1. Biologia Celular e Molecular II Cell cycle deregulation and cardiovascular diseases Ana Rita Lobo Diogo Matos Inês Matos

2. Biologia Celular e Molecular II Objectives Cell cycle o Interphase o Mitosis Control System of the Cell Cycle Cell cycle machinery and Stroke

3. Biologia Celular e Molecular II Cell Cycle The cell cycle is the series of events which take place in the cell and lead to its duplication by means of replication and duplication of its previous contents.

4. Biologia Celular e Molecular II Cell cycle’s phases The cell cycle is generally divided into two phases: o Interphase - the cell spends most of its time performing the functions that make it unique. o Mitosis - the cell divides into two daughter cells.

5. Biologia Celular e Molecular II Interphase The interphase stage of the cell cycle includes three distinctive parts: o G 1 phase- follows mitosis and is the period in which the cell is synthesizing its structural proteins and enzymes to perform its functions; o S phase- the DNA within the nucleus replicates o G 2 phase- the cell prepares for mitosis. Proteins organize themselves to form a series of fibers called the spindle, which is essential to chromosome movement during mitosis

6. Biologia Celular e Molecular II Mitotic Phase The mitotic phase is the period in the cell cycle where occurs, the nucleous division. The mitotic process includes 4 phases: o Prophase o Metaphase o Anaphase o Telophase After the mitotic phase, the cytokinesis occurs: microfilaments contract during cleavage and assist the division of the cell into two daughter cells.

7. Biologia Celular e Molecular II Control System of the Cell Cycle In the majority of eukaryotic cells, the control systems of the cell cycle activate its progression in three points.

8. Biologia Celular e Molecular II Control System of the Cell Cycle Elements involved: Cyclins-dependent kinases (CDKs) Cyclins CDK inhibitor proteins (CKIs) Retinoblastoma protein (Rb) Cyclical Proteolysis

9. Biologia Celular e Molecular II CDKs The main components of the control system of the cell cycle are a member of a kinases’ family known as cyclin- dependent kinases that can modify various protein substrates involved in cell cycle progression. CYCLINS Cyclins are a family of proteins that have no enzymatic activity of their own but activate CDKs by binding to them. Cyclin Cyclin dependt kinase (CDK)

10. Biologia Celular e Molecular II How Do CDKs Control the Cell Cycle? All cyclins are named according to the stage at which they assemble with CDKs. Common classes of cyclins include G 1 /S-phase cyclins, S- phase cyclins, and M-phase cyclins. Because cyclin-CDK complexes recognize multiple substrates, they are able to coordinate the multiple events that occur during each phase of the cell cycle.

11. Biologia Celular e Molecular II How Do CDKs Control the Cell Cycle?

12. Biologia Celular e Molecular II Inhibitor phosphorilation and CKIs(CDK inhibitor proteins) The rise and fall of the cyclin levels are the main events that control the activity of CDKs during the cell cycle. However, other mechanisms help the activity of CDKs in specific stages of the cycle.

13. Biologia Celular e Molecular II Inhibitor phosphorilation and CKIs(CDK inhibitor proteins) The phosphorilation in a pair of amino acids on the top of the kinases’ active site inhibits the cyclin-cdk complex. Cells usually use CKIs to help them in the G1-S regulation activity in the beginning of the cell cycle. There are some types of CKIs such as P15,P16,P18 and P19.

14. Biologia Celular e Molecular II Retinoblastoma protein (Rb) Important target of G1 CDKS In mid- to late- G1, Rb is phosphorylate by cyclin D1- cdk4/6 and cyclin E-cdk2 complexes Rb (hyperphosphorylated) is no longer repressed by E2F E2F binds with DP Activate genes required for S phase progression

15. Biologia Celular e Molecular II Cell cycle’s deregulation and cardiovascular diseases Hypothesis: One signal which may control ischemic neuronal death is the inappropriate activation of cell cycle regulators including cyclins, cyclin dependent kinases (CDK) and endogenous cyclin dependent kinases inhibitors (CDKI). Aberrant activation of these elements triggers neuronal death

16. Biologia Celular e Molecular II Stroke Occluded or ruptured of blood vessels and in some cases cardiac arrest. Neurons in the affected brain region are prived of oxigen and glucose. Ischemic neuronal death is determined by location, severity and duration of insult.

