O papel da via NF-kB/parkin/vegfr-1 associada à agressão hipóxico-isquêmica em amostras de matriz germinativa de pacientes recém-nascidos

Ghirelli, Eliane Amaral; Silva, Felipe Paes Gomes da; Oricil, Alessandro Gonçalves Gomes; Paula, Caroline Busatta Vaz de; Nagashima, Seigo; Oldenburg Neto, Carlos Frederico; Storti, Eduardo; Sakiyama, Fernando Yochiteru Rolim; Kayano, Rafael Martins; Sakiyama, Renata Rolim; Moreira, Vinícius da Silva; Sotomaior, Vanessa Santos; Noronha, Lucia de

Eliane Amaral Ghirelli

Study design

Data analysis

Manuscript writing

Manuscript revision

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0002-6566-7469

Felipe Paes Gomes da Silva

Data analysis

Manuscript writing

Manuscript revision

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0002-5360-3281

Alessandro Gonçalves Gomes Oricil

Data analysis

Manuscript writing

Manuscript revision

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0001-9916-3069

Caroline Busatta Vaz de Paula ^** Corresponding author: Email: carolbvaz@gmail.com (C. B. V. Paula)

Data analysis

Manuscript writing

Manuscript revision

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0002-8434-8471

Seigo Nagashima

Data analysis

Manuscript writing

Manuscript revision

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0003-0069-3823

Carlos Frederico Oldenburg Neto

Data collection

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0001-8034-2251

Eduardo Storti

Data collection

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0002-1936-7561

Fernando Yochiteru Rolim Sakiyama

Data analysis

Universidade Federal do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0003-3842-0927

Rafael Martins Kayano

Data analysis

Universidade Federal do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0001-6405-871X

Renata Rolim Sakiyama

Data analysis

Universidade Federal do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0001-6956-5083

Vinícius da Silva Moreira

Data analysis

Universidade Federal do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0002-5586-4637

Vanessa Santos Sotomaior

Data collection

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0001-5388-6384

Lucia de Noronha

Study design

Manuscript writing

Manuscript revision

Study supervision

Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil.

https://orcid.org/0000-0003-0310-7164

* Corresponding author: Email: carolbvaz@gmail.com (C. B. V. Paula)

Conflict of interests The authors declare there is no conflict of interests.

Figuras (2)
Tabelas (3)

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Figure 1.
Graphics show tissue expression of Parkin, AKT-3, VEGFR-1, and NF-kB in columns plot mean±SD. Photomicrography shows germinal matrix expressing the respective biomarker — higher expression on the left column and lower expression on the right column (200X magnification).

