Biomarker Profiling for Discrimination of High-Risk Asymptomatic Carotid Artery Stenosis Patients with Ulcerated Plaques: A Pilot Study

Abstract

Although various methods are used to treat patients with asymptomatic carotid artery stenosis (ACAS), approaches are controversial, and combining imaging of carotid plaque features with biomarkers to identify plaques prone to rupture may be crucial in identifying high-risk ACAS patients. This study aimed to investigate a blood-based biomarker for discriminating ulceration in ACAS patients by analyzing plaque surface morphology through RNA sequencing of blood samples. Peripheral blood samples were collected from ACAS patients with plaque morphology determined by Doppler ultrasonography. Then, total RNA was isolated, and RNA-Seq was performed to analyze differentially expressed genes (DEGs). The KEGG, Reactome, and Gene Ontology (GO) terms pathway enrichment analyses were performed to investigate the molecular functions and biological processes involved in plaque formation. The pilot study included 7 ACAS patients, 57.1 % exhibiting ulcerated plaques. RNA-Seq results revealed significant upregulation of genes related to immune response, cell cycle regulation, and oxidative stress in ulcerated plaques. Especially, TP73, CCL3L3, and PXDNL genes showed the highest fold changes, indicating their role in endothelial damage, immune activation, and oxidative stress. KEGG and Reactome analyses identified TNF and chemokine signaling pathways as key regulators in ulcerated plaque formation. Our findings indicate that TP73, CCL3L3, and PXDNL may be potential biomarkers for identifying high-risk ACAS patients with ulcerated plaques due to their involvement in immune system regulation and oxidative stress-related processes. Thus, these genes and the pathways may be candidate biomarkers for early diagnosis and risk stratification, improving treatment approaches for ACAS.

Keywords

• Carotid stenosis
• Carotid artery plaque
• Biomarker
• RNA-Seq
• Ulcerated plaque

Supporting Institution

Bursa Uludag University Scientific Research Projects Coordination Unit

Project Number

TAY-2022-592

Ethical Statement

The Bursa Uludag University Scientific Research Projects Coordination Unit, TAY-2022-592, supported this study.

Ülsere Plaklı Yüksek Riskli Asemptomatik Karotis Arter Stenozu Hastalarının Ayırımı İçin Biyobelirteç Profillemesi: Pilot Çalışma

Abstract

Asemptomatik karotis arter stenozu (ACAS) olan hastaların tedavisinde çeşitli yöntemler kullanılsa da yaklaşımlar tartışmalıdır ve plak rüptürü eğilimli olan hastaları belirlemek için karotis plak görüntülerinin biyobelirteçlerle birleştirilmesinin, yüksek riskli ACAS hastalarının belirlenmesinde önemli olduğu düşünülmektedir. Mevcut çalışmada, plak yüzey morfolojisini analiz ederek ACAS hastalarında ülserasyonu ayırt etmek için RNA sekanslama yoluyla kan bazlı biyobelirteç araştırılması amaçlanmıştır. Bu doğrultuda, Doppler ultrasonografi ile plak morfolojisi belirlenen ACAS hastalarından periferik kan örnekleri toplandı. Daha sonra, total RNA izole edildi ve farklı şekilde ifade edilen genleri (DEG'ler) analiz etmek için RNA-Seq gerçekleştirildi. Plak oluşumunda yer alan moleküler işlevleri ve biyolojik süreçleri araştırmak için KEGG, Reactome ve Gen Ontolojisi (GO) ile yolak zenginleştirme analizleri gerçekleştirildi. Pilot çalışmaya 7 ACAS hastası dahil edildi ve %57,1'inde ülsere plaklar olduğu görüldü. RNA-Seq sonuçları ülsere plaklarda bağışıklık tepkisi, hücre döngüsü düzenlemesi ve oksidatif stresle ilgili genlerde önemli bir artış olduğunu ortaya koydu. Özellikle TP73, CCL3L3 ve PXDNL genlerinin bu süreçlerde rol aldığı ve bu genlerin endotel hasarı, bağışıklık aktivasyonu ve oksidatif streste rolleri olduğu gösterildi. KEGG ve Reactome analizleri, ülserli plak oluşumunda anahtar düzenleyiciler olarak TNF ve kemokin sinyal yollarını tanımladı. Bulgularımız, TP73, CCL3L3 ve PXDNL'nin bağışıklık sistemi düzenlemesi ve oksidatif stresle ilişkili süreçlerdeki katılımları nedeniyle ülserli plakları olan yüksek riskli ACAS hastalarını tanımlamak için potansiyel biyobelirteçler olabileceğini göstermektedir. Bu nedenle, ilişkili genlerin ve yolakların erken tanı ve risk sınıflandırması için aday biyobelirteçler olabileceği ve ACAS için tedavi yaklaşımlarını iyileştirebileceğini düşündürmektedir.

