Surgical valve replacement is the only available therapy for calcific aortic valve stenosis. Although chronic Inflammation is believed to play a major role in the pathogenesis and progression of aortic valve lesions, the signaling mechanisms that initiate and sustain the inflammatory process in valvular cells remain unclear. Understanding of the inflammatory and osteogenic responses in valvular cells is important for prevention aortic valve lesions and their progression. We demonstrate that stimulating Toll-like receptor 4 (TLR4) with endotoxin up-regulates the expression of bone morphogenetic protein 2 (BMP-2) in human aortic valve myofibroblasts (HAVMFs) and that BMP-2 plays a major role in TLR4-induced osteogenic responses, characterized by expression of Runx2, alkaline phosphatase (ALP) and osteopontin. Further, we found that chronic stimulation of TLR4 promotes aortic valve calcification and thickening in old mice. Preliminary studies show that TLR4 signaling up-regulates the levels of Notch1 intracellular domain (NICD1) in HAVMFs. Neutralizing INF?, a TRIF-regulated type 1 interferon, abrogates the increase in cellular NICD1 and reduces BMP-2 levels following stimulation of TLR4. Further, inhibition of Notch1 also reduces BMP-2 expression following TLR4 stimulation. Together, these novel findings indicate that INF? activates Notch1 and that NICD1 enhances TLR4-induced BMP-2 expression. Interestingly, elevated levels of lNICD1 and BMP-2 coexist in human AVMFs at 24 h following TLR4 stimulation. It is likely that NICD1 plays a role not only in regulating BMP-2 expression in the early phase but also in modulating BMP-2 signaling. Indeed, preliminary studies show that activation of Notch1 enhances BMP-2-induced expression of Runx2. Thus, TLR4 signaling induces the osteogenic responses in human AVMFs, and mechanisms involve the interplay between the BMP-2 and Notch1 pathways. We formulated three interrelated aims to determine the mechanisms of TLR4-induced valvular osteogenic responses: 1) to test the hypothesis that TLR4 signaling induces aortic valve osteogenic responses through BMP-2-mediated up-regulation of Runx2, 2) to test the hypothesis that NICD1 enhances BMP-2 expression through modulation of NF-?B activity and 3) to test the hypothesis that NICD1 enhances BMP-2 signaling via interaction with Smad1/5/8. These studies will provide important insights into the molecular mechanisms by which pro-inflammatory signaling mediates AVMF osteogenic responses and aortic valve lesions.

Public Health Relevance

Aortic valve lesions (calcification and thickening) affects a large number of people and frequently progresses to calcific aortic stenosis that causes morbidity and mortality. Due to limited knowledge of the mechanisms, pharmacological intervention to prevent calcification or its progression to calcific stenosis is currently unavailable. The proposed studies will advance our knowledge and would identify therapeutic targets for suppressing the process of aortic valve lesions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL106582-04
Application #
8587498
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Evans, Frank
Project Start
2010-12-15
Project End
2015-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
4
Fiscal Year
2014
Total Cost
$339,480
Indirect Cost
$114,480
Name
University of Colorado Denver
Department
Surgery
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Song, Rui; Fullerton, David A; Ao, Lihua et al. (2017) An epigenetic regulatory loop controls pro-osteogenic activation by TGF-?1 or bone morphogenetic protein 2 in human aortic valve interstitial cells. J Biol Chem 292:8657-8666
Zhan, Qiong; Zeng, Qingchun; Song, Rui et al. (2017) IL-37 suppresses MyD88-mediated inflammatory responses in human aortic valve interstitial cells. Mol Med 23:
Zeng, Qingchun; Song, Rui; Fullerton, David A et al. (2017) Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice. Proc Natl Acad Sci U S A 114:1631-1636
Li, Fei; Yao, Qingzhou; Ao, Lihua et al. (2017) Klotho suppresses high phosphate-induced osteogenic responses in human aortic valve interstitial cells through inhibition of Sox9. J Mol Med (Berl) 95:739-751
Yao, Qingzhou; Song, Rui; Ao, Lihua et al. (2017) Neurotrophin 3 upregulates proliferation and collagen production in human aortic valve interstitial cells: a potential role in aortic valve sclerosis. Am J Physiol Cell Physiol 312:C697-C706
Li, Fei; Song, Rui; Ao, Lihua et al. (2017) ADAMTS5 Deficiency in Calcified Aortic Valves Is Associated With Elevated Pro-Osteogenic Activity in Valvular Interstitial Cells. Arterioscler Thromb Vasc Biol 37:1339-1351
Song, Rui; Fullerton, David A; Ao, Lihua et al. (2017) Altered MicroRNA Expression Is Responsible for the Pro-Osteogenic Phenotype of Interstitial Cells in Calcified Human Aortic Valves. J Am Heart Assoc 6:
Cheng, Hui; Yao, Qingzhou; Song, Rui et al. (2017) Lysophosphatidylcholine activates the Akt pathway to upregulate extracellular matrix protein production in human aortic valve cells. J Surg Res 213:243-250
Zhan, Qiong; Song, Rui; Zeng, Qingchun et al. (2015) Activation of TLR3 induces osteogenic responses in human aortic valve interstitial cells through the NF-?B and ERK1/2 pathways. Int J Biol Sci 11:482-93
Deng, Xin-Sheng; Meng, Xianzhong; Zeng, QingChun et al. (2015) Adult aortic valve interstitial cells have greater responses to toll-like receptor 4 stimulation. Ann Thorac Surg 99:62-71

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