Deep sea water prevents balloon angioplasty-induced hyperplasia through MMP-2: an in vitro and in vivo study

Abstract

Major facts about the development of restenosis include vascular smooth muscle cells (VSMCs) proliferation and migration. A previous study showed that in vitro treatment with magnesium chloride has the potential to affect the proliferation and migration of VSMCs. Magnesium is the major element in deep sea water (DSW) and is a biologically active mineral. It is unclear whether DSW intake can prevent abnormal proliferation and migration of VSMCs as well as balloon angioplasty-induced neointimal hyperplasia. Thus, we attempted to evaluate the anti-restenotic effects of DSW and its possible molecular mechanisms. Several concentrations of DSW, based on the dietary recommendations (RDA) for magnesium, were applied to a model of balloon angioplasty in SD rats. The results showed that DSW intake markedly increased magnesium content within the vascular wall and reduced the development of neointimal hyperplasia. The immunohistochemical analysis also showed that the expression of proteins associated with cell proliferation and migration were decreased in the balloon angioplasty groups with DSW supplement. Furthermore, in vitro treatment with DSW has a dose-dependent inhibitory effect on serum-stimulated proliferation and migration of VSMCs, whose effects might be mediated by modulation of mitogen-activated protein kinase (MAPK) signaling and of the activity of matrix metalloproteinase-2 (MMP-2). Our study suggested that DSW intake can help prevent neointimal hyperplasia (or restenosis), whose effects may be partially regulated by magnesium and other minerals.

Figure 1. Inhibitory effect of DSW on balloon angioplasty-induced neointimal hyperplasia.

Tissue sections from rat carotid arteries were further stained with hematoxylin-eosin to observe the thickness of neointimal layer of arterial wall (A–F). The expression levels of MMP-2 and PCNA proteins were detected with immunohistopathological analysis (H and I). The images were acquired by microscopy at 200-fold magnification. The manifestation of vessel restenosis was presented as the ratio of neointima- to-media area (N/M ratio). L, lumen; N, neointima layer; M, media layer. Red arrow is protein experssion.*P<0.05, and **P< compared with BA + water group, respectively.

Figure 2. Differences in magnesium content in the balloon-injured carotid artery.

*P<0.05 compared with BA + water group, respectively.

Figure 3. DSW-induced regulation of VSMC viability.

Cell viability was analyzed with the MTT proliferation assay. One-fold (1x) DSW was defined as water-diluted DSW containing a level of magnesium equal to that of the magnesium chloride used in the present experiment. The results are shown as % of the control. *P<0.05, and **P<0.01 compared with the 15% FBS-treated group, respectively.

Figure 4. Effects of DSW on VSMC migration. Cell migration was analyzed with transwell assays.

The images were taken at 400-fold magnification. The 1x DSW was defined as water-diluted DSW containing the same level of magnesium as the magnesium chloride used in the present experiment. The results are shown as % of the control (15% FBS).*P<0.05, and **P<0.01 compared with the 15% FBS-treated group, respectively. #P<0.05 compared with the MgCl₂-treated group.

Figure 5. Molecular regulation by DSW of cell growth and migration of VSMCs.

Inhibitory mechanisms of DSW on cell growth- and migration-associated proteins were analyzed by using the methods of Western blot (WB) and gelatin zymography. Beta-actin was used as internal control in Western blot. The results are shown as % of the control (15% FBS). The MMP-2 activity was normalized to the values of the 15% FBS group. *P<0.05, and **P<0.01 compared with the 15% FBS-treated group, respectively.