臺灣博碩士論文加值系統

心臟及血管疾病是慢性腎衰竭病患常見造成相關器官併發症及死亡的首要原因。許多研究顯示慢性腎衰竭病患因為心血管疾病死亡的比例為非慢性腎衰竭病患的10到30倍，如何減少慢性腎衰竭病患心血管疾病的發生是臨床上的重要課題。PI3K/Akt/mTOR是細胞在面臨外來刺激的主要訊息傳導路徑，而Akt(即蛋白質激酶B)在細胞代謝、細胞存活、及轉錄過程中皆扮演重要角色。以往的研究顯示Akt及其上下游的訊息傳導路徑在因慢性腎衰竭導致的心臟及血管疾病應有重要的調控功能，但相關研究仍待進行。
動脈硬化的早期病變與血管內皮細胞及平滑肌細胞的功能失調有關，腎衰竭病患血管病變的特徵是動脈血管中層的血管平滑肌細胞肥大、增生及鈣化。第一型葡萄糖轉運蛋白()可促進葡萄糖運送至血管平滑肌細胞內而第一型葡萄糖轉運蛋白的表現增強會導致血管平滑肌細胞的增生。但第一型葡萄糖轉運蛋白在因慢性腎衰竭引發的血管平滑肌細胞的增生扮演的功能及其調控機轉目前仍不清楚。
本論文的第一部分希望能釐清在慢性腎衰竭環境下，第一型葡萄糖轉運蛋白在動脈血管平滑肌細胞的表現，並研究第一型葡萄糖轉運蛋白是否會受到Akt/TSC2/mTOR/S6K訊息傳導路徑的調控。在動物實驗，我們利用外科手術方法(六分之五腎臟切除術)來建立慢性腎衰竭的大鼠動物模式並觀察其相關的血管病變現象。大鼠主動脈標本的第一型葡萄糖轉運蛋白利用反轉錄-聚合酶連鎖反應、西方免疫墨點染色法及組織免疫化學染色法來偵測。在細胞實驗，則利用被證實具有促進血管平滑肌細胞增生功能的尿毒素硫酸吲哚酚來刺激A7r5大鼠主動脈血管平滑肌細胞株，並觀察在加入或未加入第一型葡萄糖轉運蛋白抑制劑下，血管平滑肌細胞株的增生分子標記及Akt/TSC2/mTOR/S6K表現。結果顯示慢性腎衰竭的大鼠主動脈病理切片可以明顯發現其血管平滑肌細胞有增生、肥大及退化的現象而且伴隨第一型葡萄糖轉運蛋白的訊息RNA和蛋白質表現增強。我們更進一步利用尿毒素硫酸吲哚酚及A7r5血管平滑肌細胞株來證實硫酸吲哚酚可透過Akt/TSC2/mTOR/S6K此一訊息傳導路徑來調控第一型葡萄糖轉運蛋白的表現並影響血管平滑肌細胞的增生。此外，尿毒素硫酸吲哚酚除了可以顯著的引發A7r5血管平滑肌細胞株的第一型葡萄糖轉運蛋白表現增強之外，同時會導致會促進細胞增生作用的週期素D1，p21及抗凋亡的p53訊息RNA增加。而當第一型葡萄糖轉運蛋白受到抑制時，上述硫酸吲哚酚引發促進細胞增生及抗凋亡的分子標記表現都會受到抑制。除了增加血管平滑肌細胞株的第一型葡萄糖轉運蛋白，硫酸吲哚酚明顯的會在6小時及12小時後抑制Akt和 TSC2的磷酸化。我們接著用Rapamycin來抑制mTOR的下游路徑，發現S6K的磷酸化及硫酸吲哚酚對於增加第一型葡萄糖轉運蛋白的作用都受到抑制。總結以上的結果，可以發現慢性腎衰竭可促進血管平滑肌細胞的第一型葡萄糖轉運蛋白表現增強。尿毒素硫酸吲哚酚促進A7r5血管平滑肌細胞株的增生可能是透過表現增強的第一型葡萄糖轉運蛋白來作用，而Akt/TSC2/mTOR/S6K訊息傳導路徑參與了在慢性腎衰竭環境下，在血管平滑肌細胞的第一型葡萄糖轉運蛋白表現增強的調控作用。
本論文的第二部分則在探討慢性腎衰竭對於血管內皮細胞自噬作用(autophagy)的影響，自噬作用目前已被證實和維持正常的血管功能有關。高血磷是慢性腎衰竭病患常有的現象也是動脈硬化的危險因子之一。從動物實驗發現，慢性腎衰竭的大鼠主動脈血管內皮細胞的自噬作用有增強的現象。接著我們在高磷環境下，發現HMEC-1血管內皮細胞株的自噬作用會受到Akt/mTOR訊息傳導路徑的調控。在高磷環境下，若將HMEC-1細胞株同時加入自噬作用抑制劑，運用免疫墨點染色法會發現細胞凋亡標記(Apoptosis marker)的表現有上升的現象，而此細胞早期凋亡的現象可再利用流式細胞儀來得到進一步的證實。所以慢性腎衰竭高血磷導致自噬作用的增強和抑制Akt/mTOR有關並且有助於內皮細胞免於凋亡(Apoptosis)的保護效應。
本論文的第三部分希望能瞭解第一型大麻受器對於尿毒性心肌病變的作用，並進一步探討此作用是否受到Akt的調控。第一型大麻受器在心肌肥大及心肌纖維化扮演重要的角色，而此兩個特徵即為尿毒性心肌病變的臨床表現，然而第一型大麻受器是否會調控尿毒性心肌病變仍未有相關研究。在動物實驗，我們將小鼠分成三組，第一組為對照組，第二及第三組皆為接受六分之五腎臟切除術的慢性腎衰竭小鼠，其中第三組另外接受每周一次、共七次的腹腔內注射第一型大麻受器拮抗劑。接著利用心臟超音波檢查來測量小鼠的心臟大小及收縮功能變化，而心肌纖維化的嚴重程度則利用組織免疫化學染色法來比較。在細胞方面，採用H9c2心肌纖維母細胞株來進行實驗。尿毒素硫酸吲哚酚刺激H9c2細胞株後，第一型大麻受器及相關的細胞纖維化標記會在有加入或未加入第一型陰離子轉運子抑制劑及第一型大麻受器拮抗劑/促進劑的情況下，利用免疫墨點染色法來測定。磷酸化的Akt亦會同時測量以釐清H9c2細胞株在經過尿毒素硫酸吲哚酚刺激後，其第一型大麻受器和下游訊息傳導路徑的調控。結果顯示慢性腎衰竭小鼠明顯有左心室肥大及心肌纖維化現象，在經過第一型大麻受器拮抗劑的注射後，小鼠的左心室肥大及心肌纖維化有明顯改善的現象。在細胞實驗，利用尿毒素硫酸吲哚酚來刺激H9c2細胞株，發現包括第一型大麻受器、纖維化標記第一型膠原蛋白、β轉化生長因子及α平滑肌動蛋白都跟時間和劑量有正相關的現象；此纖維化標記增加的效應皆可因第一型大麻受器拮抗劑或短干擾RNA的作用而受到抑制。磷酸化Akt的表現在H9c2細胞株可因第一型大麻受器促進劑而增加，並在使用第一型大麻受器拮抗劑後而有減少的現象。總結以上的結果，我們發現第一型大麻受器拮抗劑可以減少慢性腎衰竭小鼠左心室肥大及心肌纖維化現象。細胞實驗則顯示H9c2細胞株在經過尿毒素硫酸吲哚酚刺激後，第一型大麻受器及相關的細胞纖維化標記有表現增加的現象；此現象可被第一型大麻受器拮抗劑經過Akt訊息傳導路徑的調控而被抑制。因此發展第一型大麻受器拮抗劑的藥物對於臨床上治療尿毒性心肌病變將可能有效果。
