Key Players of Nitric Oxide NO Production as

Key Players of Nitric Oxide (NO) Production as Novel Biomarkers for Coronary Artery Disease (CAD) Mohamed Z Gad, Ph. D. Professor & Chairman of Biochemistry Dept. Faculty of Pharmacy German University in Cairo 31 October 2021 Dr. Mohamed Z. Gad Cairo – Egypt 1

Statistics of CVD in Egypt Ischemic Heart Disease (IHD) represents the 1 st among top 10 causes of death in Egyptians, accounting to 21% of all deaths. WHO Mortality Country Fact Sheet 2006 A recent study conducted on 11, 731 patients in 19 countries, including 1759 Egyptians suffering from acute CAD and angina revealed that: “Egyptians are the most vulnerable to heart disease at an early age compared to people elsewhere in the world” Egyptian Society for Cardiology 2/12/2010 31 October 2021 Dr. Mohamed Z. Gad 2 2

First Person in Human History with Diagnosed Coronary Artery Disease The Egyptian princess Ahmose-Meryet-Amon, who lived in Thebes (Luxor) between 1580 and 31 October 2021 1550 BC. Dr. Mohamed Z. Gad CT scan analysis of 3, 500 -year old mummy of Ahmose. Meryet-Amon, died in her 40’s, revealed hardening (calcification) of 3 3 the coronary arteries

Nitric Oxide and Healthy Vasculature L-Arginine e. NOS Leukocyte activation NO SMC proliferation Platelet aggregation Platelet adhesion Vasorelaxation 31 October 2021 Dr. Mohamed Z. Gad 4 4

Enzymes & Regulatory Proteins that Control NO Activity/Production L-arginine -ve e. NOS X DDAH ADMA Citrulline Paraoxonase LDL NO -ve Oxidized -LDL XO 2 X Vasodilatation -ve - Endothelin-1 +ve NAD(P)H oxidase 31 October 2021 ONOO(peroxynitirte) Dr. Mohamed Z. Gad 5

The DDAH/ADMA Arm L-arginine -ve e. NOS X DDAH ADMA Citrulline Paraoxonase LDL NO -ve Oxidized -LDL XO 2 X Vasodilatation -ve - Endothelin-1 +ve NAD(P)H oxidase 31 October 2021 ONOO(peroxynitirte) Dr. Mohamed Z. Gad 6

Methylated Arginine Identified in Eukaryotes L-arginine L-NMMA SDMA 31 October 2021 ADMA 7 7

Cellular Processes Regulated by Arginine Methylation RNA Processing: For proper processing, folding, stabilization and localization of RNAs and for m. RNA translation. Transcriptional Regulation: Arginine methylation of histones regulates its gene expression (Jenuwein and Allis, 2001). It also regulates the initiation and elongation steps of transcription. Signal Transduction: Alter protein-protein interaction (Mc. Bride and Silver, 2001). DNA Repair: PRMTs play a significant role in signaling 31 October 2021 Dr. Mohamed Z. Gad DNA damage. 8 8 PRMT Substrates

ADMA : an Endogenous Competitive Inhibitor of e. NOS (Maxwell, Nitric Oxide, 2002) ADMA inhibits all 3 isoforms of NOS X 31 October 2021 ADMA X Dr. Mohamed Z. Gad Plasma ADMA determines the level of bioavailable NO 9 9

ADMA : an Independent Risk Factor for CVD 31 October 2021 Dr. Mohamed Z. Gad (Boger, 2004) 10 10

Discovery of ADMA v Methylated arginine circulate in plasma & are excreted in urine (Paik and Kim, 1967). v However, Vallance & Moncada were the first to demonstrate that endogenous ADMA antagonized endothelium-dependent vasodilatation (Vallance et al, 1992). v They observed a 9 -fold elevation of plasma ADMA in patients with renal failure. Plasma from patients with renal failure induced a vasoconstriction of vascular rings in vitro, an effect that was reversed by addition of L-arginine to the 31 October 2021 Dr. Mohamed Z. Gad 11 11

