The effects of the interaction of vascular endothelial cells and vascular smooth muscle cells on PCSK9 and NF-kB protein expression in vascular smooth muscle cells

Year 2023, Volume: 4 Issue: 3, 133 - 141, 08.08.2023

Ece Yurtseven , Kemal Baysal , Hülya Yılmaz Aydoğan , Oğuz Öztürk

Abstract

Introduction:

Atherosclerosis is the leading cause of death worldwide. Atherosclerosis is an inflammatory disease which causes significant structural and functional changes in vascular wall cells. It is known that the main cells of the vascular wall, endothelial cells, and smooth muscle cells communicate during atherosclerosis, but the mechanisms of this communication are not fully understood. PCSK9, on the other hand, is a molecule involved in the etiology of hyperlipidemia, and its levels increase in vascular wall cells during atherosclerosis, exerting direct atherosclerotic effects. In our study, we investigated the changes induced by endothelial cells exposed to atherogenic factors in smooth muscle cells and their effects on NF-kB expression, the most important molecule in inflammation, and on PCSK9 protein expression, which has recently been discovered to have direct atherosclerotic effects.

Methods:

Cells isolated from human umbilical veins (HUVEC) were incubated for 24 hours with 300 µM hydrogen peroxide (H2O2) or 40 µg/ml oxidized LDL (Ox-LDL) or 5% cigarette smoke extract (CSE). Following 24 hours, human aortic smooth muscle cells (HASMCs) were incubated with the supernatants of HUVECs which were treated with these substances after appropriate processing. After 48 hours, protein extraction was performed from smooth muscle cells and analyzed using the Western Blot method.

Results:

We observed no changes in the viability of HUVECs exposed to atherogenic substances. There was significant morphological changes in HASMCs incubated with endothelial cell supernatants exposed to 5% CSE and 300 µM H2O2. Increased expression of NF-kB (p=0.024, p=0.043) and PCSK9 protein (p=0.034, p=0.0137) was found in HASMCs incubated with supernatants exposed to 5% CSE and 300 µM H2O2, respectively. In HASMCs incubated with endothelial cell supernatant exposed to Ox-LDL, an increase in PCSK9 expression was observed (p=0.037), while no significant increase in NF-kB expression was observed (p=0.89).

Conclusion:

In our study, it was observed that endothelial cells exposed to atherogenic factors induced changes in vascular smooth muscle cells and increased PCSK9 and NF-kB expression in these cells.

Keywords

Atherosclerosis, Vascular smooth muscle cell, Vascular endothelial cell, Proprotein convertase subtilisin/kexin type 9 (PCSK9), Nuclear factor kappa B (NF-kB)

Supporting Institution

İstanbul Ünivertsitesi Bilimsel Araştırma Projeleri Birimi

Project Number

36268

Thanks

I would like to thank the Istanbul University Institute of Health Sciences and Istanbul University for providing me with the opportunity to conduct this study. This study was supported by the Scientific Research Projects Coordination Unit of Istanbul University. Project no: 36268

Vasküler endotel hücreleri ile vasküler düz kas hücrelerinin etkileşiminin, vasküler düz kas hücrelerinde PCSK9 ve NF-kB protein ekspresyonu üzerindeki etkileri.

Abstract

Giriş:

Ateroskleroz dünyada görülen en sık ölüm sebebidir. Ateroskleroz inflamatuar bir hastalık olup, damar duvarı hücrelerinde belirgin şekil ve fonksiyon değişikliklerine neden olur. Damar duvarındaki ana hücreler olan endotel hücreleri ile düz kas hücrelerinin ateroskleroz sırasında iletişim halinde oldukları bilinen bir gerçek olmakla beraber, bu iletişimin mekanizmaları net değildir. PCSK9 ise hiperlipidemi etyolojisinde yer alan bir molekül olmakla birlikte, damar duvar hücrelerinde ateroksleroz sırasında artar ve damar duvar hücrelerine direk aterosklerotik etkileri vardır. Çalışmamızda aterojenik etkenlere maruz kalan endotel hücrelerinin, düz kas hücrelerinde yaptığı değişiklikleri ve inflamasyonun en önemli molekülü olan NF-kB ve direct aterosklerotik etkileri son zamanlarda keşfedilmiş olan PCSK9 protein ekspresyonlarına olan etkilerini araştırdık.