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Figure 2.
(1) Recruitment of Parkin by inflammatory agents in the activation process of nuclear factor-kappa B (NF-kB): In the course of the hypoxic-ischemic insult, inflammatory factors are released by glial cells, such as TNF-α, activating a pathway of neural remodeling¹⁸18. Kaur C, Hao AJ, Wu CH, Ling EA. Origin of microglia. Microsc Res Tech. 2001;54:2-9. https://doi.org/10.1002/jemt.1114
https://doi.org/10.1002/jemt.1114... ,²⁰20. Supramaniam V, Vontell R, Srinivasan L, Wyatt-Ashmead J, Hagberg H, Rutherford M. Microglia activation in the extremely preterm human brain. Pediatr Res. 2013;73:301-9. https://doi.org/10.1038/pr.2012.186
https://doi.org/10.1038/pr.2012.186... . In the presence of high levels of TNF-α, it binds to its receptor, TNFR, which recruits the protein kinase interaction receptor (RIPK), which serves as a binding site for Parkin to act. This molecule promotes the activation of nuclear factor-kappa (NF-kB) by promoting the ubiquitination of the gamma (NEMO) subunit of the IkB enzyme complex (IKK)¹⁴14. Wang Y, Shan B, Liang Y, Wei H, Yuan J. Parkin regulates NF-κB by mediating site-specific ubiquitination of RIPK1. Cell Death Dis. 2018;9:732. https://doi.org/10.1038/s41419-018-0770-z
https://doi.org/10.1038/s41419-018-0770-... . Thus, Parkin promotes NEMO labeling and indirectly phosphorylation of the Beta subunit (IKKβ) of the IKK complex by an upstream kinase, activating it²¹21. Hayden MS, Ghosh S. Regulation of NF-κB by TNF family cytokines. Semin Immunol. 2014;26:253-66. https://doi.org/10.1016/j.smim.2014.05.004
https://doi.org/10.1016/j.smim.2014.05.0... ,²²22. Mulero MC, Huxford T, Ghosh G. NF-κB, IκB, and IKK: integral components of immune system signaling. Adv Exp Med Biol. 2019;1172:207-26. https://doi.org/10.1007/978-981-13-9367-9_10
https://doi.org/10.1007/978-981-13-9367-... . With this activation, IKKβ manages to phosphorylate the inhibitory protein linked to NF-kB, IkBα, which is polyubitkinated and degraded by proteosomes, thus allowing NF-kB, originating from the canonical pathway, to enter the nucleus of neuronal cells in its active form. (2) Role of NF-kB in HIF-1-α mRNA transcription: The NF-kB activated binds to the initiation site of the HIF-1-α precursor DNA molecule, which induces the transcription of the mRNA HIF-1-α²³23. Minet E, Ernest I, Michel G, Roland I, Remacle J, Raes M, et al. HIF1A gene transcription is dependent on a core promoter sequence encompassing activating and inhibiting sequences located upstream from the transcription initiation site and cis-elements located within the 5’UTR. Biochem Biophys Res Commun. 1999;261:534-40. https://doi.org/10.1006/bbrc.1999.0995
https://doi.org/10.1006/bbrc.1999.0995... .This binding allows translation of the HIF factor promoter gene, giving rise to the HIF-1-α molecule. This molecule, inside the cell cytoplasm and in the presence of oxygen, is induced to degradation by prolyl hydroxylase (PHD) enzymes and, under hypoxic conditions, allows the HIF molecule to be activated and enter the cell nucleus¹⁶16. van Uden P, Kenneth NS, Rocha S. Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochem J. 2008;412:477-84. https://doi.org/10.1042/BJ20080476
https://doi.org/10.1042/BJ20080476... . (3) Role of cell growth pathways in HIF activation and production of neural protective factors in MG such as VEGFR-1: Once the HIF molecule is produced, it needs to be stabilized and activated in order to be able to promote the production of neural protective factors such as VEGFR-1. The HIF1-α molecule in the cell cytoplasm is inactive and stabilized by normoxic conditions. For its activation to occur, several factors are related, among them, eukaryotic translation factors/growth factors, which have been shown to contribute to HIF-1-α activation by phosphorylating the molecule and allowing it to enter the nucleus²⁴24. Jiang BH, Zheng JZ, Aoki M, Vogt PK. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factors in endothelial cells. Proc Natl Acad Sci U S A. 2000;97:1749-53. https://doi.org/10.1073/pnas.040560897
https://doi.org/10.1073/pnas.040560897... ,²⁵25. Chen EY, Mazure NM, Cooper JA, Giaccia AJ. Hypoxia activates a platelet-derived growth factor receptor/phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactivation. Cancer Res. 2001;61:2429-33. PMID: 11289110. In this process, neurotrophins are released and bind to the Tyrosine Kinase C (TRK-C) receptor, which is responsible for activating the AKT-3/mTORC-1 pathway and producing eukaryotic translation factors. Inside the nucleus, HIF can produce molecules associated with cellular vascularization — VEGFR-1 — and, therefore, revitalize the tissue by guaranteeing the supply of O2.

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Table 1.
Data of antibodies used in the immunohistochemistry technique to identify the tissue immunoexpression of biomarkers.

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Table 2.
Demographic characteristics and clinical findings.

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Table 3.
Biomarkers and their correlation with clinical data.