經由以上三個研究發現，Akt在因為慢性腎衰竭導致的血管及心肌病變有重要的調控功能，同時也是未來在研發相關治療藥物的重要標的。

It has been well established that cardiovascular complications are the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). A number of epidemiologic investigations reported that the cardiovascular mortality rate in dialysis patients was 10-fold to 30-fold higher than that in the general population after stratification for age, sex, race, and the presence of diabetes. The PI3K/Akt/mTOR pathway is a major signal pathway in cellular response to various stimuli and Akt (also known as protein kinase B; PKB) appears to play an important role in regulation of metabolism, cell survival, motility and transcription. Previous studies suggested the implication of Akt signaling pathway in cardiovascular disease resulted from CKD. However, the integrity of Akt signaling in uremic vasculopathy and cardiomyopathy is not yet fully understood.
Uremic vasculopathy is characterized by medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx that leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear.
In the first part of thesis, we aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression. In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed. The results demonstrated that aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration eight weeks after 5/6 nephrectomy. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression. From in vivo and in vitro experiments, CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.
In the second part of thesis, we aimed to investigate whether autophagy is involved in the development of hyperphosphatemia-induced endothelial dysfunction and its regulatory mechanism. Hyperphosphatemia, resulting from decreased functional nephrons and dysregulated mineral metabolism, is a universal manifestation in advanced chronic kidney disease (CKD). Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction. To clarify this. the autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry. We found the in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increased in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt-mTOR signaling pathway. In HMEC-1 cells, high-Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by PFA through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy were induced by high Pi, and the inhibition of autophagy by 3-MA significantly aggravated high-Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells. We therefore concluded that hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high-Pi-induced apoptosis.
