Intermedin, a novel member of calcitonin gene-related peptide family, is an endogenous cardiovascular-protective peptide. Because intermedin exists in human atherosclerotic plaque, we studied the role of intermedin in macrophage scavenger receptor A (SR-A)-mediated foam-cell formation and atherogenesis. In an in vitro foam-cell formation model (induced by acetylated low-density lipoprotein [AcLDL]) with mouse (C57BL/6J) macrophages, intermedin reduced AcLDL uptake and binding, decreased intracellular cholesterol content, and suppressed both mRNA and protein levels of SR-A. Simultaneously, intermedin increased phosphatase and tensin homolog (PTEN) protein levels by increasing PTEN phosphorylation and inhibiting ubiquitin-mediated PTEN degradation. These effects were blocked by the intermedin receptor antagonist or cAMP-protein kinase A inhibitors. PTEN overexpression mimicked the inhibitory effects of intermedin on SR-A expression and AcLDL uptake. However, knockdown of PTEN by short-hairpin RNA completely blocked all inhibitory effects of intermedin. Furthermore, in apolipoprotein E-deficient (apoE(-/-)) mice, 6-week intermedin infusion reduced AcLDL uptake and SR-A mRNA and protein levels and increased PTEN protein level in peritoneal macrophages. PTEN level was increased and SR-A expression decreased in parallel in macrophages in atherosclerotic lesions. Thus, intermedin inhibited atherosclerosis in apoE(-/-) mice. Increased stability of FTEN by intermedin leads to SR-A inhibition in macrophages, which ameliorates foam-cell formation and atherosclerosis in apoE(-/-) mice. (c) 2012 Elsevier Ltd. All rights reserved.

Hyperphosphatemia is the major risk factor associated with vascular calcification (VC) in end-stage renal disease. As oxidative stress is increased in uremia, we studied the role of mitochondrial reactive oxygen species (ROS) and nuclear factor-kappa B signaling in phosphate-induced VC. In an in vitro calcification model (beta-glycerophosphate (BGP) induction) using bovine aortic smooth muscle cells, the production of intracellular and mitochondrial ROS, or superoxide anion, was stimulated by increased mitochondrial membrane potential. This effect was blocked by the superoxide dismutase (SOD) mimic MnTMPyP, a respiratory chain inhibitor rotenone, or a protonophore. Calcium deposition and the switch of smooth muscle cells from a contractile to an osteogenic phenotype were decreased when mitochondrial ROS generation was inhibited by the respiratory chain inhibitor, MnTMPyP, or the overexpression of SOD1 and SOD2 and uncoupling protein 2. The phosphorylation of IkK beta, I kappa B alpha degradation, and p65 nuclear translocation were increased by BGP but reversed when mitochondrial ROS production was blocked by protonophore or MnTMPyP. Knockdown of endogenous p65 or overexpression of I kappa B alpha reduced calcium deposition in the cultured cells. Furthermore, in a rat model of dietary adenine-induced chronic renal failure, MnTMPyP reduced aortic ROS levels, p65 activation, and calcium deposition. Thus, mitochondrial ROS-mediated p65 nuclear translocation is involved in phosphate-induced VC. Kidney International (2011) 79, 1071-1079; doi:10.1038/ki.2011.18; published online 2 March 2011

Autophagy is a dynamic and highly regulated process of self-digestion responsible for cell survival and reaction to oxidative stress. As oxidative stress is increased in uremia and is associated with vascular calcification, we studied the role of autophagy in vascular calcification induced by phosphate. In an in vitro phosphate-induced calcification model of vascular smooth muscle cells (VSMCs) and in an in vivo model of chronic renal failure, autophagy was inhibited by the superoxide dismutase mimic MnTMPyP, superoxide dismutase-2 overexpression, and by knockdown of the sodium-dependent phosphate cotransporter Pit1. Although phosphate-induced VSMC apoptosis was reduced by an inhibitor of autophagy (3-methyladenine) and knockdown of autophagy protein 5, calcium deposition in VSMCs was increased during inhibition of autophagy, even with the apoptosis inhibitor Z-VAD-FMK. An inducer of autophagy, valproic acid, decreased calcification. Furthermore, 3-methyladenine significantly promoted phosphate-induced matrix vesicle release with increased alkaline phosphatase activity. Thus, autophagy may be an endogenous protective mechanism counteracting phosphate-induced vascular calcification by reducing matrix vesicle release. Therapeutic agents influencing the autophagic response may be of benefit to treat aging or disease-related vascular calcification and osteoporosis.

本项目基本按照研究计划完成所有预期内容，通过本项目的研究证实了：首先高磷通过增加线粒体膜电位，促进线粒体氧自由基(ROS)产生增加，激活致炎信号NF-kB,是促使血管平滑肌向成骨/成软骨表型转化和最终导致钙化发生的重要环节之一；而另一方面，高磷引起的ROS产生增加，是引起平滑肌细胞自噬增加的重要原因，而抑制自噬通过促进基质小泡释放而加速血管钙化的发生和发展，提示自噬可能是血管平滑肌细胞对抗钙化的自我保护的机制之一；此外本研究证实在人钙化血管组织和肾衰大鼠血浆中炎症因子TNF-a及其下游NF-κB激活水平均增加;TNF-a可以通过激活NF-κB信号，促进mRNA去稳定因子TTP(Tristetraprolin)的表达增加，从而使钙化抑制因子ANKH(progressive ankylosis protein homolog)的表达减少，进而加重高磷诱导的平滑肌细胞钙沉积；最后证明IEX-1蛋白在钙化平滑肌细胞中表达下降，而过表达IEX-1可以抑制高磷诱导的平滑肌细胞ROS增加，并抑制高磷诱导的平滑肌细胞钙化和表型转换。基于本项目研究成果在kidney Int杂志(IF 6.6)共发表系列研究论文3篇，其中一篇配发了编辑述评，另一篇为该期杂志的封面文章；同时培养博士研究生3名，目前三名博士均在美国进行博士后训练。

Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-kappa B) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-kappa B by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-kappa B activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-kappa B and decreased ANKH levels. In contrast, the inhibition of NF-kappa B maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-kappa B and decreased ANKH expression. Thus, TNF-activated NF-kappa B promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion. Kidney International (2012) 82, 34-44; doi: 10.1038/ki.2012.40; published online 21 March 2012