Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC SrKmwP9jb6xRndK4KJFH34N S0066-782X2022005005206 S0066-782X2022000900372 abc.20210799 10.36660/abc.20210799 Artigo Original Significado Clínico do Domínio da Peptidase M20 Contendo 1 em Pacientes com Aterosclerose Carotídea Huang Xincheng 1 He Peiyuan 2 0000-0001-9849-6507 Wu Linling 3 Department of the Cardiovascular Medicine Fourth People’s Hospital of Chengdu Chengdu Sichuan China Department of the Cardiovascular Medicine, the Fourth People’s Hospital of Chengdu, Chengdu, Sichuan – China Health Management Center Sichuan Provincial People’s Hospital Chengdu Sichuan China Health Management Center, Sichuan Provincial People’s Hospital, Chengdu, Sichuan – China Department of the Second Ward of Acute Psychosis the Fourth People’s Hospital of Chengdu Chengdu Sichuan China Department of the Second Ward of Acute Psychosis, the Fourth People’s Hospital of Chengdu, Chengdu, Sichuan – China Correspondência: Linling Wu • Department of the Second Ward of Acute Psychosis, the Fourth People’s Hospital of Chengdu, n. 8, Huli West First Lane, Chengdu, Sichuan 610000, China. E-mail: wulinling_12@126.com

Contribuição dos autores

Concepção e desenho da pesquisa e Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Huang X, Wu L; Obtenção de dados e Análise e interpretação dos dados: Huang X, He P; Análise estatística: He P, Wu L; Redação do manuscrito: Huang X.

Potencial conflito de interesse

Não há conflito com o presente artigo.

 09 05 2022 09 2022 119 3 372 379 16 07 2021 15 11 2021 08 12 2021 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Resumo Fundamento

A aterosclerose é a principal causa da maioria das doenças cardiovasculares, e novos biomarcadores para essa condição são sempre necessários. O domínio da peptidase M20 contendo 1 (PM20D1) está associado ao metabolismo lipídico e à obesidade. No entanto, nenhum estudo se concentra no papel do PM20D1 na aterosclerose carotídea.

 Objetivo

O objetivo deste estudo foi investigar o papel do PM20D1 em pacientes com aterosclerose carotídea.

 Métodos

Estudo observacional prospectivo conduzido com um total de 231 pacientes com aterosclerose carotídea que estiveram em nosso departamento entre julho de 2018 e dezembro de 2019. Amostras de sangue e dados médicas foram obtidos de outros 231 indivíduos saudáveis com o mesmo índice de massa corporal (IMC) dos pacientes com aterosclerose carotídea. O PM20D1 sérico foi determinado por ensaio imunossorvente ligado a enzima (ELISA). As características clínicas e demográficas de todos os pacientes foram listadas, incluindo idade, sexo biológico, IMC e histórico médico. Os níveis de proteína C reativa (PCR), fator de necrose tumoral, homocisteína, colesterol total, triglicerídeos, leptina-colesterol de alta densidade e leptina-colesterol de baixa densidade foram registrados. Realizou-se análise estatística no software SPSS, com p<0,05 considerado estatisticamente significante.

 Resultados

Os níveis séricos de PM20D1 foram marcadamente mais baixos em pacientes com aterosclerose carotídea comparados aos controles saudáveis, sendo significativamente mais baixos em pacientes com aterosclerose carotídea grave e pacientes com aterosclerose carotídea/acidente vascular cerebral. Pacientes com placas instáveis apresentaram PM20D1 marcadamente menor quando comparados a pacientes com placas estáveis. Nenhuma diferença significativa foi encontrada entre pacientes com aterosclerose carotídea com diferentes IMC. Pacientes com níveis mais elevados de PM20D1 apresentaram expressão significativamente menor de PCR, fator de necrose tumoral, homocisteína, triglicerídeos, colesterol total e colesterol de baixa densidade. PM20D1 correlacionou-se negativamente com PCR, fator de necrose tumoral, homocisteína, colesterol total e leptina de baixa densidade em pacientes com aterosclerose carotídea, podendo ser usado como biomarcador para pacientes com aterosclerose carotídea grave ou com aterosclerose carotídea e acidente vascular cerebral. Sexo biológico, fator de necrose tumoral, homocisteína e PM20D1 foram considerados fatores de risco para aterosclerose carotídea.

