cyclic-gmp and Calcinosis

Calcific aortic stenosis displays some similarities to atherosclerosis including evidence of endothelial dysfunction. Whether nitric oxide (NO), which is produced by valvular endothelium, has direct protective effects extending to calcification processes in aortic valve cells has not previously been examined. In vitro calcifying nodules in porcine aortic valve interstitial cell cultures, formed in response to transforming growth factor-beta1 (TGF-beta1) 5 ng/ml, were inhibited by NO donors DETA-NONOate 5-100 microM, and sodium nitroprusside (SNP) 3 microM. Raising intracellular cGMP concentrations, via 8-bromo cGMP 1 mM or via brain natiuretic peptide and C-type natiuretic peptide 0.1 microM, inhibited TGF-beta1-induced nodule formation, potentially implicating the cGMP pathway in the NO effect. Stimulation of interstitial cells with substance P or calcium ionophone (A23187) caused NO release and increased intracellular cGMP respectively. However in the presence of TGF-beta1 basal levels of NO production via nitric oxide synthase (NOS) were insufficient to affect nodule formation. Increased dihydroethidium (DHE) fluorescence in response to TGF-beta1, which was inhibited by DETA-NONOate and TEMPOL, suggested a role for intracellular superoxide in TGF-beta1 signalling. Moreover, nodule formation was suppressed by superoxide scavengers TEMPOL, hydralazine and polyethylene glycol-superoxide dismutase (PEG-SOD), but not SOD. In conclusion, NO donors, or agents raising intracellular cGMP levels, may protect aortic valve interstitial cells from early events leading to calcification.

Topics: Animals; Aortic Valve; Calcinosis; Cells, Cultured; Cyclic GMP; Free Radical Scavengers; Ionophores; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Swine; Transforming Growth Factor beta1; Vasodilator Agents

Vascular calcification often occurs with advancing age, atherosclerosis, and metabolic disorders such as diabetes mellitus and end-stage renal disease. Vascular calcification is associated with cardiovascular events and increased mortality. Nitric oxide (NO) is crucial for maintaining vascular function, but little is known about how NO affects vascular calcification. The aim of this study was to examine the effect of NO on vascular calcification.. In this study, we examined the inhibitory effects of NO on calcification of murine vascular smooth muscle cells (VSMCs) in vitro. We measured calcium concentration, alizarin red staining, and alkaline phosphatase activity to examine the effect of NO on calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. We also determined gene expression and levels of phosphorylation of Smad2/3 by RT-PCR and western blotting. NO inhibited calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. An inhibitor of cyclic guanosine monophosphate (cGMP)-dependent protein kinase restored the inhibition by NO of osteoblastic differentiation and calcification of VSMCs. NO inhibited transforming growth factor-beta (TGF-beta)-induced phosphorylation of Smad2/3 and expression of TGF-beta-induced genes such as plasminogen activator inhibitor-1. In addition, NO inhibited expression of the TGF-beta receptor ALK5.. Our data show that NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-beta signalling through a cGMP-dependent pathway. Our findings suggest that NO may play a beneficial role in atherogenesis in part by limiting vascular calcification.

Topics: Animals; Calcinosis; Cell Differentiation; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Nitric Oxide; Osteoblasts; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases

The aim of the present study was to investigate the changes in heme-oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in clacified rat vascular smooth muscle cells (VSMCs).. Calcification of cultured rat VSMCs was induced by incubation of VSMCs with beta-glycerophosphate. Cellular calcium content, ALP activities and (45)Ca uptake were measured. HO activity, HbCO formation and content of cGMP in VSMCs were determined. Immunocytochemistry for HO-1 expression was observed.. In comparison of control VSMCs, the cellular calcium content, ALP activity and (45)Ca uptake in calcified VSMCs were obviously increased. Immunocytochemistry showed that HO-1 expression was weak and not well distributed in calcified cells as compared to non-calcified VSMCs, but interestingly, there was stronger staining in calcified nodules than in VSMCs. Compared with VSMCs, HO-1 activity in calcified cells decreased by 42.7% [36.4 +/- 2.8 pmol (mg Pr x h)(-1) vs 63.5 x 5.3 pmol (mg Pr x h)(-1), p < 0.01], and HbCO formation decreased by 39.2% (3.38 x 0.69 micromol/mg Pr vs 5.56 +/- 0.48 micromol/mg Pr, p < 0.05). The cGMP content in calcified VSMCs was 78.1% lower than that of non-calcified VSMCs (4.3 +/- 0.51 vs 19.6 +/- 1.2 pmol/mg, p < 0.01).. The results showed that HO-CO-cGMP pathway in calcified vascular cells obviously changed, which might contribute to disturbance of vascular function.

