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The Effects of 20-HETE Antagonism on Myocardial Infarction in Metabolic Syndrome Rats Corinna Lozano Pawling High School Introduction Literature Review Methodology Results Conclusion References 20-HETE levels were ~2 times greater in JCR vs. SD rats at baseline before surgery 20-HETE levels increased during the ischemic period in both SD and JCR rats Increase was more significant in JCR (~4-fold vs. NZ) than in SD (~3-fold vs. NZ) rats 20-HETE levels were somewhat increased in the NZs of SD and JCR rats Increase did not reach statistical significance Has effect of ischemia on remote myocardium 20-HETE elevated so role was assessed in myocardial infarction using 20-SOLA (antagonist) Infarct expansion (the white on the tissue images, represents how great the heart attack was throughout the heart) Resulted in MIs which involved ~50% of the LV in JCR and ~25% of the LV in SD suggests 20-HETE animals Data antagonists are able to decrease further damage to the myocardium 48-hour sample had an increase in AMPK Myocardial Infarction is another term for a heart attack (19) Blockage of the Coronary Artery Loss of Blood flow Loss of oxygen Induced in JCR and SD Rats 20% of people survive after having a heart attack More than 2 million people per year die from having a heart attack Metabolic Syndrome patients are more likely to develop Cardiovascular Disease Metabolic Syndrome is a cluster of biochemical and physiological abnormalities (16) Side effects of includes : High blood sugar Increased pressure Abnormal cholesterol or triglyceride levels Associated with the development of cardiovascular disease and type 2 diabetes Metabolic Syndrome can be correlated to 20-HETE as the functions of both are very similar (13) 20-HETE is elevated in Metabolic Syndrome Rats (13) 20-Hydroxyeicosatetraenoic acid (20-HETE) is an eicosanoid metabolite of arachidonic acid that has a wide range of effects on the vascular including the regulation of vascular tone (14) Blood flow to specific organs Sodium and fluid transport in the kidney Vascular pathway remodeling Associated with the development of stroke and heart attacks 20- HETE is a factor in the development of Cardiovascular Disease 20-SOLA is a 20-HETE Antagonist (25) Decreases 20-HETE levels Mechanism unknown Water soluble Increases blood flow Easy to administer P-Ampk alpha1/alpha2 and Ampk are part of the kinase family (19) Activated by low levels of ATP Mechanism unknown ATP levels are low AMPK promotes ATP production Protects cells from stress Energy sensing Statement of Purpose AMPK kinase is an energy sensing enzyme in which shows when cellular energy is low *D. Grahame et al 2012 20-SOLA is water soluble in which could be administered by I.V. for easy application *Victor Garcia et al 2017 20-HETE levels significantly elevated in Metabolic Syndrome rats due to poor condition of their heart *Petra Rocic et al 2017 Ischemia of the heart leads to cell death of the myocardium such as oncosis and apoptosis *University of Texas et al 2015 Heart damage in Metabolic Syndrome rats is two times greater than a control group *Petra Rocic et al 2017 Myocardial Infarction Protocol: 20-SOLA treatment: 20-HETE antagonist • I.V. placed into SD and JCR rats • Used to combat 20-HETE levels • MEASURED AT 48 HOURS,1 WEEK AND 8 WEEK • Single treatment • Cross sectional analysis of heart Western Blot: • Make Gel Electrophoresis • Isolate protein sample • BCA Protein Assay • Load and Run Gels • Transfer gels onto membrane • Wash membrane with Primary Antibody (AMPK) • Add secondary antibody • Scan on Odyssey Scanner • Compare blot samples • Image J computer program analysis • Analyze Antibodies Discussion Ischemia is an inadequate blood supply to a specific organ or part of the body , especially the heart Leads to damage of the heart Cell Death occurs Caused by heart attacks 30 minute occlusion of the left anterior descending coronary artery (LAD); After 30 minutes, blood flow is reestablished and the left ventricle is reperfused After 48 hours, 7 days, or 8 weeks, hearts are separated into IZ and NZ for following experiments Histology Protein Expression/ localization - Western Blot - Immunohistochemistry (IHC) 20-HETE levels - LC/MS/MS Normal Zone (NZ) Collateral – dependent Zone (CZ) LAD The Effects of 20-SOLA on MI Size at 48 Hours post Reperfusion It was determined that 20-HETE plays a causative role in collateral cellular growth (CCG) AMPK kinase showed a significant increase in 48-hour samples for both rats 20-SOLA was found to create an equilibrium in 20-HETE levels in all tissue samples Significantly more in JCR MI rats 20-SOLA aided the decrease in ischemia for both rats More significant in JCR MI rats When 20-HETE levels decrease ischemia also decreases Acknowledgements I would like to thank the wonderful Gillian Rinaldo, my science research teacher for supporting me through this whole course I would also like to thank all the other science research students for helping me adjust to this new course and for being great friends to talk to My amazing mentor, Petra Rocic for guiding me through my research Finally, I would like to thank all of my family members for always supporting me with whatever I do as a person and with my education Future Research p(Thr172)AMPK JCR MI JCR MI + 20-SOLA JCR MI JCR MI + 20-SOLA JCR MI JCR MI + 20-SOLA JCR control SD MI SD MI + 20-SOLA SD MI SD MI + 20-SOLA SD MI SD MI + 20-SOLA SD control 48 hours 7 days 8 weeks 48 hours 7 days 8 weeks * p<0.05 vs. control # p<0.05 vs. SD f o l d i n c r e a s e / c o n t r o l 4 3 2 1 * * * * # # * * * * $ p<0.05 vs. MI $ * $ # # # # $ $ $ Total AMPK Figure 1a shows SD (untreated) MI Size +/- 2% of LV Figure 1b shows JCR (untreated) MI Size 51 +/-+/- 2% of LV Figure 1c shows SD + 20-SOLA (10mg/kg i.v.) MI 11 Size +/- 5% of LV Figure 1d shows JCR 20-SOLA (10mg/kg i.v.) MI Size 19 +/- 3% of LV The white areas demonstrate ischemia in cross sectional analysis Figure 2a: Western blot analysis of p- AMPkalpha, p-AMPkalpha1/a2, AMPK after 48 hours shows a significant increase in both phosphorylated and total AMPK at 48 hours in SD+SOLA vs. SD and JCR+SOLA vs. JCR Figure 2b: Western blot analysis of p- AMPkalpha, p-AMPkalpha1/a2, AMPK after 1 week shows a slight increase in both phosphorylated and total AMPK at 1 week in SD+SOLA vs. SD and JCR+SOLA vs. JCR Figure 2c: Western blot analysis of p- AMPkalpha, p-AMPkalpha1/a2, AMPK after 8 weeks shows a decrease in both phosphorylated and total AMPK at 8 week in SD+SOLA vs. SD and JCR+SOLA vs. JCR Global epidemic of Cardiovascular problems and diseases that have no effective treatment option Problem 20-SOLA will decrease ischemic damage in both JCR and SD rats but will be more prevalent in the JCR rats Hypothesis Establish a more effective treatment for Myocardial Infarction in Metabolic Syndrome rats Relevant to the epidemic of cardiovascular diseases plaguing populations globally Investigate the effects 20-SOLA would have on human participants Discover the role of AMPK on other mechanisms of the body such as endothelial cell production Increase the effectiveness of 20-SOLA administration Establish an understanding on the mechanisms behind how 20-SOLA works Determine how AMPK phosphorylation sites work as the mechanism is unknown Pandey V, Garcia V, Gilani A, Mishra P, Zhang FF, Paudyal MP, Falck JR, Nasjletti A, Wang WH, Schwartzman ML. The blood pressure-lowering effect of 20-HETE blockade in Cyp4a14(-/-) mice is associated with natriuresis.J Pharmacol Exp Ther. 2017;363:412–418. doi: 10.1124/jpet.117.243618 JCR rats or James C. Russel (JCR) rat is used to represent Metabolic Syndrome Contains CP Gene which gives characteristics of Metabolic Syndrome (fatty liver and obesity) SD rats represents normal health or control group https://www.researchgate.net/figure/Androgen-driven-20-HETE-dependent-hypertension-in-mice-Mice- are-treated-with-either_fig1_236615072 http://www.clinsci.org/content/124/12/695 http:// www.cardio-research.com/ih https://www.youtube.com/watch?v=VCskwk9b1kk Molecular mechanisms and cell signaling of 20-hydroxyeicosatetraenoic acid in vascular pathophysiology. (n.d.). Retrieved from https://www.bioscience.org/2016/v21/af/4465/fulltext.php?bframe=figures.htm
Transcript of Pawling High School 48 hours 7 days 8 weeks 48 hours 7 ...€¦ · • Image J computer program...