 Conclusão

O PM20D1 estava diminuído em pacientes com aterosclerose carotídea e foi associado com gravidade, estabilidade da placa, níveis de PCR, fator de necrose tumoral, homocisteína, triglicerídeos, colesterol total e colesterol de baixa densidade em pacientes com aterosclerose carotídea.

 Doenças das Artérias Carótidas Lipídeos Índice de Massa Corporal Introdução

Como principal causa da maioria das doenças cardiovasculares, a aterosclerose pode ocorrer precocemente e permanecer assintomática por longos períodos antes de se manifestar.1,2 Dentre seus diversos tipos, acredita-se que a aterosclerose carotídea (AC) seja um preditor de acidente vascular cerebral (AVC) isquêmico.3 Relata-se que a placa carotídea é um fator de risco para acidente vascular cerebral isquêmico, que também está associado ao cádmio, enquanto o aumento do espessamento médio-intimal (EMI) da carótida e a presença de placa carotídea estão associados ao aumento do risco de acidente vascular cerebral isquêmico em indivíduos com fibrilação atrial.4,5 Muitos fatores de risco estão associados à AC, incluindo disfunção do metabolismo lipídico,6 hipertensão,7 diabetes,8 idade,9 tabagismo10 etc. No entanto, ainda é necessário um entendimento mais profundo sobre a manifestação da AC e seus resultados clínicos.

O domínio da peptidase M20 contendo 1 (PM20D1) é uma enzima secretada, recentemente identificada, que é rica em proteína de desacoplamento 1 (UCP1+), em relação a adipócitos UCP1.11 Um estudo recente mostrou que o PM20D1 está ligado ao metabolismo lipídico e pode estar associado à obesidade,12 ambos fatores de risco para AC. Verificou-se também que o PM20D1 poderia regular os desacopladores de aminoácidos lipidados das mitocôndrias e aumentar o gasto energético de PM20D1.13 No entanto, até o momento, nenhum estudo focou no papel do PM20D1 na AC.

Este é um estudo observacional prospectivo cujo intuito foi investigar o significado clínico do PM20D1 em pacientes com AC. Esta pesquisa pode fornecer evidências clínicas sobre o papel do PM20D1 na AC.

 Materiais e métodos Pacientes

Este estudo observacional prospectivo recrutou 231 pacientes com AC assistidos em nosso departamento entre julho de 2018 e dezembro de 2019. Os critérios de inclusão foram: 1) todos os pacientes diagnosticados com AC de acordo com achados da ultrassonografia com Doppler colorido dos vasos sanguíneos do pescoço e angiotomografia computadorizada intracraniana (ATC) do arco aórtico; 2) Pacientes com AC e AVC foram admitidos em até 72 horas após a manifestação, e o diagnóstico de AVC foi confirmado por ressonância magnética (RM) e tomografia computadorizada (TC); 3) os pacientes concordaram em participar da pesquisa observacional. Os critérios de exclusão foram: 1) pacientes com aneurisma cerebral, malformação arteriovenosa, dissecção, arterite ou doença de Moyamoya; 2) pacientes com embolia cerebral cardiogênica; 3) pacientes com outras doenças graves do sistema, incluindo câncer e disfunção cardíaca, renal ou hepática. A gravidade da AC foi definida da seguinte forma: 1) grupo AC leve/moderado, que apresentou espessamento médio-intimal (EMI)>1,0 mm, ou placa com estenose arterial <70% e sem AVC; 2) grupo AC grave, que apresentou uma ou mais placas com estenose arterial ≥70% e sem AVC; 3) grupo AC combinado com AVC, apresentando AC combinado com AVC isquêmico aterosclerótico de grandes artérias e estenose vascular cerebral >50%. As placas foram divididas em estáveis e instáveis, conforme amplamente aceito na prática clínica: 1) EMI≥1,2 mm foi considerado uma placa; 2) as placas estáveis possuem gradiente-eco forte uniforme ou gradiente-eco médio; 3) as placas instáveis são moles ou ulcerativas com gradiente-eco misto ou baixo.14 O EMI

foi medido por meio de um instrumento de diagnóstico por ultrassom LOGIQ C9 Color Doppler (General Electric, Estados Unidos da América) com frequência de sonda de 7~14 MHz. Foram medidos EMI da artéria carótida comum bilateral, proximal, distal e a 1 cm da bifurcação da artéria carótida comum. Todas as medidas foram realizadas pelo menos três vezes e o valor médio foi considerado o valor final do EMI.

