Functional Analysis of Genomic DNA, cDNA, and Nucleotide Sequence of the Mature C-Type Natriuretic Peptide Gene in Vascular Cells

Abstract

Objective-The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells. Methods and Results-Gene transfer was performed with cationic lipid DOCSPER or linear polyethylenimine. All 3 CNP sequences led to significant inhibition of smooth muscle cell (SMC) proliferation. In contrast, significant stimulation of cell growth was observed in endothelial cells (ECs) using CNPDNA or CNPcDNA but not CNP22aa. In a porcine restenosis model, a significant reduction in neointima hyperplasia was found 3 months after application of the CNPcDNA vector compared with the control transfection. Conclusions-The results demonstrate that the first intron in the CNP sequence does not contain any additional enhancerbinding sites. However, the signal sequence is indispensable for secretion of CNP and its appropriate physiological function. Key Words: C-type natriuretic peptide Ⅲ vascular cells Ⅲ gene transfer Ⅲ proliferation Ⅲ restenosis C -type natriuretic peptide (CNP) is a promising therapeutic agent for prevention of restenosis after angioplasty. 1-3 CNP inhibits smooth muscle cell (SMC) proliferation, decreases extracellular matrix synthesis, promotes revascularization, and positively influences vascular remodeling. 2,3 The CNP gene has been localized to chromosome 2 in the human genome, and its sequence is the most highly conserved of the natriuretic peptides across many species. 4,5 Sequence analysis reveals that there are a total of 3 exons and 2 introns. In the coding region for prepro CNP, the first 2 exons are separated by an intron. 6 Exon 3, coding for poly A, is removed during the processing pathway within the nucleus. The gene is flanked in the 5Ј-region by cis-acting regulatory elements that are not part of the other natriuretic peptide genes, suggesting that the regulation mechanisms for CNP gene transcription are different compared with the other natriuretic peptides. 6 The function of the first intron regarding transcription regulation of this gene is so far unknown. However, a number of genes are known to be regulated by enhancer elements outside the coding region, and many of them contain regulatory sequences within the first intron. 7,8 The CNP gene encodes a 126-residue precursor peptide that is processed to generate 22-and 53-aa peptides (human CNP22 and human CNP53, respectively). 9 CNP22 is more widely and abundantly expressed and is more potent than CNP53. 5,9 The amino terminal of prepro CNP contains a signal peptide sequence that allows the synthesized peptide to pass from the endoplasmic reticulum to the Golgi complex, where it is packed into granules from which CNP is then released at the cell surface by exocytosis. The signal peptide sequence is generally necessary for protein secretion. However, some proteins, such as fibroblast growth factor-2 (FGF-2), are secreted into the extracellular med...

Small Poly-<i>L</i>-Lysines Improve Cationic Lipid-Mediated Gene Transfer in Vascular Cells in vitro and in vivo

“…For flow cytometric analysis, proliferating SMCs were seeded into 12-well plates and cultivated as previously described [9, 14, 22]. Transfection with Cy5-labelled (excitation at 650 nm, emission at 670 nm) plasmid DNA (Mirus Bio Corporation, Madison, Wisc., USA), unlabelled sPLLs and DOCSPER was done as described above.…”

“…It has been shown that perivascular events significantly affect neointima: adventitial irritation results in focal intimal thickening and markers applied to the adventitia can be visualized in the intima [7, 8]. Therefore, vascular SMCs may be a suitable target using adventitial deposition of gene therapy vectors [3, 9]. …”

Comparative measurement of CNP and NT-proCNP in human blood samples: a methodological evaluation

“…In addition, CNP binds NPR3 and in turn deactivates protein kinase A by G i -protein dependent inhibition of adenylate cyclase [3]. CNP has anti-proliferative and anti-migratory properties [4] and was also identified as an endothelium derived relaxing factor [5]. Furthermore, CNP inhibits neointimal restenosis, reduces vascular constrictive remodeling and cardiac ischemia-reperfusion injury [6,7].…”

Impact of oxLDL and LPS on C-type Natriuretic Peptide System is Different between THP-1 Cells and Human Peripheral Blood Monocytic Cells

“…CNP plays an important role in bone growth [15,16], reproduction [17,18], nerve growth [19], re-endothelialisation [20], as well as renal [21], pancreatic [22], and cardiac function [23]. In addition, CNP has anti-proliferative and anti-migratory properties [11,24,25] and was also identified as an endothelium derived relaxing factor [26][27][28]. CNP stimulates selectively the human natriuretic peptide receptor 2 (NPR2), which in turn activates the cGMP-dependent kinase (cGK) by elevating intracellular level of cGMP [14].…”