Conditions with Elevated ADMA Later, elevated levels of plasma ADMA capable of inhibiting NOS have been found in: Condition Fold increase vs. Reference controls Hypertension 2 Surdacki et al, 1999 Congestive heart failure 2 -3 Hornig et al, 1998 Hypercholesterolemia 2 -3 Boger et al, 1998 Peripheral arterial disease 2 -4 Boger et al, 1997 Chronic renal failure 2 -12 Kielstein et al, 1999 Preeclampsia 2 Pettersson et al, 1998 Type 2 diabetes 2 Abbasi et al, 2001 End Stage Liver Disease 10 Tsikas et al. , 2007 Nonalcoholic fatty 31 October 2021 Disease (NAFLD) Liver 2 Dr. Mohamed Z. Gad Boga et al. , 2015 12 12

Where ADMA comes from ? ? ? 31 October 2021 Dr. Mohamed Z. Gad 13

Origin & Metabolism of ADMA (Teerlink, 2005) SAM = S-adenosylmethionine, PRMT = protein arginine methyltransferases, 31 October 2021 Dr. Mohamed Z. Gad DDAH = dimethylarginine dimethylaminohydrolase, CAT = cationic 14 14 a. a. transporters

To date, asymmetrically methylated arginine are the only product of post-translational protein modification that are known to exert substantial biological effects. 31 October 2021 Dr. Mohamed Z. Gad 15

Degradation of ADMA by DDAH ADMA DDAH Citrulline + Dimethylamine o Two isoforms of DDAH (DDAH-1 & DDAH-2) have been identified & are widely expressed in rat and human tissues. Content of isoform (% of maximum) Quantification of DDAH isoform expression in human tissues (100% = maximum level of expression of the isoform) 31 October 2021 Tissue DDAH-1 DDAH-2 Heart 15 100 Brain 56 19 Placenta 12 56 Lung 16 36 Liver 42 26 Skeletal muscle 27 25 Kidney 100 70 47 34 Pancreas Dr. Mohamed Z. Gad 16 16

DDAH-2: Properties • Localized on chromosome 6 p 21. 3 • Predominates in tissues expressing e. NOS, such as the endothelium • DDAH-2 co-localizes with e. NOS in cytosol of endothelium of BV, thus DDAH 2 may regulate e. NOS activity !! DDAH activity (DDAH-2 and/or DDAH-1 ? ? ) regulates plasma ADMA determines bioavailable NO 31 October 2021 Dr. Mohamed Z. Gad 17 17

ADMA/DDAH-2: Therapeutic Strategies ADMA DDAH For treating disorders in which NO signalling is reduced and/ or ADMA levels are increased — such as renal disease, insulin resistance and coronary artery disease 31 October 2021 AH DD A novel therapeutic target for diseases in which excessive NO production has been implicated in disease progression — e. g. septic shock, certain cancers and arthritis Dr. Mohamed Z. Gad 18

ADMA : DDAH : Vascular Health Illustrative Figure - 31 October 2021 - Dr. Mohamed Z. Gad 19 19 (Valkonen et al, 2005)

Study of DDAH-2 Gene Polymorphisms co o cti D f no H 2 DA s sm i h p or w ith s it an rre Ex se la t ru m pl or io n so in g fu nc tio f. S na an NP A l d DM pr L-a of A, rg ile SD in s in M e A, ym l o yp g D p E e g n ge on am Objectives e et Assess association of SNP profiles with risk of CVD in young susceptible 10/31/2021 31 October 2021 Dr. Mohamed Z. Gad individuals 20 20

Polymorphisms in the DDAH-2 Gene 6 polymorphisms were identified by Single-strand conformation polymorphism (SSCP) analysis and confirmed by DNA sequencing SNP 2 SNP 1 Translation start site 3 1 2 4 5 6 (Jones et al. , 2003) Non-coding exons 31 October 2021 Dr. Mohamed Z. Gad 21

Study Population 35 -50 y old PCI = Percutaneous Coronary Intervention 31 October GABG 2021 Dr. Mohamed Z. Gad Grafting = Coronary Artery Bypass 22 22

Biochemical Analysis of serum L- arginine, ADMA and SDMA: LC-MS 31 October 2021 Dr. Mohamed Z. Gad 23 23

DDAH-2 Genotyping Two SNPs: • SNP 1 (-1151 C/A, NCBI reference no. Rs 805304) • SNP 2 (-449 C/G, NCBI reference no. rs 805305) 31 October 2021 Real-Time q. PCR (ABI Prism Sequence Detection System) • SNP 1 genotypes were confirmed by sequencing PCR-amplified DNA strands using the dideoxynucleotide chain termination method. Dr. Mohamed Z. Gad 24 24