In the third part of thesis, we aimed to elucidate the role of CB1R in the development of uremic cardiomyopathy via modulation of Akt signalling. Cannabinoid receptor type 1 (CB1R) plays an important role in the development of myocardial hypertrophy and fibrosis- two pathological features of uremic cardiomyopathy. However, it remains unknown whether CB1R is involved in the pathogenesis of uremic cardiomyopathy. In vivo, the heart size and myocardial fibrosis were evaluated by echocardiography and immunohistochemical staining, respectively, in 5/6 nephrectomy chronic kidney disease (CKD) mice treated with a CB1R antagonist. In vitro, CB1R and fibrosis marker expression levels were determined by immunoblotting in H9c2 myofibroblast cells exposed to the uremic toxin indoxyl sulfate (IS), with an organic anion transporter 1 inhibitor or a CB1R antagonist or agonist. Akt phosphorylation was also assessed to examine the signaling pathways downstream of CB1R activation induced by IS in H9c2 cells. CKD mice exhibited marked left ventricular hypertrophy and myocardial fibrosis, which were reversed by treatment with the CB1R antagonist. CB1R, collagen I, transforming growth factor (TGF)-β, and α-smooth muscle actin (SMA) expression showed time- and dose-dependent upregulation in H9c2 cells treated with IS. The inhibition of CB1R by either CB1R antagonist or small interfering RNA-mediated knockdown attenuated the expression of collagen I, TGF-β, and α-SMA in IS-treated H9c2 cells, while Akt phosphorylation was enhanced by CB1R agonist and abrogated by CB1R antagonist in these cells. In summary, we conclude that CB1R blockade attenuates LVH and Akt-mediated cardiac fibrosis in a CKD mouse model. Uremic toxin IS stimulates the expression of CB1R and fibrotic markers and CB1R inhibition exerts anti-fibrotic effects via modulation of Akt signaling in H9c2 myofibroblasts. Therefore, the development of drugs targeting CB1R may have therapeutic potential in the treatment of uremic cardiomyopathy.
From these findings in this thesis, we can summarize that the PI3K/Akt/mTOR pathway is a major cellular signaling pivot in cellular response to extracellular stimuli and Akt appears to be a central player and maybe a therapeutic target in the regulation of vasculopathy and cardiomyopathy in uremic milieu.

論文電子檔著作權授權書…………………………………………………… I
論文審定同意書……………………………………………………………… II
誌謝…………………………………………………………………………… III
中文摘要……………………………………………………………………… IV-VI
英文摘要……………………………………………………………………… VII-X
目錄…………………………………………………………………………… XI
圖目錄………………………………………………………………………… XII-XIII
表目錄………………………………………………………………………… XIV
第一章 序論………………………………………………………………… 1-7
第二章 材料及方法………………………………………………………… 8-19
第三章 結果………………………………………………………………… 20-26
第四章 討論………………………………………………………………… 27-36
第五章 結論………………………………………………………………… 37-39
末來展望……………………………………………………………………… 40
參考文獻……………………………………………………………………… 41-51
圖……………………………………………………………………………… 52-75
表……………………………………………………………………………… 76
發表文章……………………………………………………………………… 77

1. Briet M, Boutouyrie P, Laurent S, London GM. Arterial stiffness and pulse pressure in CKD and ESRD. Kidney Int 2012; 82: 388–400.
2. Foley RN, Collins AJ. End-stage renal disease in the United States: An update from the United Stated Renal Data System. J Am Soc Nephrol 2007; 18: 2644-8.
3. Schiffrin EL, Lipman ML, Mann JF. Chronic kidney disease: effects on the cardiovascular system. Circulation 2007;116:85-97.
4. Shao JS, Cai J, Towler DA. Molecular mechanisms of vascular calcifica-tion: lessons learned from the aorta. Arterioscler Thromb Vasc Biol 2006;26:1423-30.
5. Shanahan CM, Cary NR, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating pro-teins in association with Monckeberg’s sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999;100: 2168-76.
6. Lehto S, Niskanen L, Suhonen M, Rönnemaa T, Laakso M. Medial artery calcification. A neglected harbinger of cardiovascular complica-tions in non-insulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol 1996;16:978-83.
7. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death,cardiovascular events, and hospitalization. N Engl J Med 2004; 351: 1296-305.
8. Smith GL, Lichtman JH, Bracken MB, Shlipak MG, Phillips CO, DiCapua P, et al. Renal impairment and outcomes in heart failure: systemic reviews and meta-analysis. J Am Coll Cardiol 2006; 47: 1987-96.
9. Amann K, Tyralla K, Gross ML, Schwarz U, Tornig J, Haas CS, et al. Cardiomyocyte loss in experimental renal failure: prevention by ramipril. Kidney Int 2003; 63: 1708-13.
10. Amann K, Kronenberg G, Gehlen F, Wessels S, Orth S, Munter K, et al. Cardiac remodelling in experimental renal failure—an immunohistochemical study. Nephrol Dial Transplant 1998; 13: 1958–66.
11. Cohn JN, Ferrari R, Sharpe N. Cardiac remodeling-concepts and clinical implications: A consensus paper from an international forum on cardiac remodeling. Behalf of an international Forum on cardiac remodeling. J Am Coll Cardiol 2000;35:568-82.
12. Nitta K, Iimuro S, Imai E, Matsuo S, Makino H, Akizawa T, et al. Risk factors for increased left ventricular hypertrophy in patients with chronic kidney disease. Clin Exp Nephrol 2013; 17: 730-42.
13. Amann K, Ritz E, Wiest G, Klaus G, Mall G. A role of parathyroid hormone for the activation of cardiac fibroblasts in uremia. J Am Soc Nephrol 1994; 4: 1814–9.