Keywords

• Karotis stenozu
• Karotis arter plağı
• Biyobelirteç
• RNA-Seq
• Ülsere plak

Project Number

TAY-2022-592

References

1. 1.Jebari-Benslaiman S, Galicia-García U, Larrea-Sebal A,Olaetxea JR, Alloza I, Vandenbroeck K, et al. Pathophysiologyof atherosclerosis. Int J Mol Sci. 2022;23(6):3346.
2. 2.Mehu M, Narasimhulu CA, Singla DK. Inflammatory cells in atherosclerosis. Antioxidants (Basel). 2022;11(2):233.
3. 3.Ismail A, Ravipati S, Gonzalez-Hernandez D, Mahmood H,Imran A, Munoz EJ, et al. Carotid artery stenosis: A look into the diagnostic and management strategies, and related complications. Cureus. 2023;15(6):e38794.
4. 4.Musialek P, Bonati LH, Bulbulia R, Halliday A, Bock B, Capoccia L, et al. Stroke risk management in carotid atherosclerotic disease: A Clinical Consensus Statement of the ESC Council on Stroke and the ESC Working Group on Aorta and Peripheral Vascular Diseases. Cardiovasc Res. 2023;119(10):2557-78.
5. 5.Qaja E, Tadi P, Theetha Kariyanna P. Symptomatic carotid artery stenosis. In: StatPearls [Internet]. Treasure Island (FL):StatPearls Publishing; 2024. Available from:https://www.ncbi.nlm.nih.gov/books/NBK442025/.
6. 6.Chatzikonstantinou A, Wolf ME, Schaefer A, Hennerici MG. Asymptomatic and symptomatic carotid stenosis: An obsolete classification? Stroke Res Treat. 2012;2012:340798.
7. 7.Liu Y, Wu B, Wu S, Liu Z, Wang P, Lv Y, et al. Comparison of stable carotid plaques in patients with mild-to-moderate carotid stenosis with vulnerable plaques in patients with significant carotid stenosis. Medicine (Baltimore). 2024;103(4):e40613.
8. 8.Fishbein MC. The vulnerable and unstable atheroscleroticplaque. Cardiovasc Pathol. 2010;19(1):6-11.

9. 9.Standish BA, Spears J, Marotta TR, Montanera W, Yang VX.Vascular wall imaging of vulnerable atherosclerotic carotid plaques: Current state of the art and potential future ofendovascular optical coherence tomography. AJNR Am J Neuroradiol. 2012;33(8):1642-50.
10. 10.Loftus I. Mechanisms of plaque rupture. In: Fitridge R, Thompson M, editors. Mechanisms of vascular disease: A reference book for vascular specialists [Internet]. Adelaide (AU): University of Adelaide Press; 2011. Available from: https://www.ncbi.nlm.nih.gov/books/NBK534259/.
11. 11.Puz P, Lasek-Bal A, Ziaja D, Kazibutowska Z, Ziaja K.Inflammatory markers in patients with internal carotid artery stenosis. Arch Med Sci. 2013;9(2):254.
12. 12.Knoflach M, Kiechl S, Mantovani A, Cuccovillo I, Bottazzi B, Xu Q, et al. Pentraxin-3 as a marker of advancedatherosclerosis: Results from the Bruneck, ARMY and ARFY studies. PLoS One. 2012;7(2):e31474.
13. 13.Yamagami H, Kitagawa K, Nagai Y, Hougaku H, Sakaguchi M, Kuwabara K, et al. Higher levels of interleukin-6 are associated with lower echogenicity of carotid artery plaques. Stroke. 2004;35(3):677-81.
14. 14.Pelisek J, Rudelius M, Zepper P, Poppert H, Reeps C, Schuster T, et al. Multiple biological predictors for vulnerable carotid lesions. Cerebrovasc Dis. 2009;28(5):601-10.
15. 15.Davis PK, Dowdy SF. p73. Int J Biochem Cell Biol.2001;33(9):935-9.
16. 16.Weiss RH, Howard LL. p73 is a growth-regulated protein invascular smooth muscle cells and is present at high levels in human atherosclerotic plaque. Cell Signal. 2001;13(11):727-33.
17. 17.Logotheti S, Richter C, Murr N, Spitschak A, Marquardt S, Pützer BM. Mechanisms of functional pleiotropy of p73 in cancer and beyond. Front Cell Dev Biol. 2021;9:737735.
18. 18.de Jager SC, Bot I, Kraaijeveld AO, Korporaal SJ, Bot M, van Santbrink PJ, et al. Leukocyte-specific CCL3 deficiency inhibits atherosclerotic lesion development by affecting neutrophil accumulation. Arterioscler Thromb Vasc Biol. 2013;33(6):e75-e83.
19. 19.Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB. Reactive oxygen species in inflammation and tissue injury. AntioxidRedox Signal. 2014;20(7):1126-67.
20. 20.Batty M, Bennett MR, Yu E. The role of oxidative stress in atherosclerosis. Cells. 2022;11(23):3843.
21. 21.Hanmer KL, Mavri-Damelin D. Peroxidasin is a novel target ofthe redox-sensitive transcription factor Nrf2. Gene. 2018;674:104-14.
22. 22.Tangeten C, Zouaoui Boudjeltia K, Delporte C, Van Antwerpen P, Korpak K. Unexpected role of MPO-oxidized LDLs in atherosclerosis: In between inflammation and its resolution. Antioxidants (Basel). 2022;11(5):874.
23. 23.Hansson GK, Libby P, Tabas I. Inflammation and plaque vulnerability. J Intern Med. 2015;278(5):483-93.
24. 24.Luo X, Zhao C, Wang S, Jia H, Yu B. TNF-α is a novel biomarker for predicting plaque rupture in patients with ST-segment elevation myocardial infarction. J Inflamm Res. 2022;15:1889-98.
25. 25.Jin M, Fang J, Wang JJ, Shao X, Xu SW, Liu PQ, et al.Regulation of toll-like receptor signaling pathways in atherosclerosis: From mechanisms to targeted therapeutics. Acta Pharmacol Sin. 2023;44(11):2358-75.
26. 26.Falck-Hansen M, Kassiteridi C, Monaco C. Toll-like receptors in atherosclerosis. Int J Mol Sci. 2013;14(7):14008-23.