A Review of the Impact of Neutrophils and Neutrophil Extracellular Traps (NETs) on the Development of Aortic Aneurysms in Animal and Human Studies

Milena Michalska; Tadeusz Grochowiecki; Tomasz Jakimowicz; Sławomir Nazarewski

doi:10.12659/MSM.935134

28 December 2021 : Review article

A Review of the Impact of Neutrophils and Neutrophil Extracellular Traps (NETs) on the Development of Aortic Aneurysms in Animal and Human Studies

Milena Michalska

^1ABCDEFG*, Tadeusz Grochowiecki^1AEFG, Tomasz Jakimowicz

^1DEF, Sławomir Nazarewski^1D

DOI: 10.12659/MSM.935134

Med Sci Monit 2021; 27:e935134

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Background Use of Animal Models in AA Pathogenesis of Aortic Aneurysm and Neutrophilic Role Inflammation Atherosclerosis and Thrombosis NETosis in Vascular Pathologies Process of Visualization and Measurement of NETs NETs in AAA NETs in Other Diseases that Can Be Risk Factors of AAA Conclusions References

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Table 2 Summary of published studies that include influence on neutrophil extracellular traps in aortic aneurysm.

No	Author	Year	Model	Population	Control group
1.	Sandrine Delbosc et al []⁶⁴	2011



2.	Humin Yan et al []⁶⁷	2016
2.	Humin Yan et al []⁶⁷	2016

3.	Akshaya K. Meher et al []⁴⁷	2018
3.	Akshaya K. Meher et al []⁴⁷	2018

4.	Michael Spinosa et al []⁶²	2018
4.	Michael Spinosa et al []⁶²	2018

5.	Silvia D. Visonà et al []⁷²	2018			–

6.	Patrick Haider et al []⁷³	2020			–
6.	Patrick Haider et al []⁷³	2020			–

7.	Wolf Eilenberg et al []⁷⁴	2021



8.	Ming Wei et al []⁷⁵	2021			–

9.	Akrivi Chrysantho-poulou et al []⁷⁷	2021



1.	Cancer, infection, and any immune-mediated disease		H1, Cit-H4, elastase	Immunofluorescence	NETs are abundant in AAA ILTsThe enlarged concentration of cf-DNA in plasma and conditioned medium between AAA patients and healthy volunteersIncreased circulating MPO-DNA complexes in AAAChronic -bacteremia prompt neutrophil recruitment in experimental AAA
			Cit-H4	Western blot
			cf-DNA	Quant- it Picogreen, dsDNA Reagent, Invitrogen
			MPO, MPO-DNA complexes, Anti-P.gingivalis Ig	ELISA
			MMP-9	Gelatin zymography
			Bacterial endotoxin released by ILT	Limulus Amebocyte Lysate (LAL) chromogenic endpoint assay)
			DNA extraction and bacterial DNA amplification	QIamp DNA Blood Midi Kit (Qiagen) PCR
			Neutrophil isolation	Immunofluorescence

2.	–		DNA-CRAMP complexes	Generation of DNA-CRAMP complexes	In elastase-induced murine aortic tissues NETs are observed, mainly in the adventitiaNeutrophil proteases and NETs play an important role in the development of AAA
			Elastin degradation,macrophage, CD3+ T cells, DCs	Immunohistochemistry	AAA is triggered by the DNA-CRAMP complexINF I and DCs promote the inflammatory responses of AAA
			IFN I, Histone H2B, MPO, CRAMP	Immunofluorescence
			DCs	Flow cytometric analysis
			Gelatinase	Zymography
			NET stimulation (by LPS or C5a)	Fluorescence microscope
			Cytokine analysis	Cytometric bead arrays

3.	–		NE	Immunofluorescence	NETosis is reduced by blocking IL-1β, in human AAA NETs coexist with IL-1β
			Cit-H3	Western blot
			Ly-6B.2 (neutrophil indicator) Alfa-smooth muscle alkaline phosphatase	Immunohistochemistry	Human neutrophils can produce ceramide by IL-1β stimulation, decreased ceramide synthesis attenuates NETosisThe initial phase of murine experimental AAA includes development of NETsFor AAA mice development, IL-1β expression in neutrophils is necessary
			cf-DNA	Quant-iT PicoGreen dsDNA Reagent ThermoFisher, Scientific
			Neutrophil culture and NETs induce, CerS6	Flow cytometry
			Ceramide isolation	Liquid chromatography-mass spectrometry