Results 31 October 2021 Dr. Mohamed Z. Gad 25 25

CVD Patients vs. Controls DDAH 2 SNP 1 (SNP 2) Allele Distribution 100% 45 56, 5 90% p=0. 02 8 80% 70% 60% 50% A allele (G allele) 40% C allele (C allele) 30% 55 20% 43, 5 10% 0% Controls (n=100) 31 October 2021 CVD patients (n=100) Dr. Mohamed Z. Gad 26 26

DDAH 2 SNP 1 (SNP 2) Genotype Distribution 100% 18 31 90% p=0. 044 80% 54 70% 52 60% AA (GG) 50% CA (CG) 40% CC (CC) 30% 28 20% 17 10% 0% Control (n=100) 31 October 2021 CVD patients (n=100) Dr. Mohamed Z. Gad 27 27

Comparison with Other Populations Hap. Map project (Healthy Population) SNP 1 Europeans Asian Egyptians (This study) Sub-Saharan African 31 October 2021 CC 8. 3% 13. 3% 28% CA 46. 7% 55. 6% 54% AA 45% 31. 1% 18% 78% 18. 6% 3. 4% Dr. Mohamed Z. Gad 28 28

Individual CVD Groups DDAH 2 SNP 1/SNP 2 Allele Distribution Percentage of subjects 100 46 54 54 54 46 46 58 68 80 60 55 46 40 A/G allele C/C allele 42 32 20 0 Controls Normal angio 31 October 2021 Med PCI CABG AMI Dr. Mohamed Z. Gad 29 29

DDAH 2 SNP 1/SNP 2 Genotype Distribution 100 18 17 26 39 36 39 64 75 55 58 54 60 AA/GG genotype 40 CA/CG genotype 27 20 CC /CC genotype 22 20 17 8 0 N AM I C AB G I PC ed or m M an al on tro gi o ls 0 C Pecentage of subjects 80 25 31 October 2021 Dr. Mohamed Z. Gad 30

Serum Biochemical Markers Acute (n=11) vs. Chronic (Med. +PCI+CABG, n=89) * 0, 750 ADMA 0000 (mmol/l) 0000 0005 0, 59 (mmol/l) 0, 850 0000 0001 * hs. CRP (mg/l) 31 October 2021 64, 3 100, 6 L-Arginine (mmol/l) * * SDMA * 0, 5 104, 1 178, 7 22, 3 160, 8 39, 6 Arg/ADMA 188, 4 Cholesterol (mg/dl) Dr. Mohamed Z. Gad * Significantly different from Chronic at p<0. 05 31 31

Conclusions • DDAH 2 SNP 1 (-1151 C/A) and SNP 2 (-449 C/G) are in complete linkage disequilibrium • A allele/AA genotype for SNP 1 and G allele/GG genotype for SNP 2 are strongly associated with CVD in male 35 -50 y Egyptian patients. • Frequency of A allele / AA genotype (SNP 1) and G allele / GG genotype (SNP 2) are directly proportional with severity of coronary insufficiency. • AMI Patients showed higher serum levels of ADMA, SDMA, and hs. CRP; and lower serum L-arginine, L-arginine/ADMA and ADMA/SDMA than chronic patients. • No direct association between DDAH 2 genotype and serum levels of 31 October 2021 Dr. Mohamed Z. Gad 32 studied biomarkers. 10/31/2021 32

