GW26-e1243 Inhibitory Effects of Hirudin on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

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Effect of Paclitaxel+Hirudin on the TLR4-MyD88 Signaling Pathway During Inflammatory Activation of Human Coronary Artery Smooth Muscle Cells and Mechanistic Analysis

Cellular Physiology and Biochemistry ◽

10.1159/000494588 ◽

2018 ◽

Vol 50 (4) ◽

pp. 1301-1317 ◽

Cited By ~ 1

Author(s):

Hongmei Li ◽

Xian Wang ◽

Anlong Xu

Keyword(s):

Smooth Muscle ◽

Coronary Artery ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Dependent Manner ◽

Human Coronary Artery ◽

Post Intervention ◽

Inflammatory Activation ◽

Myd88 Pathway

Background/Aims: Approximately 10%-20% of patients with acute cardiovascular disease who have received coronary intervention suffer restenosis and high inflammation. The stent compound paclitaxel+hirudin was prepared for the treatment of post-intervention restenosis. This study aimed to explore the anti-inflammatory and anti-restenosis mechanisms of paclitaxel+hirudin with regard to the TLR4/MyD88/NF-κB pathway. Methods: Human coronary artery smooth muscle cells (HCASMCs) at 4-6 generations after in vitro culture were used as a model. Lipopolysaccharide (LPS) was used as an inducer to maximally activate the TLR4/MyD88/NF-κB inflammation pathway. After MyD88 knockdown and selective blocking of MyD88 degradation with epoxomicin, the effects of paclitaxel+hirudin stenting on key sites of the TLR4/MyD88/NF-κB pathway were detected using ELISA, Q-PCR, and western blot analysis. Results: LPS at 1 μg/mL for 48 h was the optimal modeling condition for inflammatory activation of HCASMCs. Paclitaxel+hirudin inhibited the levels of key proteins and the gene expression, except for that of the MyD88 gene, of the TLR4-MyD88 pathway. The trend of the effect of paclitaxel+hirudin on the pathway proteins was similar to that of MyD88 knockdown. After epoxomicin intervention, the inhibitory effects of paclitaxel+hirudin on the key genes and proteins of the TLR4-MyD88 pathway were significantly weakened, which even reached pre-intervention levels. Paclitaxel+hirudin affected the MyD88 protein in a dosage-dependent manner. Conclusion: The paclitaxel+hirudin compound promotes MyD88 degradation in the TLR4/MyD88/NF-κB pathway to reduce the activity of TLR4 and NF-κB p65 and to weaken the LPS-initiated inflammatory reactions of IL-1β, IL-6, and TNF-α.

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Differential regulation of pregnancy associated plasma protein-A in human coronary artery endothelial cells and smooth muscle cells

Growth Hormone & IGF Research ◽

10.1016/j.ghir.2007.09.001 ◽

2008 ◽

Vol 18 (3) ◽

pp. 213-220 ◽

Cited By ~ 34

Author(s):

Cheryl A. Conover ◽

Sean C. Harrington ◽

Laurie K. Bale

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Coronary Artery ◽

Smooth Muscle Cells ◽

Plasma Protein ◽

Protein A ◽

Muscle Cells ◽

Differential Regulation ◽

Human Coronary Artery

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Intimal Smooth Muscle Cells of Porcine and Human Coronary Artery Express S100A4, a Marker of the Rhomboid Phenotype In Vitro

Circulation Research ◽

10.1161/01.res.0000262654.84810.6c ◽

2007 ◽

Vol 100 (7) ◽

pp. 1055-1062 ◽

Cited By ~ 76

Author(s):

Anne C. Brisset ◽

Hiroyuki Hao ◽

Edoardo Camenzind ◽

Marc Bacchetta ◽

Antoine Geinoz ◽

...

Keyword(s):

Smooth Muscle ◽

Coronary Artery ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Human Coronary Artery

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Inhibitory effects of resveratrol on MCP-1, IL-6, and IL-8 production in human coronary artery smooth muscle cells

Naunyn-Schmiedeberg s Archives of Pharmacology ◽

10.1007/s00210-013-0877-9 ◽

2013 ◽

Vol 386 (9) ◽

pp. 835-839 ◽

Cited By ~ 7

Author(s):

Ichiro Wakabayashi ◽

Yuji Takeda

Keyword(s):

Smooth Muscle ◽

Coronary Artery ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Inhibitory Effects ◽

Human Coronary Artery

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Abstract 180: The Myristolated Alanine-Rich C Kinase Substrate (MARCKS) Differentially Regulates Proliferation of Vascular Smooth Muscle Cells and Endothelial Cells through Affecting Protein Stability and Proteolytic Processing of the Kinase Interacting with Stathmin (KIS)

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.180 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Dan Yu ◽

Charles Drucker ◽

Rajabrata Sarkar ◽

Dudley K Strickland ◽

Thomas S Monahan

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Coronary Artery ◽

Smooth Muscle Cells ◽

Protein Stability ◽

Muscle Cells ◽

Cell Types ◽

Proteolytic Processing ◽

Functional Domain ◽

Human Coronary Artery

Objective: Presently, the antiproliferative agents used in drug eluting stents and drug coated balloons inhibit both VSMC and endothelial cell (EC) proliferation, and thus these patients require dual antiplatelet therapy indefinitely. Identification of a VSMC-specific target to prevent proliferation represents a significant unmet clinical need. Previously we found that knockdown of MARCKS arrests VSMC proliferation through a p27 kip1 -dependent mechanism. Interestingly MARCKS knockdown increases EC proliferation. p27 kip1 is phosphorylated by KIS allowing it to exit the nucleus and be degraded. Here we seek to understand how MARCKS influences KIS protein expression in these two cell types. Approach and Results: We performed siRNA-mediated knock down of MARCKS in human coronary artery endothelial cells (CAECs) and human coronary artery smooth muscle cells (CASMCs). MARCKS knockdown did not affect KIS mRNA expression as determined with RT-PCR in either cell type. KIS protein stability was evaluated in the presence of cyclohexamide with Western blot. In CAECs, MARCKS knockdown increased KIS stability, however, in CASMCs, MARCKS knockdown significantly decreased KIS protein stability. In CASMCs, MARCKS knockdown significantly increased KIS ubiquitinization where as in CAECs, MARCKS knockdown decreased KIS ubiquitinization. Interestingly, the well-studied functional domain of MARCKS(ED domain) is not directly involved in KIS regulation. MARCKS mutants (S4G and S4D) rescued proliferation in VSMCs. MARCKS knockdown in vivo in the murine femoral wire injury model resulted in decreased medial bromodeoxyuridine (BrdU) integration and neointima formation. MARCKS knockdown enhanced endothelial barrier function recovery four days after injury as assessed by Evans Blue integration. Conclusions: MARCKS differentially regulates the protein stability and proteolytic processing of KIS in VSMCs and ECs. The differential interaction of MARCKS and KIS likely explains the observed difference in proliferation observed with MARCKS knockdown in these two cell types.

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