Além disso, os pacientes foram divididos em diferentes grupos de índice de massa corporal (IMC): grupo normal, com IMC<24 kg/m2; grupo com sobrepeso, com 28 kg/m2>IMC≥24 kg/m2; e grupo obesidade, com IMC>28 kg/m2.15 Também foram obtidas amostras de sangue e características médicas de 231 indivíduos saudáveis que compareceram ao exame físico de rotina, tendo a mesma distribuição de IMC dos pacientes com AC. Todos os pacientes assinaram o termo de consentimento livre e informado. Este estudo foi aprovado pelo comitê de ética do Fourth People’s Hospital of Chengdu (CDH-2018-057).

 Medição do PM20D1

As amostras de sangue de todos os casos foram coletadas em até 24 horas após a admissão ou chegada ao ambulatório. Resumidamente, 5 ml de sangue foram coletados em tubos sem qualquer anticoagulante. Após uma hora, a centrifugação foi realizada a 2.000 g por 15 minutos, em temperatura ambiente, e foram obtidas as amostras de soro. Os níveis séricos de PM20D1 foram medidos por ensaio imunoenzimático (ELISA), usando um kit PM20D1 (MYBioSource, cat. nº MBS280518), em total acordo com as instruções do fabricante.

 Coleta e medição de dados

As características clínicas e demográficas de todos os pacientes foram coletadas, incluindo idade, sexo biológico, IMC e histórico médico. Foi realizado hemograma total de encaminhamento com um analisador bioquímico automático (Hitachi 7600, Hitachi Corporation, Japão), bem como dosados os níveis de proteína C reativa (PCR), fator de necrose tumoral (TNF-α), homocisteína (Hcy), colesterol total (CT), triglicerídeos (TG), colesterol de leptina de alta densidade (HDL-ch) e colesterol de leptina de baixa densidade (LDL-ch).

 Análise estatística

Os dados com distribuição normal foram expressos por média ± desvio padrão (DP), e os dados com distribuição não normal foram expressos como mediana (intervalo interquartil). As variáveis categóricas foram apresentadas como número (taxas). A distribuição dos dados foi analisada pelo método de Kolmogorov-Smirnov. Para dados com distribuição normal, foi realizada a comparação entre dois grupos pelo teste t não pareado e por análise de variância unidirecional (ANOVA), seguidos pelo teste post hoc de Tukey, usado para comparação entre três ou mais grupos. Para dados com distribuição não normal, utilizou-se o teste de Mann-Whitney para comparação entre dois grupos e o teste de Kruskal-Wallis para comparação entre três ou mais grupos, seguidos do teste post hoc de Dunn. As taxas foram analisadas pelo teste qui-quadrado. A correlação entre PM20D1, Hcy, metabolismo lipídico e fatores inflamatórios foi analisada por meio da correlação de Pearson. Uma curva receiver operating characteristic (ROC) foi usada para análise de PM20D1 em pacientes com AC. A regressão logística foi utilizada para análise de risco de AC. O valor de p<0,05 foi considerado estatisticamente significativo. Todo o cálculo foi realizado no programa SPSS 25.0 (SPSS Inc., Chicago, Estados Unidos da América) ou no GraphPad 6.0 (GraphPad Software, San Diego, Califórnia, Estados Unidos da América).

 Resultados Características clínicas basais de todos os pacientes

Esta pesquisa incluiu 231 pacientes com AC no total, sendo 152 casos de AC leve/moderada, 50 casos de AC grave e 29 casos de AC com AVC. A razão da soma dos pacientes com sobrepeso e obesidade foi significativamente maior nos pacientes com AC grave quando comparada à dos pacientes leves/moderados (Tabela 1). A proporção de placa instável foi significativamente maior em pacientes com AC grave e AC/AVC. Os níveis de PCR, TNF-α e Hcy foram notadamente maiores em pacientes com AC grave e pacientes com AC/AVC em comparação aos pacientes leves/moderados. Apenas TG, TC e LDL-ch estavam mais altos em pacientes com AC grave e pacientes com AC/AVC em relação aos casos leves/moderados. Além disso, os controles saudáveis apresentaram níveis significativamente mais baixos de PCR, TNF-α e Hcy, bem como TG, CT e LDL-ch em relação a todos os pacientes com AC. Nenhuma outra diferença significativa foi encontrada.