Topics: Animals; Calcinosis; Calcium; Carbon Monoxide; Carboxyhemoglobin; Cell Differentiation; Cells, Cultured; Cyclic GMP; Glycerophosphates; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Osteoblasts; Rats; Rats, Inbred Strains

The aim of the present study was to investigate the change in heme oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in vascular calcification. Vascular calcification model was established in rats by using vitamin D(3) and nicotine. Vascular calcium content, alkaline phosphatase (ALP) activity, HO activity, HbCO formation and content of cGMP in vessels were measured. Immunochemistry (IH) for HO 1 expression and in situ hybridization (ISH) for HO 1 mRNA were observed. Compared to those of control rats, the aortic calcium content and vascular ALP activity in rats of the calcified group (VDN group) were obviously increased, but HO 1 activity, CO concentration and cGMP content in vessels of rats in VDN group were markedly decreased. Expressions of HO-1 protein and mRNA were significantly decreased compared to control rats. Vascular calcification might induce a down regulation in vascular HO-CO-cGMP pathway.

Topics: Alkaline Phosphatase; Animals; Aorta, Thoracic; Calcinosis; Calcium; Carbon Monoxide; Cyclic GMP; Disease Models, Animal; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Immunoenzyme Techniques; In Situ Hybridization; Male; Rats; Rats, Wistar; RNA, Messenger

To clarify the regulating mechanism of vascular calcification, the investigators observed the effects of three vasoactive peptides, adrenomedullin (ADM), C-type natriuretic peptide (CNP), and parathyroid hormone-related peptide (PTHrP) on calcification in rat vascular smooth muscle cells (VSMCs). Beta-glycerophosphate stimulated growth and calcification in VSMCs. Adrenomedullin and CNP lowered beta-glycerophosphate-induced increase in VSMC growth. All three vasoactive peptides attenuated the increases of 45Ca accumulation, calcium content, and alkaline phosphatase activity in calcified VSMCs. As for comparing the inhibitory effects, the strongest was PTHrP. Both ADM and PTHrP increased cyclic adenosine monophosphate (cAMP) content in calcified VSMCs, but CNP upregulated cyclic guanosine monophosphate (cGMP) content. The PKA inhibitor PKAI completely reversed the inhibition of ADM on cell growth and all inhibitory effects of PTHrP on the parameters of calcification. The PKG inhibitor H8, however, strongly antagonized all the inhibitory effects of CNP on calcification. These data suggested that beta-glycerophosphate-induced calcification in VSMCs was inhibited by ADM, CNP, and PTHrP. Adrenomedullin and PTHrP inhibited VSMC calcification partially through the cAMP/PKA pathway, whereas CNP inhibited VSMC calcification through the cGMP/PKG pathway. This study could be of help in understanding the pathogenesis of vascular calcification, and providing new target for clinical treatment of cardiovascular diseases associated with vascular calcification.

Topics: Adrenomedullin; Alkaline Phosphatase; Animals; Aorta; Calcinosis; Calcium; Cell Count; Cell Division; Cells, Cultured; Culture Media; Cyclic AMP; Cyclic GMP; Glycerophosphates; Male; Muscle, Smooth, Vascular; Natriuretic Peptide, C-Type; Parathyroid Hormone-Related Protein; Peptides; Rats; Rats, Sprague-Dawley