14. Creager MA, Lüscher TF, Cosentino F, Beckman JA. Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part I. Circulation 2003;108:1527-32.
15. Mazzone T, Chait A, Plutzky J. Cardiovascular disease risk in type 2 diabetes mellitus: insights from mechanistic studies. Lancet 2008;371: 1800-9.
16. Hattori Y, Hattori S, Sato N, Kasai K. High-glucose-induced nuclear factor kappaB activation in vascular smooth muscle cells. Cardiovasc Res 2000;46:188-97.
17. Srivastava AK. Hyperglycemia-induced protein kinase signaling path-ways in vascular smooth muscle cells: implications in the pathogenesis of vascular dysfunction in diabetes. Adv Exp Med Biol 2001;498:311-8.
18. Igarashi M, Wakasaki H, Takahara N, Ishii H, Jiang ZY, Yamauchi T, et al. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest 1999;103:185-95.
19. Amiri F, Venema VJ, Wang X, Ju H, Venema RC, Marrero MB. Hyperglycemia enhances angiotensin II-induced Janus-activated kinase/STAT signaling in vascular smooth muscle cells. J Biol Chem 1999;274:32382-6.
20. Suzuki LA, Poot M, Gerrity RG, Bornfeldt KE. Diabetes accelerates smooth muscle accumulation in lesions of atherosclerosis: lack of direct growth-promoting effects of high glucose levels. Diabetes 2001;50: 851-60.
21. White MF. Insulin signaling in health and disease. Science 2003;302: 1710-1.
22. Avena R, Mitchell ME, Neville RF, Sidawy AN. The additive effects of glucose and insulin on the proliferation of infragenicular vascular smooth muscle cells. J Vasc Surg 1998;28:1033-8; discussion: 1038-9.
23. Hruz PW, Mueckler MM. Structural analysis of the GLUT1 facilitative glucose transporter (review). Mol Membr Biol 2001;18:183-93.
24. Kaiser N, Sasson S, Feener EP, Boukobza-Vardi N, Higashi S, Moller DE, et al. Differential regulation of glucose transport and transporters by glucose in vascular endothelial and smooth muscle cells. Diabetes 1993;42:80-9.
25. Buller CL, Loberg RD, Fan MH, Zhu Q, Park JL, Vesely E, et al. A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression. Am J Physiol Cell Physiol 2008;295: C836-43.
26. Loberg RD, Vesely E, Brosius FC 3rd. Enhanced glycogen synthase kinase-3beta activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metab-olism. J Biol Chem 2002;277:41667-73.
27. Vesely ED, Heilig CW, Brosius FC 3rd. GLUT1-induced cFLIP ex-pression promotes proliferation and prevents apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol 2009;297:C759-65.
28. Zhou J, Deo BK, Hosoya K, Terasaki T, Obrosova IG, Brosius FC 3rd, et al. Increased JNK phosphorylation and oxidative stress in response to increased glucose flux through increased GLUT1 expression in rat retinal endothelial cells. Invest Ophthalmol Vis Sci 2005;46:3403-10.
29. Hall JL, Chatham JC, Eldar-Finkelman H, Gibbons GH. Upregulation of glucose metabolism during intimal lesion formation is coupled to the inhibition of vascular smooth muscle cell apoptosis. Role of GSK3beta. Diabetes 2001;50:1171-9.
30. Chen NX, Duan D, O’Neill KD, Moe SM. High glucose increases the expression of Cbfa1 and BMP-2 and enhances the calcification of vascular smooth muscle cells. Nephrol Dial Transplant 2006;21: 3435-42.
31. Rufino M, Hernández D, Barrios Y, Salido E. The GLUT-1 XbaI gene polymorphism is associated with vascular calcifications in nondiabetic uremic patients. Nephron Clin Pract 2008;108:c182-7.
32. Proud CG. MTOR signalling in health and disease. Biochem Soc Trans 2011;39:431-6.
33. Craver L, Marco MP, Martinez I, Rue M, Borras M, Martin ML, et al. Mineral metabolism parameters throughout chronic kidney disease stages 1-5--achievement of K/DOQI target ranges. Nephrol Dial Transplant 2007; 22: 1171-1176.
34. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med 2004; 351: 1296-1305.
35. Ganesh SK, Stack AG, Levin NW, Hulbert-Shearon T, Port FK. Association of elevated serum PO(4), Ca x PO(4) product, and parathyroid hormone with cardiac mortality risk in chronic hemodialysis patients. J Am Soc Nephrol 2001; 12: 2131-2138.
36. Block GA, Klassen PS, Lazarus JM, Ofsthun N, Lowrie EG, Chertow GM. Mineral metabolism, mortality, and morbidity in maintenance hemodialysis. J Am Soc Nephrol 2004; 15: 2208-2218.
37. Shanahan CM, Cary NR, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999; 100: 2168-2176.
38. Moe SM, Chen NX. Pathophysiology of vascular calcification in chronic kidney disease. Circ Res 2004; 95: 560-567.
39. Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thrombo Vas Biol 2003; 23: 489-494.