– Características clínicas basais de todos os pacientes
Variáveis Todas as ACs, n=231 AC leve/moderada, n=152 AC grave, n=50 AC/AVC, n=29 Saudáveis, n=231 P1* P2#
Idade, anos 57,85±9,04 58,17±9,35 58,32±8,90 55,37±7,37 58,36±8,97 0,543 0,289
Sexo, feminino (%) 105 (45,45) 64 (42,11) 25 (50,00) 16 (55,17) 108 (46,75) 0,854 0,177
IMC, n (%) 0,968 0,176
<24 kg/m2 98 (42,42) 65 (42,76) 15 (30,00) 8 (27,59) 102 (44,16)
28 kg/m2>IMC≥24 kg/m2 76 (32,90) 48 (31,58) 20 (40,00) 11 (37,93) 73 (31,60)
>28 kg/m2 57 (24,68) 39 (25,66) 15 (30,00) 10 (34,48) 56 (24,24)
Complicações, n (%) - 0,993
Diabetes 62 (26,84) 39 (25,66) 15 (30,00) 8 (27,59) - 0,790
Hipertensão 114 (49,35) 75 (49,34) 24 (48,00) 15 (51,72) - 0,868
Histórico de doença cardíaca coronária 41 (17,75) 27 (17,76) 9 (18,00) 5 (17,24) - 0,990
Histórico de AVC 12 (5,19) 7 (4,61) 3 (6,00) 2 (6,90) - 0,804
Tabagismo 129 (55,84) 87 (57,24) 24 (48,00) 18 (62,07) 97 (41,99) 0,090 0,126
Placa, n (%) - - <0,001
Estável 103 (44,59) 81 (53,29) 17 (34,00) 5 (17,24) -
Instável 128 (55,41) 71 (46,71) 33 (66,00) 24 (82,76) -
PCR, mg/L 6,91 (0,52~34,87) 5,13 (0,52~9,99) 17,01 (2,92~34,87) 13,40 (3,01~33,64) 4,23 (0,57~10,01) <0,001 <0,001
TNF-α, pg/ml 24,18±12,99 17,45±4,40 37,01±14,70 37,34±13,59 14,81±2,81 <0,001 <0,001
Hcy, μmol/L 12,28±3,22 10,78±2,34 15,08±2,72 15,33±2,61 7,46±1,45 <0,001 <0,001
TC, mmol/L 4,26±0,78 4,00±0,66 4,80±0,72 4,66±0,84 3,96±0,68 <0,001 <0,001
TG, mmol/L 1,48±0,51 1,37±0,54 1,65±0,40 1,77±0,36 1,28±0,45 <0,001 <0,001
LDL-ch, mmol/L 3,01±0,55 2,87±0,44 3,25±0,61 3,33±0,66 2,74±0,41 <0,001 <0,001
HDL-ch, mmol/L 1,15±0,08 1,15±0,07 1,16±0,08 1,15±0,09 1,16±0,08 0,473 0,735

*Comparação P1 entre todas as ateroscleroses carotídeas (ACs) e o controle saudável. #P2 comparação entre pacientes leves/moderados, graves e AC/AVC. A análise de variância unidirecional seguida pelo teste post hoc de Tukey foi usada para dados com distribuição normal. Para dados com distribuição não normal, o teste de Kruskal-Wallis foi usado para comparação entre pacientes leves/moderados, graves e AC/AVC, seguido do teste post hoc de Dunn. As taxas foram analisadas por meio do teste qui-quadrado. AVC: acidente vascular cerebral; IMC: índice de massa corporal; PCR: proteína C reativa; TNF-α: fator de necrose tumoral; Hcy: homocisteína; CT: colesterol total; TG: triglicérides; LDL-ch: colesterol de leptina de baixa densidade; HDL-ch: colesterol de leptina de alta densidade.

