Contribution of bone morphogenetic protein-2 to aortic valve calcification in aged rat.

Although aging is well established as an important risk factor for aortic stenosis, the mechanism of age-related aortic valve calcification is yet unknown. Here, we investigated this mechanism in tissue and cellular levels using middle-aged rats. Aortic valve specimens were obtained by dissecting from 9-week-old (young) and 30-week-old (aged) male Wistar rats. In the aged rats, the main risk factors for aortic stenosis in plasma were still in the normal range; however, their number of calcified specimens was significantly increased in comparison with the young rats. Aortic valve interstitial cells (AVICs) obtained from explants of aortic valve specimens were cultured for 14 days after reaching confluence. Spontaneous calcification, the expressions of calcigenic genes, that is, BMP-2, alkaline phosphatase (ALP), and osterix (osteogenic transcription factor) and ALP enzyme activity in AVICs from aged rats were enhanced in comparison with those from young rats. However, neither typical calcification inducing reagents (dexamethasone, ß-glycerophosphate, and high concentration of phosphate) nor tumor necrosis factor-α (an inflammatory cytokine) accelerated the spontaneous calcification of AVICs from aged rats. These results suggest that aortic valve calcification progresses with age partly through an activation of the BMP-2 pathway.

Opposite effects of two resveratrol (trans-3,5,4'-trihydroxystilbene) tetramers, vitisin A and hopeaphenol, on apoptosis of myocytes isolated from adult rat heart.

It has been reported that resveratrol (trans-3,5,4'-trihydroxystilbene) from Vitis plants has various cardioprotective effects. Vitis plants also include various resveratrol tetramers. The aim of our study is to clarify the pharmacological properties of resveratrol tetramers. We isolated two resveratrol tetramers as major products of Vitis plants. One is vitisin A, a complex of two resveratrol dimers, (+)-epsilon-viniferin and ampelopsin B, and the other is hopeaphenol, composed of 2 mol ampelopsin B. Vitisin A (30-300 nM) unexpectedly dose-dependently facilitated swelling and depolarization of mitochondria and cytochrome c release from mitochondria, which are indices of cardiomyocyte apoptosis. Furthermore, vitisin A induced apoptosis in the primary culture of adult rat ventricular myocytes. On the other hand, hopeaphenol (1-10 microM) dose-dependently inhibited Ca(2+) (30 microM)-induced mitochondrial depolarization and cytochrome c release from mitochondria but had not affected mitochondrial swelling. Moreover, hopeaphenol inhibited vitisin A-induced apoptosis. In structural and functional studies, we further confirmed that vitisin B, one of the resveratrol tetramers having (+)-epsilon-viniferin unit, induces mitochondrial swelling and cytochrome c release from mitochondria like vitisin A and that vitisifuran A, one of the resveratrol tetramers having the ampelopsin B unit, inhibits Ca(2+)-induced cytochrome c release from mitochondria like hopeaphenol. These results show that resveratrol tetramers have at least two opposite effects on cardiomyocytes; the one having the (+)-epsilon-viniferin unit induces cardiomyocyte apoptosis, and the other having ampelopsin B but not (+)-epsilon-viniferin unit inhibits it.

Calcification of aortic smooth muscle cells isolated from spontaneously hypertensive rats.

Although hypertension and vascular calcification are well established as important risk factors for several cardiovascular diseases, the relationship between them is unknown. Here, we investigated whether hypertension is relevant to vascular calcification by examining aortic smooth muscle cells (SMCs) isolated from the descending thoracic aortas of Wistar Kyoto rats (WKY) as normotensive rats and spontaneously hypertensive rats (SHR), a typical rat model of hypertension. Cells were cultured in DMEM containing 10% FBS for 6 days after reaching confluence. Von Kossa staining revealed that the positively stained calcified area of aortic SMCs from SHR increased rapidly compared to that from WKY. The gene expressions of calcification-regulating proteins including msh homeobox homolog 2, Osterix (a master transcription factor for osteogenesis), and alkaline phosphatase (ALP) (a marker of vascular calcification) were significantly increased in aortic SMCs from SHR compared to SMCs from WKY. On the other hand, Runx2, another osteogenic transcription factor, did not upregulate. Furthermore, we confirmed that ALP activity was strongly increased in aortic SMCs from SHR compared to SMCs from WKY. These results suggest that aortic SMCs from SHR tend to become easily calcified via an Msx2-Osterix signaling pathway.

Possible role of extracellular nucleotides in ectopic ossification of human spinal ligaments.

To reveal the involvement of extracellular nucleotides in the ossification process in ossification of the posterior longitudinal ligament of the spine (OPLL), the mRNA expression profiles of P2 purinoceptors, mechanical stress-induced ATP release, and ATP-stimulated expression of osteogenic genes were analyzed in ligament cells derived from the spinal ligament of OPLL patients (OPLL cells) and non-OPLL cells derived from the spinal ligaments of cervical spondylotic myelopathy patients as a control. The extracellular ATP concentrations of OPLL cells in static culture were significantly higher than those of non-OPLL cells, and this difference was diminished in the presence of ARL67156, an ecto-nuclease inhibitor. Cyclic stretch markedly increased the extracellular ATP concentrations of both cell types to almost the same level. P2Y1 purinoceptor subtypes were intensively expressed in OPLL cells, but only weakly expressed in non-OPLL cells. Not only ATP addition but also cyclic stretch raised the mRNA levels of alkaline phosphatase and osteopontin in OPLL cells, which were blocked by MRS2179, a selective P2Y1 antagonist. These increases in the expression of osteogenic genes were not observed in non-OPLL cells. These results suggest an important role of P2Y1 and extracellular ATP in the progression of OPLL stimulated by mechanical stress.