The Effects of 20-HETE Antagonism on Myocardial Infarction in Metabolic Syndrome Rats
Corinna Lozano
Pawling High School
Introduction
Literature Review
Methodology
Add your information, graphs and images to this section.
Results
Add your information, graphs and images to this section.
Conclusion
References
20-HETE levels were ~2 times greater in JCR vs. SD rats at baseline before surgery 20-HETE levels increased during the ischemic period in both SD and JCR rats
Increase was more significant in JCR (~4-fold vs. NZ) than in SD (~3-fold vs. NZ) rats
20-HETE levels were somewhat increased in the NZs of SD and JCR rats
Increase did not reach statistical significance Has effect of ischemia on remote myocardium
20-HETE elevated so role was assessed in myocardial infarction using 20-SOLA (antagonist) Infarct expansion (the white on the tissue images, represents how great the heart attack was throughout
the heart) Resulted in MIs which involved ~50% of the LV in JCR and ~25% of the LV in SD
suggests 20-HETE animals
Data antagonists are able to decrease further damage to the myocardium 48-hour sample had an increase in AMPK
Myocardial Infarction is another term for a heart attack (19)
Blockage of the Coronary Artery Loss of Blood flow Loss of oxygen Induced in JCR and SD Rats
20% of people survive after having a heart attack More than 2 million people per year die from having a heart attack Metabolic Syndrome patients are more likely to develop Cardiovascular Disease
Metabolic Syndrome is a cluster of biochemical and physiological abnormalities (16) Side effects of includes :
High blood sugar Increased pressure Abnormal cholesterol or triglyceride levels Associated with the development of cardiovascular disease and type 2 diabetes
Metabolic Syndrome can be correlated to 20-HETE as the functions of both are very similar (13)
20-HETE is elevated in Metabolic Syndrome Rats (13)
20-Hydroxyeicosatetraenoic acid (20-HETE) is an eicosanoid metabolite of arachidonic acid that has a wide range of effects on the vascular including the regulation of vascular tone (14)
Blood flow to specific organs Sodium and fluid transport in the kidney Vascular pathway remodeling Associated with the development of stroke and heart
attacks 20- HETE is a factor in the development of Cardiovascular Disease
20-SOLA is a 20-HETE Antagonist (25) Decreases 20-HETE levelsMechanism unknownWater soluble Increases blood flow Easy to administer
P-Ampk alpha1/alpha2 and Ampk are part of the kinase family (19)Activated by low levels of ATPMechanism unknownATP levels are low AMPK promotes ATP production Protects cells from stressEnergy sensing
Statement of Purpose
AMPK kinase is an energy sensing enzyme in which shows when cellular energy is low
*D. Grahame et al 2012
20-SOLA is water soluble in which could be administered by I.V. for easy application
*Victor Garcia et al 2017
20-HETE levels significantly elevated in Metabolic Syndrome rats due to poor condition of their heart
*Petra Rocic et al 2017
Ischemia of the heart leads to cell death of the myocardium such as oncosis and apoptosis
*University of Texas et al 2015
Heart damage in Metabolic Syndrome rats is two times greater than a control group
*Petra Rocic et al 2017
Myocardial Infarction Protocol:
20-SOLA treatment: 20-HETE antagonist
• I.V. placed into SD and JCR rats • Used to combat 20-HETE levels• MEASURED AT 48 HOURS,1 WEEK AND 8 WEEK• Single treatment • Cross sectional analysis of heart
Western Blot:• Make Gel Electrophoresis• Isolate protein sample• BCA Protein Assay• Load and Run Gels• Transfer gels onto membrane • Wash membrane with Primary Antibody (AMPK)• Add secondary antibody • Scan on Odyssey Scanner • Compare blot samples• Image J computer program analysis• Analyze Antibodies
Discussion
Ischemia is an inadequate blood supply to a specific organ or part of the body , especially the heart
Leads to damage of the heart Cell Death occurs Caused by heart attacks