Metod:
İnsan umblikal veninden izole edilen hücreler (HUVEC) ayrı ayrı 24 saat boyunca 300 µM hidrojen peroksit (H2O2), 40 µg/ml okside LDL (Ox-LDL) %5 sigara duman ekstraktı (SDE) ile inkübe edildi, 24 saat sonra aterojenik etkenlere maruz kalan endotel hücrelerinin süpernatantları uygun işlemlerden sonra insan aortic düz kas hücrelerinin (HAVSMC) inkübe edilmesi için kullanıldı. Düz kas hücrelerinden 48 saat sonra protein ekstraksiyonu yapılarak Western Blot yöntemiyle analiz edildi.

Bulgular:
Aterojenik maddelere maruz kalan HUVEC’lerde canlılığın korunduğu saptandı. Bu maddelere maruz kalan endotel hücre süpernatantları ile inkübe edilen HASMC’de belirgin bir morfolojik değişiklik saptandı. %5 SDE ve 300 µM H2O2’ye maruz kalan endotel hücre süpernatantları ile inkübe edilen HASMC’lerde NF-kB (sırasıyla p=0.024,p=0.043) ve PCSK9 protein (p=0.034, p=0.0137) ekspresyonları artmış bulundu. Ox-LDL’ye maruz kalan endotel hücre süpernatantı ile inkübe edilen HASMC’lerde ise PCSK9 ekspresyonu artmış olup (p=0.037), NF-kB ekspresyonunda anlamlı bir artış izlenmemiştir. (p=0.89)

Sonuç:

Çalışmamızın sonucunda aterojenik etkenlere maruz kalan endotel hücrelerinin, damar düz kas üzerinde değişikliklere neden olduğu,damar düz kas hücrelerindeki PCSK9 ve NF-kB ekspresyonunu arttırdığı gözlenmiştir.

Keywords

Ateroskleroz, Vasküler düz kas hücresi, Vasküler endotel hücresi, Proprotein konvertaz subtilisin/keksin tip 9 (PCSK9), Nüleer Faktör Kappa B (NF-kB)