40. Dhingra R, Sullivan LM, Fox CS, Wang TJ, D'Aqostino RB Sr, Gaziano JM, et al. Relations of serum phosphorus and calcium levels to the incidence of cardiovascular disease in the community. Arch Intern Med 2007; 167: 879-885.
41. Di Marco GS, Hausberg M, Hillebrand U, Rustemeyer P, Wittkowski W, Lang D, et al. Increased inorganic phosphate induces human endothelial cell apoptosis in vitro. Am J Physiol Renal Physiol 2008; 294: F1381-1387.
42. Peng A, Wu T, Zeng C, Rakheja D, Zhu J, Ye T, et al. Adverse effects of simulated hyper- and hypo-phosphatemia on endothelial cell function and viability. PLoS One 2011; 6: e23268.
43. Shuto E, Taketani Y, Tanaka R, Harada N, Isshiki M, Sato M, et al. Dietary phosphorus acutely impairs endothelial function. J Am Soc Nephrol 2009; 20: 1504-1512.
44. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000; 290: 1717-1721.
45. Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med 2013; 368: 1845-1846.
46. Martinet W, Knaapen MW, Kockx MM, De Meyer GR. Autophagy in cardiovascular disease. Trends Mol Med 2007; 13: 482-491.
47. Nemchenko A, Chiong M, Turer A, Lavandero S, Hill JA. Autophagy as a therapeutic target in cardiovascular disease. J Mol Cell Cardiol 2011; 51: 584-593.
48. Lavandero S, Troncoso R, Rothermel BA, Martinet W, Sadoshima J, Hill JA. Cardiovascular autophagy: Concepts, controversies and perspectives. Autophagy 2013; 9: (Epub ahead of print).
49. Schrijvers DM, De Meyer GR, Martinet W. Autophagy in atherosclerosis: a potential drug target for plaque stabilization. Arterioscler Thromb Vasc Biol 2011; 31: 2787-2791.
50. Martinet W, De Meyer GR. Autophagy in atherosclerosis: a cell survival and death phenomenon with therapeutic potential. Circ Res 2009; 104: 304-317.
51. Yang Z, Klionsky DJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol 2010; 22: 124-131.
52. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 2009; 43: 67-93.
53. Chong ZZ, Shang YC, Maiese K. Cardiovascular disease and mTOR signaling. Trends Cardiovasc Med 2011; 21: 151-155.
54. Lin CY, Hsu SC, Lee HS, et al. Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) pathway in experimental renal failure. J Vasc Surg 2013; 57: 475-485.
55. Fayard E, Tintignac LA, Baudry A, Hemmings BA. Protein kinase B/Akt at a glance. J Cell Sci 2005; 118: 5657-8.
56. LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updat 2008; 11: 32-50.
57. Sekulic A, Hudson CC, Homme JL, Yin P, Otterness DM, Kamitz LM, et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 2000;60:3504–13.
58. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006;124:471–84.
59. Brito PM, Devillard R, Negre-Salvayre A, Almeida LM, Dinis TC, Salvayre R, et al. Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells. Atherosclerosis 2009; 205: 126-34.
60. Block GA, Klassen PS, Lazarus JM, Ofsthun N, Lowrie EG, Chertow GM. Mineral metabolism, mortality, and morbidity in maintenance hemodialysis. J Am Soc Nephrol 2004; 15: 2208-18.
61. Shanahan CM, Cary NR, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 1999; 100: 2168-76.
62. Moe SM, Chen NX. Pathophysiology of vascular calcification in chronic kidney disease. Circ Res 2004; 95: 560-7.
63. Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thrombo Vas Biol 2003; 23: 489-94.
64. Dhingra R, Sullivan LM, Fox CS, Wang TJ, D'Aqostino RB Sr, Gaziano JM, et al. Relations of serum phosphorus and calcium levels to the incidence of cardiovascular disease in the community. Arch Intern Med 2007; 167: 879-85.
65. Hsu YJ, Hsu SC, Huang SM, Lee HS, Lin SH, Tsai CS, et al. Hyperphosphatemia induces protective autophagy in endothelial cells through the inhibition of Akt/mTOR signaling. J Vasc Surg. 2015;62:210-21.
66. Siedlecki AM, Jin X, Muslin AJ: Uremic cardiac hypertrophy is reversed by rapamycin but not by lowering of blood pressure. Kidney Int 2009; 75: 800–8.
67. Semple D, Smith K, Bhandari S, Seymour AM. Uremic cardiomyopathy and insulin resistance: A critical role for Akt? J Am Soc Nephrol 2011; 22:207-15.
68. Silberberg JS, Barre PE, Prichard SS, Sniderman AD. Impact of left ventricular hypertrophy on survival in end-stage renal disease. Kidney Int 1989; 36: 286–90.
69. Levin A, Thompson CR, Ethier J, Carlisle EJ, Tobe S, Mendelssohn D,et al. Left ventricular mass index increase in early renal disease: Impact of decline in hemoglobin. Am J Kidney Dis 1999; 34: 125–34.
