animals. Although the class I HDAC inhibitor only modestly reduced right ventricular hypertrophy, it had multiple beneficial effects on the right ventricle, which included suppression of pathological gene expression, inhibition of proapoptotic caspase activity, and repression of proinflammatory protein expression. Key Words: histone deacetylase Ⅲ pulmonary hypertension Ⅲ proliferation Ⅲ gene expression Ⅲ signaling pathways I n patients with pulmonary hypertension (PH), restricted blood flow through the pulmonary arterial circulation due to increased pulmonary vascular resistance often results in right-sided heart failure. Despite recent advances in the treatment of PH, the 5-year mortality rate for individuals with this disease still approaches 50%, which highlights an urgent need for novel therapeutics. 1 Current standards of care for patients with PH typically involve the use of vasoactive drugs, including endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and prostacyclins. More effective therapeutic strategies will likely be based on the combined use of vasodilators and agents that target distinct pathogenic mechanisms in PH, such as pulmonary vascular inflammation and fibrosis, as well as aberrant proliferation of smooth muscle cells, endothelial cells, and fibroblasts in the lung vasculature. 2 Additionally, because right ventricular (RV) failure is the cause of death in the majority of PH patients, 3,4 and it is unclear whether standards of care for left ventricular Original received October 8, 2011; revision received January 11, 2012; accepted January 18, 2012. In December 2011 HDACs control cell proliferation, inflammation, and fibrosis by catalyzing removal of acetyl groups from lysine residues in a variety of proteins. The 18 mammalian HDACs are encoded by distinct genes and are grouped into 4 classes. 6 Two broad-spectrum HDAC inhibitors are approved for the treatment of cancer. One of these compounds,...