Figure 2. (1) Recruitment of Parkin by inflammatory agents in the activation process of nuclear factor-kappa B (NF-kB): In the course of the hypoxic-ischemic insult, inflammatory factors are released by glial cells, such as TNF-α, activating a pathway of neural remodeling¹⁸18. Kaur C, Hao AJ, Wu CH, Ling EA. Origin of microglia. Microsc Res Tech. 2001;54:2-9. https://doi.org/10.1002/jemt.1114
https://doi.org/10.1002/jemt.1114... ,²⁰20. Supramaniam V, Vontell R, Srinivasan L, Wyatt-Ashmead J, Hagberg H, Rutherford M. Microglia activation in the extremely preterm human brain. Pediatr Res. 2013;73:301-9. https://doi.org/10.1038/pr.2012.186
https://doi.org/10.1038/pr.2012.186... . In the presence of high levels of TNF-α, it binds to its receptor, TNFR, which recruits the protein kinase interaction receptor (RIPK), which serves as a binding site for Parkin to act. This molecule promotes the activation of nuclear factor-kappa (NF-kB) by promoting the ubiquitination of the gamma (NEMO) subunit of the IkB enzyme complex (IKK)¹⁴14. Wang Y, Shan B, Liang Y, Wei H, Yuan J. Parkin regulates NF-κB by mediating site-specific ubiquitination of RIPK1. Cell Death Dis. 2018;9:732. https://doi.org/10.1038/s41419-018-0770-z
https://doi.org/10.1038/s41419-018-0770-... . Thus, Parkin promotes NEMO labeling and indirectly phosphorylation of the Beta subunit (IKKβ) of the IKK complex by an upstream kinase, activating it²¹21. Hayden MS, Ghosh S. Regulation of NF-κB by TNF family cytokines. Semin Immunol. 2014;26:253-66. https://doi.org/10.1016/j.smim.2014.05.004
https://doi.org/10.1016/j.smim.2014.05.0... ,²²22. Mulero MC, Huxford T, Ghosh G. NF-κB, IκB, and IKK: integral components of immune system signaling. Adv Exp Med Biol. 2019;1172:207-26. https://doi.org/10.1007/978-981-13-9367-9_10
https://doi.org/10.1007/978-981-13-9367-... . With this activation, IKKβ manages to phosphorylate the inhibitory protein linked to NF-kB, IkBα, which is polyubitkinated and degraded by proteosomes, thus allowing NF-kB, originating from the canonical pathway, to enter the nucleus of neuronal cells in its active form. (2) Role of NF-kB in HIF-1-α mRNA transcription: The NF-kB activated binds to the initiation site of the HIF-1-α precursor DNA molecule, which induces the transcription of the mRNA HIF-1-α²³23. Minet E, Ernest I, Michel G, Roland I, Remacle J, Raes M, et al. HIF1A gene transcription is dependent on a core promoter sequence encompassing activating and inhibiting sequences located upstream from the transcription initiation site and cis-elements located within the 5’UTR. Biochem Biophys Res Commun. 1999;261:534-40. https://doi.org/10.1006/bbrc.1999.0995
https://doi.org/10.1006/bbrc.1999.0995... .This binding allows translation of the HIF factor promoter gene, giving rise to the HIF-1-α molecule. This molecule, inside the cell cytoplasm and in the presence of oxygen, is induced to degradation by prolyl hydroxylase (PHD) enzymes and, under hypoxic conditions, allows the HIF molecule to be activated and enter the cell nucleus¹⁶16. van Uden P, Kenneth NS, Rocha S. Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochem J. 2008;412:477-84. https://doi.org/10.1042/BJ20080476
https://doi.org/10.1042/BJ20080476... . (3) Role of cell growth pathways in HIF activation and production of neural protective factors in MG such as VEGFR-1: Once the HIF molecule is produced, it needs to be stabilized and activated in order to be able to promote the production of neural protective factors such as VEGFR-1. The HIF1-α molecule in the cell cytoplasm is inactive and stabilized by normoxic conditions. For its activation to occur, several factors are related, among them, eukaryotic translation factors/growth factors, which have been shown to contribute to HIF-1-α activation by phosphorylating the molecule and allowing it to enter the nucleus²⁴24. Jiang BH, Zheng JZ, Aoki M, Vogt PK. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factors in endothelial cells. Proc Natl Acad Sci U S A. 2000;97:1749-53. https://doi.org/10.1073/pnas.040560897
https://doi.org/10.1073/pnas.040560897... ,²⁵25. Chen EY, Mazure NM, Cooper JA, Giaccia AJ. Hypoxia activates a platelet-derived growth factor receptor/phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactivation. Cancer Res. 2001;61:2429-33. PMID: 11289110. In this process, neurotrophins are released and bind to the Tyrosine Kinase C (TRK-C) receptor, which is responsible for activating the AKT-3/mTORC-1 pathway and producing eukaryotic translation factors. Inside the nucleus, HIF can produce molecules associated with cellular vascularization — VEGFR-1 — and, therefore, revitalize the tissue by guaranteeing the supply of O2.

Antibody	Type	Clone/Code	Dilution	Source	Species reactivity
Anti-AKT-3	Monoclonal/Mouse	66C1247.1	1:400	Thermo Fisher	Human, Rat, Mouse
Anti-NF-kB p100/52	Polyclonal/Rabbit	ab7972	1:400	Abcam	Human, Rat, Mouse
Anti-PARK 2	Monoclonal/Mouse	PRK8	1:100	Abcam	Human, Rat, Mouse
Anti-TRK-C	Monoclonal/Mouse	75219	1:200	Leinco	Human
Anti-VEGFR-1	Polyclonal/Rabbit	PA1-21731	1:50	Thermo Fisher	Human, Rat, Mouse