Project Number

36268

References

• 1. Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, De Ferranti SD, Floyd J, Fornage M, Gillespie C, Isasi CR, Jim’nez MC, Jordan LC, Judd SE, Lackland D, Lichtman JH, Lisabeth L, Liu S, Longenecker CT, MacKey RH, Matsushita K, Mozaffarian D, Mussolino ME, Nasir K, Neumar RW, Palaniappan L, Pandey DK, Thiagarajan RR, Reeves MJ, Ritchey M, Rodriguez CJ, Roth GA, Rosamond WD, Sasson C, Towfghi A, Tsao CW, Turner MB, Virani SS, Voeks JH, Willey JZ, Wilkins JT, Wu JHY, Alger HM, Wong SS, Muntner P (2017) Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation 135:e146–e603 . https://doi.org/10.1161/CIR.0000000000000485
• 2. Libby P, Buring JE, Badimon L, Hansson GK, Deanfield J, Bittencourt MS, Tokgözoğlu L, Lewis EF (2019) Atherosclerosis. Nat Rev Dis Prim 5:56 . https://doi. org/10.1038/s41572-019-0106-z
• 3. Abifadel M, Benjannet S, Farnier M, Seidah NG, Moulin P, Wickham L, Devillers M, Junien C, Cruaud C, Lecerf J-M, Chambaz J, Ouguerram K, Bruckert E, Krempf M, Varret M, Derré A, Chanu B, Allard D, Luc G, Weissenbach J, Beucler I, Rabès J-P, Prat A, Villéger L, Erlich D, Boileau C (2003) Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 34:154–156 . https:// doi.org/10.1038/ng1161
• 4. Rosenson RS, Hegele RA, Fazio S, Cannon CP (2018) The Evolving Future of PCSK9 Inhibitors. J Am Coll Cardiol 72:314–329 . https://doi.org/10.1016/J. JACC.2018.04.054
• 5. Lagace TA (2014) PCSK9 and LDLR degradation: regulatory mechanisms in circulation and in cells. Curr Opin Lipidol 25:387–393 . https://doi.org/10.1097/MOL.0000000000000114
• 6. Karagiannis AD, Liu M, Toth PP, Zhao S, Agrawal DK, Libby P, Chatzizisis YS (2018) Pleiotropic Anti-atherosclerotic Effects of PCSK9 InhibitorsFrom Molecular Biology to Clinical Translation. Curr. Atheroscler. Rep. 20
• 7. Bennett MR, Sinha S, Owens GK (2016) Vascular smooth muscle cells in atherosclerosis. Circ Res 118:692 . https://doi.org/10.1161/CIRCRESAHA.115.306361
• 8. Ragusa R, Basta G, Neglia D, De Caterina R, Del Turco S, Caselli C (2021) PCSK9 and atherosclerosis: Looking beyond LDL regulation. Eur J Clin Invest 51: . https:// doi.org/10.1111/ECI.13459
• 9. Liu T, Zhang L, Joo D, Sun SC (2017) NF-κB signaling in inflammation. Signal Transduct Target Ther 2017 21 2:1–9 . https://doi.org/10.1038/sigtrans.2017.23
• 10. Cheng W, Deng C, Jin L, Shao L, Xu X, Shu C (2015) [Role of NF-κB/survivin signal pathway on intima hyperplasia of rat carotid balloon injury restenosis model]. Zhonghua Xin Xue Guan Bing Za Zhi 43:248–253
• 11. Yoshida T, Yamashita M, Horimai C, Hayashi M (2013) Smooth muscle-selective inhibition of nuclear factor-κB attenuates smooth muscle phenotypic switching and neointima formation following vascular injury. J Am Heart Assoc 2: . https:// doi.org/10.1161/JAHA.113.000230
• 12. Tang Z-H, Peng J, Ren Z, Yang J, Li T-T, Li T-H, Wang Z, Wei D-H, Liu L-S, Zheng X-L, Jiang Z-S (2017) New role of PCSK9 in atherosclerotic inflammation promotion involving the TLR4/NF-κB pathway. Atherosclerosis 262:113–122 . https:// doi.org/10.1016/j.atherosclerosis.2017.04.023
• 13. Méndez‐barbero N, Gutiérrez‐muñoz C, Blanco-Colio LM (2021) Cellular Crosstalk between Endothelial and Smooth Muscle Cells in Vascular Wall Remodeling. Int J Mol Sci 22: . https://doi.org/10.3390/IJMS22147284
• 14. Gellner CA, Reynaga DD, Leslie FM (2016) Cigarette Smoke Extract: A preclinical model of tobacco dependence. Curr Protoc Neurosci 77:9.54.1 . https://doi.org/10.1002/CPNS.14
• 15. Liu M, Samant S, Vasa CH, Pedrigi RM, Oguz UM, Ryu S, Wei T, Anderson DR, Agrawal DK, Chatzizisis YS (2023) Co-culture models of endothelial cells, macrophages, and vascular smooth muscle cells for the study of the natural history of atherosclerosis. PLoS One 18: . https://doi.org/10.1371/JOURNAL. PONE.0280385
• 16. Graham DJ, Alexander JJ, Miguel R (1991) Aortic endothelial and smooth muscle cell co-culture: an in vitro model of the arterial wall. J Invest Surg 4:487–494 . https://doi.org/10.3109/08941939109141179
• 17. Sandoo A, Zanten JJC. V van, Metsios GS, Carroll D, Kitas GD (2010) The Endothelium and Its Role in Regulating Vascular Tone. Open Cardiovasc Med J 4:302 . https://doi.org/10.2174/1874192401004010302
