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Presentation summer 2011

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Presentation summer 2011

  1. 1. MCP-1 AND IL-8 EXPRESSION BY MONOCYTES IN CLOTS Paola A. Arias-Mendoza, MS1 University of Puerto Rico, Medical Sciences Campus Robert Campbell, Ph.D. Andrew S. Weyrich, Ph.D. Program in Molecular Medicine University of Utah
  2. 2. INTRODUCTION• Cardiovascular Disease is the leading cause of death in US. • Arterial thrombosis – Myocardial infarction and stroke accounted for 37% of all deaths in 2007. • Venous thrombosis – Effects more than a million a year with 30% mortality within the first 30 days.
  3. 3. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  4. 4. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  5. 5. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  6. 6. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  7. 7. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  8. 8. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  9. 9. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  10. 10. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  11. 11. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  12. 12. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  13. 13. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  14. 14. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  15. 15. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  16. 16. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  17. 17. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  18. 18. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  19. 19. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  20. 20. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  21. 21. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  22. 22. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  23. 23. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  24. 24. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  25. 25. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  26. 26. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  27. 27. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  28. 28. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  29. 29. COAGULATION Red Blood Cell Leukocytes Platelets Coagulation Factors Thrombin (IIa) Fibrinogen
  30. 30. BACKGROUND• Activated platelets interact with monocytes promoting chemokine secretion involved in inflammatory response. (A.S. Weyrich 1996) • Monocyte Chemotactic Protein 1 (MCP-1) • Released by monocytes after induction by RANTES and P-selectin expressed by platelets. • Interleukin-8 (IL-8) also “neutrophil chemotatic factor” • Known chemokine and angiogenic factor• IL-8 and MCP-1 both needed for migration and proliferation of leukocytes in subendothelial layers (Z. Franks 2010)• IL-8 and MCP-1 have been found elevated in patients with stroke ( Z. Franks 2010)
  31. 31. IN VITRO SYSTEM Form clot from donors plasma Add 20 mM CaCl2 and tissue factorMonocytes
or
Monocytes
+
 Allow clot to form for 30 minutes platelets Overlay clot with monocytes or Plasma
Clot monocytes with platelets Collect supernatant at designated times and analyze for different cytokines
  32. 32. BACKGROUND 120000 100000 S Counts per Minutes 80000• Plasma Clots promotes an increase in the synthesis of 60000 proteins. 40000 35 20000 0 NT IIa LPS Clot
  33. 33. RESULTSIL‐8 MCP‐1 DAPI
=
Blue,
CD14
=
Green,
IL‐8
or
MCP‐1
=
RED
  34. 34. OBJECTIVE• Characterize MCP-1 and IL-8 mRNA expression in monocytes associated with clots.
  35. 35. MATERIALS AND METHODS• Blood peripheral monocytes isolation• In vitro Clot system experiments• RNA Isolation• cDNA Preparation• Real Time PCR
  36. 36. RESULTS
  37. 37. SUMMARY• MCP-1 and IL-8 mRNA and protein are increased in monocytes associated with clots• Our data suggest clinicians should consider the inflammatory consequences of thrombi in patients. Treatments focused on the inflammatory response may results in better outcomes.• Future Direction: • Identification of other chemokines and other inflammatory genes. • Determine mechanism of how clots induce inflammatory response.
  38. 38. ACKNOWLEDGEMENTS• PI: Andrew S. Weyrich, Ph.D.• Mentor: Robert A. Campbell, Ph.D.• Medical Research Program At University of Utah • Janet Basset• “Summer Family”
  39. 39. ACKNOWLEDGEMENTS• PI: Andrew S. Weyrich, Ph.D.• Mentor: Robert A. Campbell, Ph.D.• Medical Research Program At University of Utah • Janet Basset• “Summer Family”

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