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Cystathionine-beta-synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells
Utpal Sen1, Neetu Tyagi, Munish Kumar
1Department of Physiology & Biophysics, University of Louisville School of Medicine, Louisville, KY 40202, USA.
Insights
Elevated homocysteine (Hcy) from high methionine intake increases vascular inflammation and remodeling by upregulating adhesion molecules and matrix proteins. Gene therapy with cystathionine-beta-synthase (CBS) effectively reduced these effects.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Elevated homocysteine (Hcy), or hyperhomocysteinemia (HHcy), is linked to vascular inflammation and remodeling.
- The precise mechanisms underlying Hcy-mediated vascular damage remain incompletely understood.
- Matrix metalloproteinases (MMPs) and adhesion molecules are critical in vascular remodeling processes.
Purpose of the Study:
- To investigate the hypothesis that HHcy induces vascular inflammation and remodeling by increasing adhesion molecules and matrix protein expression.
- To elucidate the role of methionine metabolism in Hcy-induced vascular changes.
- To evaluate potential therapeutic interventions for HHcy-induced vascular damage.
Main Methods:
- Endothelial cells were treated with high methionine to induce Hcy accumulation, measured by HPLC.
- Nitric oxide (NO) bioavailability was assessed using a NO probe.
- Protein expression (ICAM-1, VCAM-1, collagen type-1) was quantified via Western blot.
- MMP-9 activity was determined using gelatin-gel zymography.
- Interventions included treatment with 3-deazaadenosine (DZA) and transfection with the cystathionine-beta-synthase (CBS) gene.
Main Results:
- High methionine supplementation led to increased Hcy levels, ICAM-1, VCAM-1, collagen type-1 synthesis, and MMP-9 activity.
- DZA treatment attenuated methionine-induced increases in adhesion molecules and collagen type-1.
- CBS gene transfection significantly reduced Hcy accumulation, inflammatory markers (ICAM-1, VCAM-1), collagen type-1 synthesis, and MMP-9 activity.
Conclusions:
- Methionine-induced Hcy accumulation promotes vascular inflammation and matrix remodeling.
- Adhesion molecules (ICAM-1, VCAM-1), collagen type-1 synthesis, and MMP-9 activity are key mediators in this process.
- In vitro interventions like DZA and CBS gene therapy show promise in mitigating HHcy-induced vascular damage within the methionine metabolism pathway.
Abstract:
Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-beta-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway.
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