70. London GM, Pannier B, Guerin AP, Blacher J, Marchais SJ, Darne B, et al. Alterations of left ventricular hypertrophy in and survival of patients receiving hemodialysis: Follow-up of an interventional study. J Am Soc Nephrol 2001; 12:2759–67.
71. Scheid MP, Woodgett JR. PKB/AKT: functional insights from genetic models. Nat Rev, Mol Cell Biol 2001; 2: 760– 8.
72. Scheid MP, Woodgett JR. Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett 2003; 546: 108–12.
73. Vanhaesebroeck B, Alessi DR. The P13K–PDK1 connection: more than just a road to PKB. Biochem J 2000; 346: 561–76.
74. Leevers SJ. Growth control: invertebrate insulin surprises! Curr Biol 2001;11:R209–12.
75. Leevers SJ, Weinkove D, MacDougall LK, Hafen E, Waterfield MD. The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J 1996; 15: 6584– 94.
76. Shioi T, McMullen JR, Kang PM, Douglas PS, Obata T, Franke TF, et al. Akt/protein kinase B promotes organ growth in transgenic mice. Mol Cell Biol 2002; 22: 2799– 809.
77. Condorelli G, Drusco A, Stassi G, Bellacosa A, Roncarati R, Iaccarino G, et al. Akt induces myocardial contractility and cell size in vivo in transgenic mice. Proc Natl Acad Sci U S A 2002; 99: 12333 –8.
78. Matsui T, Li L, Wu JC, Cook SA, Nagoshi T, Picard MH, et al. Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J Biol Chem 2002; 277: 22901–6.
79. Everett AD, Stoops TD, Nairn AC, Brautigan D. Angiotensin II regulates phosphorylation of translation elongation factor-2 in cardiac myocytes. Am J Physiol 2001; 281: H161–7.
80. Pacher P, Bátkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 2006; 58: 389–462.
81. Engeli S. Dysregulation of the endocannabinoid system in obesity. J Neuroendocrinol 2008; 20: S110–5.
82. Di Marzo V. The endocannabinoid system in obesity and type 2 diabetes. Diabetologia 2008; 51: 1356–67.
83. Engeli S, Böhnke J, Feldpausch M, Gorzelniak K, Janke J, Bátkai S, et al. Activation of the peripheral endocannabinoid system in human obesity. Diabetes 2005; 54: 2838–43.
84. Silvestri C, Ligresti A, Di Marzo V. Peripheral effects of the endocannabinoid system in energy homeostasis: adipose tissue, liver and skeletal muscle. Rev Endocr Metab Disord 2011; 12: 153–62.
85. Mukhopadhyay P, Bátkai S, Rajesh M, Czifra N, Harvey-White J, Hasko G, et al. Pharmacological inhibition of CB1 cannabinoid receptor protects against doxorubicin-induced cardiotoxicity. J Am Coll Cardiol 2007; 50: 528–36.
86. Mukhopadhyay P, Rajesh M, Bátkai S, Patel V, Kashiwaya Y, Liaudel L, et al. CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicininduced cardiomyopathy and in human cardiomyocytes. Cardiovasc Res 2010; 85: 773–84.
87. Mukhopadhyay P, Horváth B, Rajesh M, Matsumoto S, Saito K, Bátkai S, et al. Fatty acid amide hydrolase is a key regulator of endocannabinoid-induced myocardial tissue injury. Free Radic Biol Med 2011; 50: 179–95.
88. Rajesh M, Bátkai S, Kechrid M, Mukhopadhyay P, Lee WS, Horváth B, et al. Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy. Diabetes 2012; 61:716-27.
89. Siedlecki AM, Jin X, Muslin AJ: Uremic cardiac hypertrophy is reversed by rapamycin but not by lowering of blood pressure. Kidney Int 2009; 75: 800–8.
90. Lusis AJ. Atherosclerosis. Nature 2000;407:233–41.
91. Remmele W, Stegner HE. [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe 1987; 8:138-40.
92. Yang HC, Zuo Y, Fogo AB. Models of chronic kidney disease. Drug Discov Dis Models 2010; 7: 13-9.
93. Kennedy DJ, Elkareh J, Shidyak A, Shapiro AP, Smaili S, Mutgi K, et al. Partial nephrectomy as a model for uremic cardiomyopathy in the mouse. Am J Physiol Renal Physiol. 2008; 294: F450–4.
94. Berti G, Fossati P, Tarenghi G, Musitelli C, Melzi d’Eril GV. Enzymatic colorimetric method for the determination of inorganic phosphorus in serum and urine. J Clin Chem Clin Biochem 1988; 26: 399-404.
95. Cheng TH, Shih NL, Chen SY, Wang DL, Chen JJ. Reactive oxygen species modulate endothelin-I_induced c-fos gene expression in cardiomyocytes. Cardiovasc Res 1999; 41: 654-62.
96. Yamamoto H, Tsuruoka S, Ioka T, Ando H, Ito C, Akimoto T, et al. Indoxyl sulfate stimulates proliferation of rat vascular smooth muscle cells. Kidney Int 2006;69:1780-5.