4.	–		RvD1IL-1beta	RvD1 ELISA	In the Ang II mouse model, RvD1 reduced AAAs and decreased Cit-H3
			CitH3	Western blot	Treatment RvD1 displays lower stain for markers of NETosis
			NE	Immunohistochemistry	Treatment RvD1 displays lower stain for markers of NETosis
			MMP-2MMP-9	Gelatin zymography	In the treatment group RvD1, IL-1β is decreased, whereas IL-10 is increased

5.	Chronic aortitis and periaortitis, purely traumatic dissections, incomplete sampling		α-smooth muscle alpha-actin (SMA) MPOCit-H3α-CD3, α-CD68 (macrophages identificator)CD31 and CD34 (EC identificator)	Immunohistochemistry	The presence of NEU and NETs were increased significantly in subacute infiltering dissections but decreased in early organized and late scarring groupsThere were high numbers of MPO neutrophils in acute dissection but the Cit-H3 NETS levels were relatively little
5.			Dissection age (4 phases)	Histological

6.	–		Cit-H4MPOCD68Ly6GPolarization of macrophages	Immunofluorescence	NETs develop voluntarily in human blood clotDifferently polarized macrophages can degrade NETs by DNA uptake by individuals’ mechanismsNETs were found in all vessels aortic wall; most NET were found in the abluminal section and then in the luminal part
			DNA uptake from blood clots by macrophages	Flow cytometry
			Degradation of NETs in vitro	Fluorescence	DNase1L3 can be produced by generated macrophages
			Protein quantification (DNase)	ELISA and flow cytometry	Blockage of macropinocytosis cause enlarge NET burden and decreased thrombus degradation
			DNase 1L1	Immunofluorescence	Reginal concertation of macrophages is inversely connected with NETs in human AAA
			Apoptosis assessment	Flow cytometry
			DNA extraction	PCR and electrophoresis

7.	Recent tumors (<1 year), chemotherapy, systemic autoimmune or hematological disease, organ transplantation		Cit-H3MPOcf-DNA-histone complexesIL-1beta	ELISA	The level of Cit-H3 in plasma can be a biomarker of AAA enlargementAfter repairing AAA level of Cit-H3 is decreased
			Ly6GCit-H4	Immunofluorescence	Blocking the citrullination in histone can prevent the development of AAA in an Ang II-induced model
			NGALelastase	Mass spectrometers (Luminex MagPix)

8.			MPO	Immunohistochemistry	In vitro and in vivo NET formation is enhanced by Ang IIIn vitro PAD4 inhibition (by YW3-56) reduced Ang II-induced NET formationPAD4 inhibition prevent AAA rapture and decline VSMC apoptosisBy p38/JNK pathway NET enhance VSMC apoptosis
			Cit-H3Ly6GNE	Immunofluorescence
			dsDNAPAD4	Quant-iT PicoGreen dsDNA Assay Kit Thermo Fisher, Scientific
			Depletion of VSMC	Annexin V/PI apoptosis detection kit (556547, BD biosciencesFlow cytometry
			Neutrophil isolation	Immunofluorescence
			p38p-JNK	Western blot

9.	Age<18 years, presence of secondary HT, presence of pregnancy, CVD, DM, renal disease, infection, inflammatory or neoplastic disease, antihypertensive therapy, vaccination at least 4 weeks before		MPONECit-H4	Immunofluorescence	The development of NETs is induced by Ang II in a ROS/autophagy-dependent mechanismIn fibrotic renal and aneurysmal aortic tissue are noticeable NETs from NEU with tissue factor expression
			Cit-H3	ELISA
			TF	TF activity assay
			MPO/DNA complexes	ELISA
			RNA isolation	PCR
			Neutrophil isolation and viability	Flow cytometry

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A Review of the Impact of Neutrophils and Neutrophil Extracellular Traps (NETs) on the Development of Aortic Aneurysms in Animal and Human Studies

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