PM20D1 associado à gravidade e estabilidade da placa em pacientes com AC

Em seguida, foi determinada a expressão sérica de PM20D1 em pacientes com AC. Verificaram-se níveis séricos de PM20D1 marcadamente mais baixos em pacientes com AC quando comparados ao controle saudável (Figura 1). Pacientes com AC grave e com AC/AVC apresentaram níveis significativamente mais baixos de PM20D1 em comparação aos pacientes leves/moderados. No entanto, nenhuma diferença significativa foi encontrada entre pacientes graves e AC/AVC. Pacientes com placas instáveis apresentaram expressão marcadamente mais baixa de PM20D1 do que pacientes com placas estáveis.

– Níveis séricos de PM20D1 em pacientes com aterosclerose carotídea (AC) com gravidades e estabilidade da placa diferentes. ***p<0,001, **p<0,01. AVC: acidente vascular cerebral. AVC: acidente vascular cerebral.

PM20D1 não associado a pacientes com aterosclerose carotídea e diferentes índices de massa corporal

Para investigar mais detalhadamente o papel do PM20D1 em pacientes com AC, também avaliamos os níveis de PM20D1 em pacientes com AC e diferentes distribuições de IMC. Não foi observada diferença significativa (Figura 2).

– Níveis séricos de PM20D1 em pacientes com aterosclerose carotídea e diferentes índices de massa corporal (IMC).

Correlação entre PM20D1, metabolismo lipídico e fatores inflamatórios

Em seguida, os pacientes foram divididos em grupo de alta expressão de PM20D1 e grupo de baixa expressão, de acordo com o nível médio de PM20D1 (4,53 ng/ml). Pacientes com níveis mais elevados de PM20D1 apresentaram expressão significativamente menor de PCR, TNF-α, Hcy, TG, CT e LDL-ch (Tabela 2). Foi feita a correlação de Pearson e mostrou que o PM20D1 se correlacionou negativamente com PCR, TNF-α, Hcy, TC e LDL-ch em pacientes com AC (Tabela 3).

– Expressão do metabolismo lipídico e fatores inflamatórios em pacientes com diferentes níveis de PM20D1
Variáveis PM20D1 alto, n=117 PM20D1 baixo, n=114 p*
PCR, mg/L 5,27 (0,52~33,64) 15,16 (0,68~34,87) <0,001
TNF-α, pg/ml 18,78±7,32 29,72±15,08 <0,001
Hcy, μmol/L 11,26±2,66 13,34±3,41 <0,001
CT, mmol/L 4,03±0,70 4,49±0,80 <0,001
TG, mmol/L 1,41±0,54 1,56±0,47 0,031
LDL-ch, mmol/L 2,90±0,48 3,12±0,59 0,003
HDL-ch, mmol/L 1,14±0,08 1,16±0,08 0,269

*O teste t de Student foi usado para comparar pacientes leves/moderados, graves e AC/AVC para dados normalmente distribuídos. Para dados com distribuição não normal, utilizou-se o teste de Mann-Whitney. As taxas foram analisadas pelo teste qui-quadrado. PCR: proteína C reativa; TNF-α: fator de necrose tumoral; Hcy: homocisteína; CT: colesterol total; TG: triglicérides; LDL-ch: colesterol de leptina de baixa densidade; HDL-ch: colesterol de leptina de alta densidade.

– Correlação entre PM20D1, homocisteína (Hcy), metabolismo lipídico e fatores inflamatórios
Variáveis Correlação de Pearson p
PCR, mg/L -0,514 <0,001
TNF-α, pg/ml -0,585 <0,001
Hcy, μmol/L -0,598 <0,001
TC, mmol/L -0,254 <0,001
TG, mmol/L -0,059 0,198
LDL-ch, mmol/L -0,071 0,126
HDL-ch, mmol/L 0,022 0,622

PCR: proteína C reativa; TNF-α: fator de necrose tumoral; Hcy: homocisteína; CT: colesterol total; TG: triglicérides; LDL-ch: colesterol de leptina de baixa densidade; HDL-ch: colesterol de leptina de alta densidade.