97. Weiss RH, Joo A, Randour C. p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J Biol Chem 2000;275:10285-90.
98. Mercer J, Figg N, Stoneman V, Braganza D, Bennett MR. Endogenous p53 protects vascular smooth muscle cells from apoptosis and reduces atherosclerosis in ApoE knockout mice. Circ Res 2005;96:667-74.
99. Amann K, Wolf B, Nichols C, Törnig J, Schwarz U, Zeier M, et al. Aortic changes in experimental renal failure: hyperplasia or hypertrophy of smooth muscle cells? Hypertension 1997;29:770-5.
100. Cai L, Kang YJ. Oxidative stress and diabetic cardiomyopathy: a brief review. Cardiovasc Toxicol 2001;1:181-93.
101. Park JL, Heilig CW, Brosius FC 3rd. GLUT1-deficient mice exhibit impaired endothelium-dependent vascular relaxation. Eur J Pharmacol 2004;496:213-4.
102. Liao R, Jain M, Cui L, D’Agostino J, Aiello F, Luptak I, et al. Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice. Circulation 2002;106:2125-31.
103. Adhikari N, Basi DL, Carlson M, Mariash A, Hong Z, Lehman U, et al. Increase in GLUT1 in smooth muscle alters vascular contractility and increases inflammation in response to vascular injury. Arterioscler Thromb Vasc Biol 2011;31:86-94.
104. Luptak I, Yan J, Cui L, Jain M, Liao R, Tian R. Long-term effects of increased glucose entry on mouse hearts during normal aging and ischemic stress. Circulation 2007;116:901-9.
105. Muteliefu G, Enomoto A, Niwa T. Indoxyl sulfate promotes prolifera-tion of human aortic smooth muscle cells by inducing oxidative stress. J Ren Nutr 2009;19:29-32.
106. Lin Z, Weinberg JM, Malhotra R, Merritt SE, Holzman LB, Brosius FC 3rd. GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation. Am J Physiol Endocrinol Metab 2000;278:E958-66.
107. Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL. Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res 2000;87:1055-62.
108. Zhou QL, Jiang ZY, Holik J, Chawla A, Hagan GN, Leszyk J, et al. Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes. Biochem J 2008;411:647-55.
109. Nowicki M, Zabirnyk O, Duerrschmidt N, Borlak J, Spanel-Borowski K. No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy. Eur J Cell Biol 2007; 86: 605-616.
110. Shen W, Tian C, Chen H, Yang Y, Zhu D, Gao P, et al. Oxidative stress mediates chemerin-induced autophagy in endothelial cells. Free Radic Biol Med 2013; 55: 73-82.
111. Wang Q, Liang B, Shirwany NA, Zou MH. 2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase. PLoS One2011; 6: e17234.
112. Dong Z, Wang L, Xu J, Li Y, Zhang Y, Zhang S, et al. Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells. Toxicol In Vitro 2009; 23: 105-110.
113. Xie Y, You SJ, Zhang YL, Han Q, Cao YJ, Xu XS, et al. Protective role of autophagy in AGE-induced early injury of human vascular endothelial cells. Mol Med Rep 2011; 4: 459-464.
114. Han J, Pan XY, Xu Y, Xiao Y, An Y, Tie L, et al. Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage. Autophagy 2012; 8: 812-825.
115. Khan MJ, Rizwan Alam M, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, et al. Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells. J Biol Chem 2012; 287: 21110-21120.
116. Hernando N, Forster IC, Biber J, Murer H. Molecular characteristics of phosphate transporters and their regulation. Exp Nephrol 2000; 8: 366-375.
117. Virkki LV, Biber J, Murer H, Forster IC. Phosphate transporters: a tale of two solute carrier families. Am J Physiol Renal Physiol 2007; 293: F643-654.
118. Li X, Giachelli CM. Sodium-dependent phosphate cotransporters and vascular calcification. Curr Opin Nephrol Hypertens 2007; 16: 325-328.
119. Li X, Yang HY, Giachelli CM. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification. Circ Res 2006; 98: 905-912.
120. Di Marco GS, Konig M, Stock C, Wiesinger A, Hillebrand U, Reiermann S, et al. High phosphate directly affects endothelial function by downregulating annexin II. Kidney Int 2013; 83: 213-222.
121. LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updat 2008; 11: 32-50.
122. Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M, Ohsumi Y. Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J Cell Biol 2000; 150: 1507-1513.
123. Neufeld TP. TOR-dependent control of autophagy: biting the hand that feeds. Curr Opin Cell Biol 2010; 22: 157-168.
124. Martinet W, Schrijvers DM, Timmermans JP, Bult H. Interactions between cell death induced by statins and 7-ketocholesterol in rabbit aorta smooth muscle cells. Br J Pharmacol 2008; 154: 1236-1246.
125. Hamacher-Brady A, Brady NR, Gottlieb RA. The interplay between pro-death and pro-survival signaling pathways in myocardial ischemia/reperfusion injury: apoptosis meets autophagy. Cardiovasc Drugs Ther 2006; 20: 445-462.
126. Hamacher-Brady A, Brady NR, Logue SE, Sayen MR, Jinno M, Kirshenbaum LA, et al. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ 2007; 14: 146-157.