• 18. Dowling P, Clynes M (2011) Conditioned media from cell lines: a complementary model to clinical specimens for the discovery of disease-specific biomarkers. Proteomics 11:794–804 . https://doi.org/10.1002/PMIC.201000530
• 19. Messner B, Bernhard D (2014) Smoking and cardiovascular disease: mechanisms of endothelial dysfunction and early atherogenesis. Arterioscler Thromb Vasc Biol 34:509–515 . https://doi.org/10.1161/ATVBAHA.113.300156
• 20. Barua RS, Ambrose JA, Srivastava S, DeVoe MC, Eales-Reynolds LJ (2003) Reactive Oxygen Species Are Involved in Smoking-Induced Dysfunction of Nitric Oxide Biosynthesis and Upregulation of Endothelial Nitric Oxide Synthase. Circulation 107:2342–2347 . https://doi.org/10.1161/01.CIR.0000066691.52789.BE
• 21. Bernhard D, Csordas A, Henderson B, Rossmann A, Kind M, Wick G (2005) Cigarette smoke metal-catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules. FASEB J 19:1096–1107 . https:// doi.org/10.1096/FJ.04-3192COM
• 22. Sundar IK, Chung S, Hwang J woong, Lapek JD, Bulger M, Friedman AE, Yao H, Davie JR, Rahman I (2012) Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-κB-dependent Genes. PLoS One 7:e31378 . https://doi.org/10.1371/JOURNAL.PONE.0031378
• 23. Ma B, Wang X, Zhang R, Niu S, Rong Z, Ni L, Di X, Han Q, Liu C (2021) Cigarette smoke extract stimulates PCSK9 production in HepG2 cells via ROS/NF-κB signaling. Mol Med Rep 23: . https://doi.org/10.3892/mmr.2021.11970
• 24. Ferri N, Marchianò S, Tibolla G, Baetta R, Dhyani A, Ruscica M, Uboldi P, Catapano AL, Corsini A (2016) PCSK9 knock-out mice are protected from neointimal formation in response to perivascular carotid collar placement. Atherosclerosis 253:214–224 . https://doi.org/10.1016/j.atherosclerosis.2016.07.910
• 25. Madamanchi NR, Vendrov A, Runge MS (2005) Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 25:29–38 . https://doi.org/10.1161/01. ATV.0000150649.39934.13
• 26. Su LJ, Zhang JH, Gomez H, Murugan R, Hong X, Xu D, Jiang F, Peng ZY (2019) Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis. Oxid Med Cell Longev 2019: . https://doi.org/10.1155/2019/5080843
• 27. Ungvari Z, Orosz Z, Labinskyy N, Rivera A, Xiangmin Z, Smith K, Csiszar A (2007) Increased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries. Am J Physiol Heart Circ Physiol 293: . https:// doi.org/10.1152/AJPHEART.01346.2006
• 28. Cyrne L, Oliveira-Marques V, Marinho HS, Antunes F (2013) H2O2 in the Induction of NF-κB-Dependent Selective Gene Expression. Methods Enzymol 528:173–188 . https://doi.org/10.1016/B978-0-12-405881-1.00010-0
• 29. Ding Z, Liu S, Wang X, Deng X, Fan Y, Sun C, Wang Y, Mehta JL (2015) Hemodynamic Shear Stress via ROS Modulates PCSK9 Expression in Human Vascular Endothelial and Smooth Muscle Cells and Along the Mouse Aorta. Antioxid Redox Signal 22:760–771 . https://doi.org/10.1089/ars.2014.6054
• 30. Safaeian L, Mirian M, Bahrizadeh S (2022) Evolocumab, a PCSK9 inhibitor, protects human endothelial cells against H2O2-induced oxidative stress. Arch Physiol Biochem 128:1681–1686 . https://doi.org/10.1080/13813455.2020.1788605
• 31. Cochain C, Vafadarnejad E, Arampatzi P, Pelisek J, Winkels H, Ley K, Wolf D, Saliba AE, Zernecke A (2018) Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis. Circ Res 122:1661–1674 . https://doi.org/10.1161/CIRCRESAHA.117.312509
• 32. Ding Z, Liu S, Wang X, Deng X, Fan Y, Shahanawaz J, Shmookler Reis RJ, Varughese KI, Sawamura T, Mehta JL (2015) Cross-talk between LOX-1 and PCSK9 in vascular tissues. Cardiovasc Res 107:556–567 . https://doi.org/10.1093/cvr/cvv178
• 33. Chen L, Yang G, Zhang X, Wu J, Gu Q, Wei M, Yang J, Zhu Y, NanpingWang, Guan Y (2009) Induction of MIF expression by oxidized LDL via activation of NF-kappaB in vascular smooth muscle cells. Atherosclerosis 207:428–433 . https://doi.org/10.1016/J.ATHEROSCLEROSIS.2009.05.021
• 34. Robbesyn F, Salvayre R, Negre-Salvayre A (2004) Dual role of oxidized LDL on the NF-kappaB signaling pathway. Free Radic Res 38:541–551 . https://doi.org/10.1080/10715760410001665244