Valor diagnóstico de PM20D1 na aterosclerose carotídea

Por fim, uma curva ROC foi desenhada para observar o valor diagnóstico de PM20D1 para AC, bem como AC grave ou AC/AVC. Verificou-se que o PM20D1 poderia ser um potencial biomarcador diagnóstico de AC com valor de corte de 5,94 ng/ml, área sob a curva ROC (AUC) 0,876, IC95% (0,845~0,906), sensibilidade de 80,1% e especificidade de 73,6% (Figura 3). O PM20D1 também pode ser usado como biomarcador para pacientes com AC grave ou AC/AVC, com valor de corte de 3,99 ng/ml, AUC 0,917, IC95% (0,883~0,951), sensibilidade de 81,6%, especificidade de 77,2%. A regressão logística binária adicional, que incluiu todos os fatores de idade, IMC, PCR, TNF-α, Hcy, TC, TG, LDL-ch, HDL-ch, PM20D1, bem como sexo biológico, diabetes, hipertensão, histórico de doença cardíaca coronária, histórico de AVC e tabagismo, estabeleceu sexo, TNF-α, Hcy e PM20D1 como fatores de risco para AC (Tabela 4).

– Curva receiver operating characteristic (ROC) para valor diagnóstico de PM20D1 de aterosclerose carotídea (AC), AC grave ou AC/AVC. AVc: acidente vascular cerebral.

– Regressão logística binária para fatores de risco de aterosclerose carotídea
Variáveis Wald Odds ratio IC95% p
Idade 0,011 1,003 0,935~1,078 0,914
Sexo 4,210 0,208 1,073~21,401 0,040
IMC 0,524 1,057 0,909~1,231 0,469
Diabetes <0,001 <0,001 0,000 0,995
Hipertensão <0,001 <0,001 0,000 0,993
Histórico de doença cardíaca coronária <0,001 <0,001 0,000 0,995
Histórico de AVC <0,001 0,001 0,000 0,999
Tabagismo <0,001 0,844 0,232~3,075 0,798
PCR 0,004 1,008 0,777~1,310 0,947
TNF-α 11,820 0,664 0,526~0,839 0,001
Hcy 22,852 0,155 0,073~0,334 <0,001
CT 0,462 0,697 0,248~1,967 0,496
TG 1,403 0,411 0,095~1,787 0,236
LDL-ch 1,559 0,340 0,063~1,847 0,212
HDL-ch 1,216 0,008 1,797E-6~40,532 0,270
PM20D1 18,152 8,485 3,173~22,693 <0,001

IMC: índice de massa corporal; AVC: acidente vascular cerebral; PCR: proteína C reativa; TNF-α: fator de necrose tumoral; Hcy: homocisteína; CT: colesterol total; TG: triglicérides; LDL-ch: colesterol de leptina de baixa densidade; HDL-ch: colesterol de leptina de alta densidade.

Discussão

O diagnóstico precoce e predição da AC podem melhorar muito a eficácia e o prognóstico do tratamento. Assim, biomarcadores para diagnóstico e prognóstico da AC são de grande importância. Neste estudo, demonstramos que PM20D1 estava mais baixo em pacientes com AC e se associou com a gravidade e condição de placa dos pacientes com AC. Além disso, o PM20D1 correlacionou-se negativamente com PCR, TNF-α, Hcy, TC e LDL-ch nesses pacientes.

O metabolismo lipídico e a obesidade estão associados ao desenvolvimento da AC. Verificou-se que os níveis de TG, TC e LDL-ch estavam significativamente maiores em pacientes com placa na artéria carótida.16 Outra pesquisa constatou que o tratamento com atorvastatina ou ezetimiba diminuiu os níveis de TC, TG e LDL-ch em pacientes com AC.17 Em estudo recente, Pan et al. mostraram que, em residentes rurais de baixa renda na China, LDL-ch e TC foram fatores de risco para aterosclerose em estágio inicial e foram ligados ao risco aumentado de placa carotídea em 24% e 62% para cada aumento de 1 mmol/L em TC e LDL-ch.18 Em nossa pesquisa, também encontramos que TG, TC e LDL-ch foram expressos em níveis altos em pacientes com AC, principalmente em casos graves, corroborando as pesquisas supracitadas. Também observamos que fatores inflamatórios como PCR, TNF-α e expressão de Hcy estavam elevados em pacientes com AC, o que também foi demonstrado em diversas pesquisas.19-22

