About

Atherosclerosis
Presented to Dr. Jalil Daher

By Leona Antoun
 TABLE OF CONTENTS

01 Background 03 Procedure

02 Literature review 04 Results

05 Future Perspectives*
 01
ABOUT THE DISEASE
• Atherosclerosis: Complex cardiovascular disease with arterial plaque
buildup, leading to severe complications.

• Endothelial Dysfunction: Initial damage to arterial lining, promoting

inflammation and monocyte adhesion.

• Mox-LDL: Oxidized LDL induced by myeloperoxidase (MPO),

contributes to endothelial dysfunction and atherosclerosis.
 01 THE PROCESS

• Mox-LDL uptake by macrophages results in foam cell formation, characteristic of

early atherosclerotic lesions.

• Mox-LDL triggers an inflammatory response, promoting immune cell recruitment

and perpetuating inflammation.

• Mox-LDL promotes smooth muscle cell migration and proliferation in the arterial
intima, leading to plaque development.

• Unstable plaque fibrous cap rupture leads to thrombus formation, potentially

blocking arteries.
 01 FIBRINOLYSIS

• Fibrinolysis dissolves blood clots, vital for cardiovascular health.

• Plasmin is generated from plasminogen by activators like tPA and uPA, and it
breaks down fibrin.

• Regulation by inhibitors: PAIs and alpha-2-antiplasmin.

• Atherosclerosis disrupts the balance between coagulation and fibrinolysis.

• Mox LDL is associated with decreased fibrinolysis in endothelial cells, and this
mechanism needs further study.
 SCOPE OF THE PROJECT
Article 1:Impact of Myeloperoxidase Modified LDL on Aortic Endothelial Cells Study

Objective: investigate the effect of Mox-LDL on aortic endothelial cells in bovine and human subjects.

Findings: about mox-LDL

• Surprising Results: Mox-LDL did not directly influence the expression of major fibrinolytic factors (t-PA and PAI-
1) at the protein level.

• mRNA levels: No significant changes in fibrinolytic factors observed through quantitative real-time polymerase
chain reaction (qRT-PCR).

• Induction of Oxidative Stress: Mox-LDL induced oxidative stress on endothelial cells.

• Endothelial Dysfunction: Increased oxidative stress may contribute to endothelial dysfunction, a critical factor in
atherosclerosis development.
 SCOPE OF THE PROJECT
Article 2: "Exposure of Endothelial Cells to Physiological Levels of Myeloperoxidase-Modified LDL Delays
Pericellular Fibrinolysis“

Study Objective:
• Investigate the impact of Mox-LDL on fibrinolysis, using the EA.hy926 endothelial cell line.
• Explore the potential link between modified LDL, fibrin deposition, and atherosclerosis plaque development.

Findings:
• Mox-LDL treatment decreased fibrinolysis at the endothelial cell surface.
• No significant changes in mRNA levels of fibrinolytic factors, including plasminogen, a2-antiplasmin, a2-
macroglobulin, FXIII, FXI, FXII, and kallikrein.
• No alterations in protein levels of PAI-1, t-PA, and membrane or soluble uPAR as determined by ELISA.

Strong evidence from both studies indicates that Mox-LDL affects fibrinolysis in endothelial cells independently of known
fibrinolytic factors' expression.
The underlying molecular mechanisms responsible for this effect require further investigation.
PROPOSED METHOD +
EXPECTED RESULTS
 03 PROPOSAL AND EXPECTED RESULTS

1. Mox-LDL Uptake Assay:

1. Utilize fluorescence-labeled Mox-LDL to track internalization by endothelial cells.

2. Confocal Microscopy:
1. Confocal microscopy is a powerful imaging technique that provides high-resolution, 3D
visualization of cellular structures.
2. Cells treated with fluorescence-labeled Mox-LDL will display punctate fluorescent signals in
the cytoplasm, indicating Mox-LDL localization

These will show fluorescent signals within the endothelial cells' cytoplasm, indicating Mox-LDL
uptake, so we can better understand the mechanism of action and localization of this latter
 03 PROPOSAL AND EXPECTED RESULTS

3. Proteomic Analysis:
 Utilize mass spectrometry to analyze the cellular protein profile altered by Mox-LDL treatment.
 Identify potential molecular targets involved in regulating fibrinolysis.

 Potential Candidate Proteins:

 Cell Surface Receptors
 Signaling Molecules
 Proteins involved in Cell Adhesion and Migration

 Example Candidate Proteins:

 Annexins: Involved in membrane remodeling and lipid binding.
 Integrins: Mediate cell-extracellular matrix interactions.
 Matrix Metalloproteinases (MMPs): Involved in extracellular matrix remodeling.

 Quantification of Fold Changes:

 Measure fold changes in expression of candidate proteins to assess their differential regulation.
 Example: Two-fold upregulation of a receptor or three-fold downregulation of a signaling protein in
response to Mox-LDL treatment.
 03 PROPOSAL AND EXPECTED RESULTS

4. siRNA Knockdown:
• Silencing candidate protein expression in endothelial cells.
• Investigate if altered proteins influence Mox-LDL effects.

• Experimental Procedure: a. Deliver siRNA molecules targeting candidates. b. Confirm

successful knockdown using Western blot analysis.
• Real-time Fibrinolysis Monitoring:
• Observe fibrinolysis in siRNA-treated cells.
• Compare results with control cells.

• Outcomes:
• Decreased fibrinolysis time suggests candidate proteins regulate fibrinolysis.
• No significant effect indicates alternative mechanisms.

• Significance:
• Understand protein functions in Mox-LDL effects
 03 PROPOSAL AND EXPECTED RESULTS

5. Examine endothelial dysfunction markers in Mox-LDL-treated cells:

• Adhesion Molecules: Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1
(VCAM-1)
• Cytokines: Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α)
• Oxidative Stress: Reactive Oxygen Species (ROS) levels

• Methods:
• Enzyme-Linked Immunosorbent Assay (ELISA) or Immunostaining:
• ELISA: Quantitative measurement of protein levels using specific antibodies.
• Immunostaining: Visualization of protein expression and localization using fluorescent antibodies.
• Cells will be treated with Mox-LDL and control (normal LDL).

• Results:
• Increased expression levels of ICAM-1, VCAM-1, IL-6, and TNF-α in Mox-LDL-treated cells compared to
control cells.
• Elevated ROS levels in Mox-LDL-treated cells, indicating oxidative stress.
• Confirmation of Mox-LDL-induced endothelial dysfunction
 Future
Perspectives
• Proposing a drug that acts as an annexin inhibitor

• Studies have suggested that certain annexins, particularly annexin A2,

may contribute to endothelial dysfunction and impaired fibrinolysis

• However, further research is needed to fully understand the specific roles

of annexins in atherosclerosis and to develop targeted therapies
effectively.
Thank You!