Data	Category	Number of patients (%)	Mean±standard deviation
Gender	Male	66 (55.9)	--
Gender	Female	52 (44.1)	--
Birth weight (g)	≤1000	34 (28.8)	171±975
	1001-1499	29 (24.6)
	≥1500	55 (46.6)
Gestational age (weeks)	≤30	56 (47.5)	--
	31 a 36	36 (30.5)
	≥37	26 (22.0)
Small for gestational age (SGA)	No	101 (85.6)	--
Small for gestational age (SGA)	Yes	17 (14.4)	--
Acidemia	No	39 (33.1)	--
Acidemia	Yes	79 (66.9)	--
Asphyxia (apgar score in the fifth minute ≤3)	No	84 (71.2)	5.6±2.8
Asphyxia (apgar score in the fifth minute ≤3)	Yes	34 (28.8)	5.6±2.8
Survival time (hours)	Until 24h	38 (32.2)	4.4±8.0
	25–72h	48 (40.7)
	More than 72	32 (27.1)

	Gender	p-value	Weight (g)	p-value	GA (weeks)	p-value	SGA	p-value	CNS (IG ≤30)	p-value	Acidemia (pH≤7,2)	p-value	Asphyxia (Apgar ≤3)	p-value	Survival (hours)	p-value
AKT-3	Male 8727±5578	0.094*	<1000 7483±3067	0.089*	≤30 8713±5119	0.089*	No 7956±4978	0.451*	No 7001±4550	0.032*	No 8612±6162	0.662*	No 7538±4864	0.300*	Until 24h 7202±4515	0.666*
	Male 8727±5578		1000 a <1500 9778±6714		31 a 36 7302±4811		No 7956±4978		No 7001±4550		No 8612±6162		No 7538±4864		25–72h 8116±5228
	Female 6641±3503		≥1500 7033±4504		≥37 6609±4245		Yes 6967±4280		Yes 8713±5119		Yes 7446±4122		Yes 8526±4948		Over 72h 8157±4889
NF-Kb	Male 7401±3974	0.342*	<1000 8536±435	0.054*	≤30 8521±4112	0.092*	No 8000±4154	0.194*	No 7023±3787	0.045*	No 7707±4588	0.735*	No 7970±4126	0.402*	Until 24h 6374±3200	0.037**
	Male 7401±3974		1000 a <1500 8329±3313		31 a 36 7381±3858		No 8000±4154		No 7023±3787		No 7707±4588		No 7970±4126		25–72h 8064±3958
	Female 8195±4034		≥1500 6920±4025		≥37 6572±3720		Yes 6103±2243		Yes 8521±4112		Yes 7775±3725		Yes 7775±3725		Over 72h 8899 ± 4529
Parkin	Male 570±440	0.726*	<1000 750±420	<0.001*	≤ 30 690±460	<0.001*	No 510±400	0.014*	No 420±300	<0.001*	No 630±430	0.355*	No 520±380	0.179*	Until 24h 550±420	0.988*
	Male 570±440		1000 a <1500 600±450		31 a 36 490±350		No 510±400		No 420±300		No 630±430		No 520±380		25–72h 510±340
	Female 510±360		≥1500 390±310		≥ 37 310±190		Yes 730±410		Yes 690±460		Yes 510±390		Yes 610±460		Over 72h 490±490
TRK-C	Male 870±560	0.179	<1000 1090±68	0.073*	≤30 1040±680	0.137*	No 910±600	0.266*	No 850±510	0.098*	No 1050±720	0.197*	No 960±600	0.481*	Until 24h 870±570	0.545*
	Male 870±560		1000 a <1500 1000±660		31 a 36 920±520		No 910±600		No 850±510		No 1050±720		No 960±600		25–72h 1010±63
	Female 1020±650		≥1500 800±490		≥37 740±470		Yes 1090±570		Yes 1040±680		Yes 880±530		Yes 870±610		Over 72h 900±590
VEGFR1	Male 629±1021	0.221*	<1000 881±1427	0.668*	≤ 30 908±1397	0.528*	No 648±1014	0.637*	No 551±1010	0.286*	No 899±1625	0.928*	No 778±1247	0.025*	Until 24h 697±1343	0.180*
	Male 629±1021		1000 a <1500 894±1312		31 a 36 574±1252		No 648±1014		No 551±1010		No 899±1625		No 778±1247		25–72h 941±1418
	Female 823±1412		≥1500 526±996		≥37 521±576		Yes 1125±2026		Yes 908±1397		Yes 632±963		Yes 572±1137		Over 72h 387±293

[1] * Corresponding author: Email: carolbvaz@gmail.com (C. B. V. Paula)

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O papel da via NF-kB/parkin/vegfr-1 associada à agressão hipóxico-isquêmica em amostras de matriz germinativa de pacientes recém-nascidos

RESUMO

Objetivo:

Métodos:

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