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Prevention of intima hyperplasia by mitogen-activated protein kinase antisense oligodeoxynucleotide
1Laboratory of Molecular Pharmacology, Hu-nan Medical University, Changsha 410078, China.
Aim:
To investigate the preventive effect of Ca(2+)-calmodulin dependent kinase (CCDPK) (formerly: mitogen-activated protein kinase or MAPK) antisense phosphorothioate oligodeoxynucleotide (ODN) on vascular smooth muscle cell (VSMC) proliferation in vitro and on intima hyperplasia after injury in vivo.
Methods:
Liposomal transfection was used to introduce phosphorothioate-protected 17-mer antisense CCDPK ODN directed against the initiation of translation sites of the p42 and p44 CCDPK isoforms into cultured rat VSMC to deplete CCDPK and DNA synthesis induced by endothelin-1 (ET) or platelet derived growth factor (PDGF). A 17-mer sense and a random sequence CCDPK ODN were used as controls. CCDPK protein p44 and p42 levels were measured by Western blot. DNA synthesis was measured by [3H]thymidine incorporation. In in vivo study, rat balloon angioplasty was performed by a 2F Fogarty catheter. The antisense CCDPK ODN 200 micrograms was administered to the adventitial surface of the injured carotid artery by pluronic gel 30% (w/v) solution. Two weeks after vascular injury, carotid arteries were removed and cross sections were made and stained with hematoxylin/eosin for patho-histological examination. Fluorecein isothiocynate (FITC)-labeled and phosphorothioate-protected ODN was used to detect the uptake of ODN in vitro and in vivo.
Results:
CCDPK antisense ODN (0.4 mumol.L-1) reduced p42/p44 protein expression and inhibited VSMC [3H]thymidine incorporation stimulated by ET and PDGF. Antisense CCDPK ODN treatment at 2 wk after injury resulted in a significant inhibition of intima hyperplasia, compared with untreated vessels.
Conclusion:
The p42/p44-CCDPK antisense ODN inhibits in vitro stimulated rat VSMC proliferation and in vivo injured arterial intima hyperplasia.
Insights
Antisense oligodeoxynucleotides targeting Ca(2+)-calmodulin dependent kinase (CCDPK) effectively inhibited vascular smooth muscle cell proliferation and reduced intimal hyperplasia after vascular injury. This demonstrates CCDPK
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Biochemistry
Background:
- Vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia are key processes in cardiovascular disease.
- Ca(2+)-calmodulin dependent kinase (CCDPK), formerly known as mitogen-activated protein kinase (MAPK), plays a role in cellular signaling pathways.
- Targeting specific kinases offers a potential therapeutic strategy for vascular disorders.
Purpose of the Study:
- To evaluate the preventive efficacy of CCDPK antisense oligodeoxynucleotides (ODN) against VSMC proliferation in vitro.
- To assess the impact of CCDPK antisense ODN on intimal hyperplasia following vascular injury in vivo.
Main Methods:
- In vitro studies utilized cultured rat VSMCs, employing liposomal transfection to deliver antisense CCDPK ODN targeting p42 and p44 isoforms.
- In vivo studies involved rat balloon angioplasty, with antisense CCDPK ODN administered to the injured carotid artery adventitia.
- Western blot analysis measured protein levels, [3H]thymidine incorporation assessed DNA synthesis, and histological examination evaluated intimal hyperplasia.
Main Results:
- CCDPK antisense ODN significantly reduced p42/p44 protein expression and inhibited ET-1 and PDGF-stimulated VSMC DNA synthesis in vitro.
- In vivo, antisense CCDPK ODN treatment markedly inhibited intimal hyperplasia in injured carotid arteries two weeks post-injury.
- Uptake of FITC-labeled ODN was confirmed in both in vitro and in vivo models.
Conclusions:
- p42/p44-CCDPK antisense ODN effectively inhibits VSMC proliferation stimulated in vitro.
- Antisense CCDPK ODN demonstrates a significant preventive effect on intimal hyperplasia in an in vivo model of vascular injury.
- Targeting CCDPK with antisense ODN presents a promising therapeutic approach for preventing vascular smooth muscle cell proliferation and intimal hyperplasia.