17. Biologia Celular e Molecular II Cell cycle machinery and Stroke In vitro evidence

18. Biologia Celular e Molecular II Cell cycle machinery and Stroke In vitro evidence J. Rasidian, G.O. Iyirhiaro, D.S. Park, Cell cycle machinery and stroke, BBA – Molecular Basis of Disease (2006), doi: 10.1016 / j.bbadis.2006.11.009

19. Biologia Celular e Molecular II Cell cycle machinery and Stroke In vitro evidence Normal conditions: The levels of activity of key cell cycle are downregulated; More activity of CKIs; The levels of hypophosphorylated Rb increases resulting in greater E2F sequestration.

20. Biologia Celular e Molecular II Cell cycle machinery and Stroke In vitro evidence Ischemic injury conditions: Lose of CKIs example: CDK inhibitor p27 is reported loss of CKI following oxygen glucose deprivation (stroke) Increase in Cyclins example: cyclin D1 protein levels and activation of Cdk2 after stroke Rb is phosphorylated following hypoxia/reoxygenation Increase in E2F mRNA transcription Activation of the cell cycle

21. Biologia Celular e Molecular II Cell cycle machinery and Stroke This only showed the activation of cell cycle components and does not adresss the issue of whether this signal is required for death

22. Biologia Celular e Molecular II Cell cycle machinery and Stroke J. Rasidian, G.O. Iyirhiaro, D.S. Park, Cell cycle machinery and stroke, BBA – Molecular Basis of Disease (2006), doi: 10.1016 / j.bbadis.2006.11.009

23. Biologia Celular e Molecular II Cell cycle machinery and Stroke Proceeding 1:use drugs that inactivate the cell cycle, such as CDK inhibitors like flavopiridol Conclusion 1: the cortical neurons have been protected

24. Biologia Celular e Molecular II Cell cycle machinery and Stroke Proceeding 2: genetic manipulation of components of the cell cycle Conclusion 2: in mice with expressing kinase dead cdk4 or null for its regulator cyclin (D1) are resistent to hypoxia mediated ischemic death

25. Biologia Celular e Molecular II Cell cycle machinery and Stroke Proceeding 3: envision of the cell cycle pathway with E2F null Conclusion 3:cortical neurons derived from E2F null mice are less susceptible to death by hypoxia Furthermore E2F deficiency improves the recovery of neurons from loss of synaptic transmission

26. Biologia Celular e Molecular II Cell cycle machinery and Stroke Taken together, these in vitro evidence strongly implicate the reactivation of cell cycle components in ischemic neuronal death.

27. Biologia Celular e Molecular II The atherosclerotic plaque is due to an overproliferation of endothelial, smooth muscle, and inflammatory cells and macrophages The division of these cells is activated by a Allograft Inflammatory Factor (AIF-1) Cell cycle role in atherosclerosis

28. Biologia Celular e Molecular II AIF-1 is not present in normal arteries, and it is produced by VSMC when they are stimulated. Constitutive expression of AIF-1 results in a shorter cell cycle, and aberrant expression of cell cycle proteins. AIF-1 actively participates in the upregulation of VSMC’s proliferation This happens because AIF-1, alongside with calmodulin, shortens the G1 and antecipates the S phase Cell cycle role in atherosclerosis

29. Biologia Celular e Molecular II This is important to cope with the inflammatory processes. But if this happens in ordinary situations, it may cause a overproduction of VSM cells, obtructing crucial arteries of the cardiovascular system. Atherosclerotic Restenosis Cell cycle role in atherosclerosis

30. Biologia Celular e Molecular II Bibliography Alberts B., Bray D., Lewis J., M. Raff, Roberts K., & Watson J.D. (1994) Molecular Biology of the Cell, 3rd Ed. Garland Publishing, Inc. New York. Netgraphy http://www.nature.com/scitable/topicpage/cdk-14046166 http://atvb.ahajournals.org/content/21/9/1421.short https://sites.google.com/site/kefalikinisi/home/fisiologia-humana-1/celula-e-celulas/ciclo-celular http://scholar.google.pt/scholar?start=10&q=deregulation+in+cell+cycle+and+cardiovascular+disea ses&hl=pt-PT&as_sdt=0 http://scholar.google.pt/scholar?start=10&q=deregulation+in+cell+cycle+and+cardiovascular+disea ses&hl=pt-PT&as_sdt=0 Articles J. Rasidian, G.O. Iyirhiaro, D.S. Park, Cell cycle machinery and stroke, BBA – Molecular Basis of Disease (2006), doi: 10.1016 / j.bbadis.2006.11.009 Michael V. Autieri, Christopher M. Carbone - Overexpression of Allograft Inflammatory Factor-1 Promotes Proliferation of Vascular Smooth Muscle Cells by Cell Cycle Deregulation (Arterioscler Thromb Vasc Biol. 2001;21:1421-1426.)