127. Rudolph A, Abdel-Aty H, Bohl S, Boye P, Zagrosek A, Dietz R, et al. Noninvasive detection of fibrosis applying contrast-enhanced cardiac magnetic resonance in different forms of left ventricular hypertrophy: relation to remodeling. J Am Coll Cardiol 2009; 53: 284–91.
128. Mark PB, Johnston N, Groenning BA, Foster JE, Blyth KG, Martin TN, et al. Redefinition of uremic cardiomyopathy by contrast-enhanced cardiac magnetic resonance imaging. Kidney Int 2006; 69:1839-45.
129. Edwards NC, Ferro CJ, Townend JN, Steeds RP. Aortic distensibility and arterial-ventricular coupling in early chronic kidney disease: a pattern resembling heart failure with preserved ejection fraction. Heart 2008; 94: 1038-43.
130. Tyralla K, Amann K. Cardiovascular changes in renal failure. Blood Purif 2002; 20: 462-5.
131. Tyralla K, Amann K. Morphology of the heart and arteries in renal failure. Kidney Int 2003; 63: S80–3.
132. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008; 214: 199-210.
133. Lijnen PJ, Petrov VV, Fagard RH. Induction of cardiac fibrosis by transforming growth factor-beta(1). Mol Genet Metab 2000; 71: 418-35.
134. Lijnen P, Petrov V. Induction of cardiac fibrosis by aldosterone. J Mol Cell Cardiol 2000; 32: 865-79.
135. Pohlers D, Brenmoehl J, Loffler I, Muller CK, Leipner C, Schultze-Mosqau S, et al. TGF-β and fibrosis in different organs-molecular pathway imprints. Biochim Biophys Acta 2009; 1792: 746-56.
136. London GM. Cardiovascular disease in chronic renal failure: pathophysiologic aspects. Semin Dial 2003; 16: 85-94.
137. Alhaj E, Alhaj N, Rahman I, Niazi TO, Berkowitz R, Klapholz M. Uremic cardiomyopathy: an underdiagnosed disease. Congest Heart Fail 2013; 19: E40-5.
138. Lekawanvijit S, Adrahtas A, Kelly DJ, Kompa AR, Wang BH, Krum H. Does indoxyl sulfate, a uraemic toxin, have direct effects on cardiac fibroblasts and myocytes? Eur Heart J 2010; 31:1771-9.
139. Lekawanvijit S, Kompa AR, Manabe M, Wang BH, Langham RG, Nishijima F, et al. Chronic kidney disease-induced cardiac fibrosis is ameliorated by reducing circulating levels of a non-dialysable uremic toxin, indoxyl sulfate. PLoS One 2012; 7: e41281.
140. Barreto FC, Barreto DV, Liabeuf S, Meert N, Glorieux G, Temmar M, et al. European Uremic Toxin Work Group (EUTox). Serum indoxyl sulfate is associated with vascular disease and mortality in chronic kidney disease patients. Clin J Am Soc Nephrol 2009; 4:1551-8.
141. Shimazu S, Hirashiki A, Okumura T, Yamada T, Okamoto R, Shinoda N, et al. Association between indoxyl sulfate and cardiac dysfunction and prognosis in patients with dilated cardiomyopathy. Circ J 2013; 77: 390-6.
142. Walsh K. Akt signaling and growth of the heart. Circulation 2006; 113:2032-4.
143. DeBosch B, Sambandam N, Weinheimer C, Courtois M, Muslin AJ: Akt2 regulates cardiac metabolism and cardiomyocyte survival. J Biol Chem 2006; 281: 32841-51.
144. Hescheler J, Meyer R, Plant S, Plant S, Krautwurst D, Rosenthal W, Schultz G. Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ Res 1991; 69: 1476–86.
145. Bugyei-Twum A, Advani A, Advani SL, Zhang Y, Thai K, Kelly DJ, et al. High glucose induces Smad activation via the transcriptional coregulator p300 and contributes to cardiac fibrosis and hypertrophy. Cardiovasc Diabetol 2014;13:89.
146. Zong J, Zhang DP, Zhou H, Bian ZY, Deng W, Dai J, et al. Baicalein protects against cardiac hypertrophy through blocking MEK-ERK1/2 signaling. J Cell Biochem 2013; 114: 1058-65.
147. Bai Y, Cui W, Xin Y, Miao X, Barati MT, Zhang C, et al. Prevention by sulforaphane of diabetic cardiomyopathy is associated with up-regulation of Nrf2 expression andtranscription activation. J Mol Cell Cardiol 2013; 57: 82-95.
148. Singla DK, Singla RD, Lamm S, Glass C. TGF-β2 treatment enhances cytoprotective factors released from embryonic stem cells and inhibits apoptosis in infarcted myocardium. Am J Physiol Heart Circ Physiol 2011; 300: H1442-50.
149. Ghiggeri GM, Oleggini R, Musante L, Caridi G, Gusmano R, Ravazzolo R. A DNA element in the alpha1 type III collagen promoter mediates a stimulatory response by angiotensin II. Kidney Int 2000; 58:537-48.