1. Antenatal Maternal Stress: Epigenetic Mechanisms of Developmental Programming of Diseases Ravi Goyal, MD, PhD Assistant Professor Center for Perinatal Biology School of Medicine Loma Linda University

2. Developmental Origin Hypothesis The “Developmental Origin of Adult Health and Disease” proposes that under-nutrition in-utero permanently changes body functions and metabolism leading to an increased risk of diseases in adult life Barker’s Hypothesis Fetal programming of adult health and disease Barker et al, 1991

3. The Dutch Hunger Winter World War II The Dutch government (exile in London) called a general railway strike in the northern and western Netherlands Germans retaliated All food transport to western and northern Netherlands was interdicted

4. The Hunger Winter Winter of 1944/1945 was unusually harsh Canals rapidly froze over and became impassable for barges Lasted 6 months, from November 1944- May 5, 1945, when Holland was liberated from the German occupation

5. Adult rations - 400-800 calories a day (less than a quarter of the recommended adult caloric intake)

6. The Dutch Hunger Winter: Birth Cohorts Ravelli, G.-P., et al. N Engl J Med 1976; 295:349-353

7. Barker et al., Fetal origins of coronary heart disease. BMJ, 311: 1995

8. Epidemiological Studies Adult cohorts show that low infant weight is strongly associated with an increased disease risk – Barker DJP Lancet 1989 – Eriksson JG Diabetologia 2003;46:190 – Bhargava S New Eng J Med 2004;350:865

9. The effects of the famine Increased incidence of – Hypertension – Type -2 DM – Cardiovascular mortality – Schizophrenia – Obesity Baby born during the hunger winter First 2 trimesters - 80% higher prevalence of overweight (p<0.0005)

10. The Thrifty Phenotype As a result of poor nutritional conditions, a pregnant female can modify the development of her unborn child such that it will be prepared for survival in an environment in which resources are likely to be short, resulting in a thrifty phenotype (Hales & Barker, 1992)

11. Our studies Mice (FVB/NJ) Control, 50% protein diet and 33% protein diet (isocaloric) Mice consumed approximately equal amount of food

12. Research Questions How does this developmental programming occurs? What are the molecular mechanisms? What are the genetic and epigenetic changes in specific signal transduction pathways?

13. Maternal Protein Deprivation IUGR Baby born during the hunger winter

14. Birth Weight was reduced but not the litter size Isocaloric diet with reduced protein intake can lead to IUGR

15. What if a thrifty phenotype child is given excess nutrition? Rapid catch-up growth – Obesity – Hypertension – Diabetes

16. Maternal Protein Deprivation IUGR Rapid Catch-up Growth

17. Rapid catch-up growth

19. Maternal Protein Deprivation IUGR Rapid Catch-up Growth Obesity

20. Adipose Volume in Adult Offspring

21. Maternal Protein Deprivation IUGR Rapid Catch-up Growth Hypertension

22. Mean Arterial Blood Pressure in Females

23. Mean Arterial Blood Pressure in Males

24. Maternal Protein Deprivation IUGR Rapid Catch-up Growth Blood Sugar Obesity Hypertension

25. Prenatal protein malnutrition and blood glucose levels in adult life Insulin levels were unchanged

26. Maternal Protein Deprivation Hypertension Altered gene expression Renin – Angiotensin System Altered gene expression Renin – Angiotensin System

27. Renin-Angiotensin System (Systemic & Local) Angiotensiongen (AGT) Angiotensin 1 Angiotensin 2 Angiotensin 2 Type 1 Receptor (AT1) Renin (REN) Angiotensin 1 Converting Enzyme (ACE1) Ang 1-7 Angiotensin 2 Type 2 Receptor (AT2) Angiotensin 2 Converting Enzyme (ACE2)

28. Angiotensin 1 Converting Enzyme (Brain)

29. Protein Deprivation Maternal Hypoxia Caloric Excess Epigenetic Changes

30. Maternal Protein Deprivation Hypertension Altered gene expression Epigenetic Changes

31. Epigenetics Heritable changes in phenotype or gene expression caused by mechanisms other than changes in DNA sequence per se Transcriptional Regulation DNA methylation Histone Modifications DNA methylation Histone Modifications miRNA, lnRNA-mediated regulation Epigenetic Changes Translational Regulation

32. Methylated CpG Islands in Promoter Region mRNA DNA Methylation – Transcriptional Modification 5’3’ RNA Polymerase 5’3’ CH 3 RNA Polymerase Higher amount of mRNA Transcription Hindered – Less mRNA Hypomethylated CpG Islands

33. Angiotensin 1 Converting Enzyme (Fetal Brain) is ACE promoter hypomethylated?

34. Maternal Low Protein Diet Fetal Brain ACE1 – DNA Methylation Normal Diet Hypomethylated CpG Islands in ACE1 Upregulates Gene Expression

35. Question to Consider The programming occurs during fetal life and mRNA are increased during fetal life, then why does blood pressure remains normal until adulthood?

36. Angiotensin 1 Converting Enzyme (Brain) Control Diet MLPD Although, the mRNA levels of ACE1 are increased, the protein expression remains low..

37. microRNA - Post-Transcriptional regulation mRNA 3’ 5’ UTR microRNA ACE1 Protein Translation ACE1 Protein Increased microRNA causes decreased protein expression

38. Micro RNAs identified miRNA putatively regulating ACE translation mmu-mir-27a and mmu-mir-27b

39. ACE1 microRNAs (Brain) Increased microRNA targeted for ACE1 leads to decreased protein expression

40. MLPD Hypo-methylation of ACE promoter Hypo-methylation of ACE promoter Increased ACE mRNA Up-regulation of miRNA Programming of Hypertension Hypertension Reduced/normal ACE protein expression

41. Current Project CBAP scAAV.CBAP.27a.eGFP scAAV.CBAP.27a scAAV viral vector-mediated production of miRNA 27a in hypertensive mice lungs CBAP 27a

42. Conclusion Maternal Stress during Pregnancy Epigenetic Changes Changes in Gene Products Physiological/Pathological Changes

43. Acknowledgements – Lawrence D. Longo, MD – Ciprian Georghe, MD, PhD – Dipali Goyal, BS – Andre Obenaus, PhD – Nina Chu, BS – Andrew Gallfy, BS – E Eun Jang, MD – Toni-An Wright, B.S.

44. Thank You