O PM20D1 é um fator associado tanto ao metabolismo lipídico quanto à obesidade. Verificou-se que camundongos com aumento de PM20D1 circulante apresentaram aumento da frequência respiratória e aumento sérico dos aminoácidos N-acil, o que melhorou a homeostase da glicose e aumentou o consumo de energia e, portanto, pode regular a obesidade.13 Benson et al. demonstraram que a diminuição do PM20D1 estava associada à relação genética entre obesidade e doenças neurodegenerativas em humanos.23 Li et al. descobriram que a inibição do microRNA miR-324-5p aumentou o consumo de oxigênio das células primárias dos tecidos adiposos branco e marrom, aumentou o consumo de gordura e, assim, reduziu o peso dos camundongos, aumentando os níveis de PM20D1.24 Long et al. observaram que, em camundongos com deficiência de PM20D1, a atividade da hidrolase/sintetase do N-acetil aspartato estava diminuída no soro e nos tecidos, bem como uma variedade de fenótipos metabólicos e de dor, incluindo resistência à insulina, mudanças de temperatura fria e comportamento antinociceptivo, que estão associados à obesidade, diabetes e outras doenças.12 Assim, podemos especular que níveis mais altos de PM20D1 podem ser benéficos para a obesidade e redução de lipídios. Embora não haja nenhum estudo que demonstre o papel de PM20D1 na AC, a regulação diminuída de PM20D1 em pacientes com AC em nosso estudo pode se dever, em parte, à disfunção do metabolismo lipídico e obesidade de pacientes com AC. Como os pacientes apresentaram níveis mais baixos de PM20D1 do que os controles saudáveis com mesma distribuição de IMC, e os pacientes com AC e IMC diferente não apresentaram diferença significativa de PM20D1; ou seja, o IMC pode não estar associado ao nível de PM20D1 em pacientes com AC. A correlação negativa entre PM20D1 e Hcy, TC e LDL-ch pode ser uma das razões para a expressão anormal de PM20D1 em pacientes com AC. No entanto, para fundamentar todas essas especulações, são necessários mais estudos para obter mais evidências.

O presente estudo teve, no entanto, algumas limitações. Primeiro, trata-se de um estudo observacional de um único centro, com apenas 231 casos. Assim como o mecanismo molecular do PM20D1 exercendo influência no desenvolvimento da AC ainda não está claro.

 Conclusão

Em conclusão, este estudo observacional demonstrou que a diminuição de PM20D1 estava associada à gravidade, condição da placa e expressão de PCR, TNF-α, Hcy, TG, TC e LDL-ch em pacientes com AC. Este estudo pode fornecer um novo alvo de pesquisa para PM20D1 em relação à AC.

