A Novel p38 Mitogen-activated Protein Kinase/Elk-1 Transcription Factor-dependent Molecular Mechanism Underlying Abnormal Endothelial Cell Proliferation in Plexogenic Pulmonary Arterial Hypertension

Abstract

Background: Plexiform lesions comprising proliferative endothelial cells are hallmarks of pulmonary arterial hypertension. Results: Granzyme B cleaves intersectin-1s and generates a fragment with endothelial cell proliferative potential via phosphorylation of p38MAPK and Elk-1 transcription factor. Conclusion: Granzyme B cleavage of intersectin-1s and subsequent p38 MAPK /Elk-1 activation are critical for endothelial cell proliferation. Significance: The novel pathogenic p38 MAPK /Elk-1 signaling may explain the formation of plexiform lesions.

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed

“…In addition, the Dharmacon ONTARGETplus siRNA reagents and the siRNA concentration used allowed us to minimize the off-target effects and provide a highly specific on-target gene knockdown, while reducing not only the off-target gene modulation but also the extent of ITSN knockdown in other organs. 12 Myc-EH ITSN DNA (amino acids 1 to 271), cloned into the pReceiver/myc-M43 vector, as in the study by Patel et al, 11 was delivered to mouse lungs similar to siRNA. DNAliposome complexes were prepared at a ratio of 8 nmoles liposomes/1 mg myc-EH ITSN DNA, a ratio found in pilot studies to induce efficient protein expression without pulmonary toxicity.…”

“…After the 2-hour incubation, proteinase K was denatured at 95 C for 5 minutes. The tubes were then centrifuged at 9,500 Â g for 10 minutes, and 1 mL of each extracted DNA sample was used for conventional PCR, along with the ITSN primers, as in the study by Patel et al 11 The PCR products were resolved onto a 1.2% agarose gel and visualized using ethidium bromide.…”

“…11 The nuclear extracts were then analyzed by enzyme-linked immunosorbent assay (TransAM Kit; Active Motif, Carlsbad, CA), with colorimetric readout quantifiable by spectrophotometry, in a 96-well plate containing the immobilized Elk-1 consensus site oligonucleotide. Activated Elk-1 was detected via an Ab against phosphorylated Elk-1, followed by a horseradish peroxidaseeconjugated reporter Ab.…”

“…11 Moreover, lung tissue of PAH animal models as well as human PAH lung tissue and pulmonary artery ECs isolated from PAH subjects are deficient of ITSN compared to controls and express the EH ITSN product. 11 Given these findings, we hypothesized that the EH ITSN may be responsible for angioproliferation in advanced human PAH.…”

“…11 Moreover, lung tissue of PAH animal models as well as human PAH lung tissue and pulmonary artery ECs isolated from PAH subjects are deficient of ITSN compared to controls and express the EH ITSN product. 11 Given these findings, we hypothesized that the EH ITSN may be responsible for angioproliferation in advanced human PAH. Thus, in the present study, we addressed the in vivo effects of EH ITSN expression on lung vascular remodeling using two murine models of ITSN-1 deficiency, the ITSN knockdown mouse (KD ITSN ) 12 and the ITSN knockout/heterozygous mouse (K0 ITSNþ/À ), 13 both transduced with a myc-tagged EH ITSN plasmid.…”