Summary of Results of the EGY-NO-MI Study Healthy Controls P MI Patients Number / Age Mal= 170 Fem= 32 / 22 -56 y Mal =169 Fem = 35 / 25 -55 y e. NOS (298 G/T) 58. 4 % 33. 7 % 7. 9% TT 51. 7% 42% GT 6. 3% TT GG GT GG DDAH 2 -1151 C/A) 18% AA 54% AC 28% CC 31% AA 52% AC 17% CC DDAH 2 - 449 C/G) 18% GG 54% CG 28% CC 31% GG 52% CG 17% CC NADPH oxid. (242 27. 7 % 71. 3 % 0. 99%TT 40. 2 % 59. 8% 0% TT C/T) NO (m. M) CC 30. 26 CT CC 44. 66 CT Serum + 0. 16 + 1. 97 PON 1 ADMA (192 Q/R) 66. 7% 0. 68 25% QR 8. 3% RR 38. 2% 0. 67 49. 0% Serum (m. M) + 0. 019 + 0. 026 12. 8% QQ 0. 47 + 0. 01 QQ 0. 53 QR Serum SDMA (m. M) + 0. 025 RR ET-1 Lys 198 Asn 62. 2%GG 32. 4%G 63. 4 97. 28 32. 9%G Serum L-arginine 87. 39 + 4. 07 5. 4%TT + 4. 643. 7%TT T %GG T (m. M) Serum L-arg. /ADMA 136. 5 + 7. 11 171. 8 + 8. 82 Serum hs-CRP 5. 98 + 1. 1 23. 37 + 3. 22 (mg/l) Serum ET-1 (pg/ml) 5. 95 + 0. 192 10. 8 + 0. 61 Serum Ox-LDL 31 October 2021 (ng/ml) 113. 2 + 4. 9 Dr. Mohamed Z. Gad 203. 4 + 21. 33 0. 388 0. 044 0. 0259 0. 0001 0. 0006 0. 75 0. 026 0. 9 0. 11 0. 002 2 x 10 -5 2 x 1011 8 x 10 -5 33

Publications 1. Gad M. Z. , Hassanein S. I. , Abdel-Maksoud S. M. , Shaban, G. M. , Abou-Aisha K. , Hossam A. Elgabarty H. A. (2010) Assessment of serum levels of asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and L-arginine in coronary artery disease. Biomarkers 15(8): 746 -752. 2. Gad M. Z. , Hassanein S. I. , Abdel-Maksoud S. M. , Shaban G. M. , Abou-Aisha K. (2011) Association of DDAH 2 Gene Polymorphism with Cardiovascular Disease in Egyptian Patients. J Genet 90(1): 161 -163. 3. Gad M. Z. , Abdel Rahman M. F. , Hashad I. M. , Abdel-Maksoud S. M. , Farag N. M. , Abou-Aisha K. (2012) Endothelial Nitric Oxide Synthase (G 894 T) Gene Polymorphism in a Random Sample of Egyptian Population: Comparison with Myocardial Infarction Patients. Genet Test Mol Biomarkers 16(7): 695 -700 4. Sahar Abdel-Maksoud, Sally Ibrahim, Feeby Samir, Khaled Abou-Aisha, Mohamed Z. Gad (2013) Correlation of Glu 298 Asp e. NOS Polymorphism with Serum NO Levels in Egyptian Patients with Coronary Artery Disease British Journal of Medicine and Medical Research (BJMMR) 3(4): 1678 -1687. 5. Ingy M. Hashad , Mohamed F. Abdel Rahman, Sahar M. Abdel-Maksoud, Khalda S. Amr, Laila K. Effat, Gamal M. Shaban, Mohamed Z. Gad (2014) C 242 T polymorphism of NADPH oxidase p 22 phox gene reduces the risk of coronary artery disease in a random sample of Egyptian population. Mol Biol Rep 41(4): 2281 -6 6. Mohamed F. Abdel Rahman, Ingy M. Hashad, Khaled Abou-Aisha, Sahar M. Abdel-Maksoud, Mohamed Z. Gad (2015) Addressing the Link between Paraoxonase-1 Gene Variants and the Incidence of Early Onset Myocardial Infarction Archives of Medical Science (AMS) 11(3): 513 -520. 31 October 2021 Research. Gate – Mohamed Gad Dr. Mohamed Z. Gad 34 Google Scholar Citations- Mohamed Gad

Acknowledgments • • • Dr. Sally Hassanein Dr. Sahar Abdel-Maksoud Dr. Khaled Abou-Aisha Dr. Ingy Hashad Dr. Mohamed Farouk Dr. Hossam El-Gabarty • Prof. Rainer Boger • Dr. N. Lueneburg • Dr. E. Shwedhelm National Research Center, Egypt National Heart Institute, Egypt • Prof. Gamal Shaban • Prof. Yehia Gad This study was supported by the Science and Technology 31 October 2021 Dr. Mohamed Z. Gad 35 10/31/2021 35 Development Fund (STDF) grant No. 2951.