30 minute occlusion of the left anterior descending coronary artery (LAD); After 30 minutes, blood flow is reestablished and the left ventricle is reperfused
After 48 hours, 7 days, or 8 weeks, hearts are separated into IZ and NZ for following experiments
Histology
Protein Expression/ localization- Western Blot- Immunohistochemistry (IHC)
20-HETE levels- LC/MS/MS
Normal Zone (NZ)
Collateral –dependent Zone (CZ)
LAD
The Effects of 20-SOLA on MI Size at 48 Hours post Reperfusion It was determined that 20-HETE plays a causative role in collateral cellular growth
(CCG)
AMPK kinase showed a significant increase in 48-hour samples for both rats
20-SOLA was found to create an equilibrium in 20-HETE levels in all tissue samples
Significantly more in JCR MI rats
20-SOLA aided the decrease in ischemia for both rats
More significant in JCR MI rats
When 20-HETE levels decrease ischemia also decreases
AcknowledgementsI would like to thank the wonderful Gillian Rinaldo, my science research teacher for supporting me through this whole course
I would also like to thank all the other science research students for helping me adjust to this new course and for being great friends to talk to
My amazing mentor, Petra Rocic for guiding me through my research
Finally, I would like to thank all of my family members for always supporting me with whatever I do as a person and with my education
Future Research
p(Thr172)AMPK
JCR MI JCR MI
+
20-SOLA
JCR MI JCR MI
+
20-SOLA
JCR MI JCR MI
+
20-SOLA
JCR
control
SD MI SD MI
+
20-SOLA
SD MI SD MI
+
20-SOLA
SD MI SD MI
+
20-SOLA
SD
control
48 hours 7 days 8 weeks 48 hours 7 days 8 weeks
* p<0.05 vs. control
# p<0.05 vs. SD
fold increase/control
4
3
2
1
*
*
*
* #
#
*
*
* *
$ p<0.05 vs. MI
$
* $ #
#
## $ $ $
Total AMPK
Figure 1a shows SD (untreated) MI Size +/-2% of LV
Figure 1b shows JCR (untreated) MI Size 51 +/-+/- 2% of LV
Figure 1c shows SD + 20-SOLA (10mg/kg i.v.) MI 11 Size +/- 5% of LV
Figure 1d shows JCR 20-SOLA (10mg/kg i.v.) MI Size 19 +/- 3% of LV
The white areas demonstrate ischemia in cross sectional analysis
Figure 2a: Western blot analysis of p-AMPkalpha, p-AMPkalpha1/a2, AMPK after 48 hours shows a significant increase in both phosphorylated and total AMPK at 48 hours in SD+SOLA vs. SD and JCR+SOLA vs. JCR
Figure 2b: Western blot analysis of p-AMPkalpha, p-AMPkalpha1/a2, AMPK after 1 week shows a slight increase in both phosphorylated and total AMPK at 1 week in SD+SOLA vs. SD and JCR+SOLA vs. JCR
Figure 2c: Western blot analysis of p-AMPkalpha, p-AMPkalpha1/a2, AMPK after 8 weeks shows a decrease in both phosphorylated and total AMPK at 8 week in SD+SOLA vs. SD and JCR+SOLA vs. JCR
Global epidemic of Cardiovascular problems
and diseases that have no effective treatment option
Problem
20-SOLA will decrease ischemic damage in both JCR and SD rats but will
be more prevalent in the JCR rats
Hypothesis
Establish a more effective treatment for Myocardial Infarction in Metabolic Syndrome rats
Relevant to the epidemic of cardiovascular diseases plaguing populations globally
Investigate the effects 20-SOLA would have on human participants Discover the role of AMPK on other mechanisms of the body such as endothelial cell
production
Increase the effectiveness of 20-SOLA administration Establish an understanding on the mechanisms behind how 20-SOLA works
Determine how AMPK phosphorylation sites work as the mechanism is unknown
Pan
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JCR rats or James C. Russel (JCR)rat is used to represent Metabolic Syndrome
Contains CP Gene which gives characteristics of Metabolic Syndrome (fatty liver and obesity)
SD rats represents normal health or control group
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36615072
http://www.clinsci.org/content/124/12/695
http://www.cardio-research.com/ih
https://www.youtube.com/watch?v=VCskwk9b1kk
Molecular mechanisms and cell signaling of 20-hydroxyeicosatetraenoic acid in vascular pathophysiology. (n.d.). Retrieved from https://www.bioscience.org/2016/v21/af/4465/fulltext.php?bframe=figures.htm