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Long JZ Roche AM Berdan CA Louie SM Roberts AJ Svensson KJ et al Ablation of PM20D1 Reveals N-acyl Amino Acid Control of Metabolism and Nociception Proc Natl Acad Sci U S A 2018 115 29 E6937 E6945 10.1073/pnas.1803389115. 13.Long JZ, Svensson KJ, Bateman LA, Lin H, Kamenecka T, Lokurkar IA, et al. The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria. Cell. 2016;166(2):424-35. doi: 10.1016/j.cell.2016.05.071. Long JZ Svensson KJ Bateman LA Lin H Kamenecka T Lokurkar IA et al The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria Cell 2016 166 2 424 435 10.1016/j.cell.2016.05.071 14.Funakoshi Y, Imamura H, Tani S, Adachi H, Fukumitsu R, Sunohara T, et al. Safety and Efficacy of an Open-cell Stent and Double-balloon Protection for Unstable Plaques: Analysis of 184 Consecutive Carotid Artery Stentings. J Neurointerv Surg. 2020;12(8):758-62. doi: 10.1136/neurintsurg-2019-015393. Funakoshi Y Imamura H Tani S Adachi H Fukumitsu R Sunohara T et al Safety and Efficacy of an Open-cell Stent and Double-balloon Protection for Unstable Plaques: Analysis of 184 Consecutive Carotid Artery Stentings J Neurointerv Surg 2020 12 8 758 762 10.1136/neurintsurg-2019-015393 15.Wang Y, Wang K, Han T, Zhang P, Chen X, Wu W, et al. Exposure to Multiple Metals and Prevalence for Preeclampsia in Taiyuan, China. Environ Int. 2020;145:106098. doi: 10.1016/j.envint.2020.106098. Wang Y Wang K Han T Zhang P Chen X Wu W et al Exposure to Multiple Metals and Prevalence for Preeclampsia in Taiyuan, China Environ Int 2020 145 106098 10.1016/j.envint.2020.106098. 16.Feng S, Zhu Y, Yan C, Wang Y, Zhang Z. Retinol Binding Protein 4 Correlates with and is an Early Predictor of Carotid Atherosclerosis in Type 2 Diabetes Mellitus Patients. J Biomed Res. 2015;29(6):451–5. doi: 10.7555/JBR.29.20140087. Feng S Zhu Y Yan C Wang Y Zhang Z Retinol Binding Protein 4 Correlates with and is an Early Predictor of Carotid Atherosclerosis in Type 2 Diabetes Mellitus Patients J Biomed Res 2015 29 6 451 455 10.7555/JBR.29.20140087. 17.Wang J, Ai XB, Wang F, Zou YW, Li L, Yi XL. Efficacy of Ezetimibe Combined with Atorvastatin in the Treatment of Carotid Artery Plaque in Patients with Type 2 Diabetes Mellitus Complicated with Coronary Heart Disease. Int Angiol. 2017;36(5):467-73. doi: 10.23736/S0392-9590.17.03818-4. Wang J Ai XB Wang F Zou YW Li L Yi XL Efficacy of Ezetimibe Combined with Atorvastatin in the Treatment of Carotid Artery Plaque in Patients with Type 2 Diabetes Mellitus Complicated with Coronary Heart Disease Int Angiol 2017 36 5 467 473 10.23736/S0392-9590.17.03818-4. 18.Pan J, Liu J, Wang H, Li W, Du X, Lin Q, et al. Association of Carotid Atherosclerosis With Lipid Components in Asymptomatic Low-Income Chinese: A Population-Based Cross-Sectional Study. Front Neurol. 2020;11:276. doi: 10.3389/fneur.2020.00276. Pan J Liu J Wang H Li W Du X Lin Q et al Association of Carotid Atherosclerosis With Lipid Components in Asymptomatic Low-Income Chinese: A Population-Based Cross-Sectional Study Front Neurol 2020 11 276 10.3389/fneur.2020.00276 19.Wu MM, Chiou HY, Hsueh YM, Hong CT, Su CL, Chang SF, et al. Effect of Plasma Homocysteine Level and Urinary Monomethylarsonic Acid on the Risk of Arsenic-associated Carotid Atherosclerosis. Toxicol Appl Pharmacol. 2006;216(1):168-75. doi: 10.1016/j.taap.2006.05.005. Wu MM Chiou HY Hsueh YM Hong CT Su CL Chang SF et al Effect of Plasma Homocysteine Level and Urinary Monomethylarsonic Acid on the Risk of Arsenic-associated Carotid Atherosclerosis Toxicol Appl Pharmacol 2006 216 1 168 175 10.1016/j.taap.2006.05.005. 20.Lorenz MW, Karbstein P, Markus HS, Sitzer M. High-sensitivity C-reactive Protein is not Associated with Carotid Intima-media Progression: The Carotid Atherosclerosis Progression Study. Stroke. 2007;38(6):1774-9. doi: 10.1161/STROKEAHA.106.476135. Lorenz MW Karbstein P Markus HS Sitzer M High-sensitivity C-reactive Protein is not Associated with Carotid Intima-media Progression: The Carotid Atherosclerosis Progression Study Stroke 2007 38 6 1774 1779 10.1161/STROKEAHA.106.476135 21.Fawzy RM, Hammad GA, Egila SE, Elkasas AN, Fouad NA. Association of Tumor Necrosis factor-α (TNF-α)− 308A/G (rs1800629) Gene Polymorphism with Carotid Artery Atherosclerosis in Rheumatoid Arthritis Patients. Egypt Rheumatol. 2020;42(3):177-81. doi: 10.1016/j.ejr.2020.05.004 Fawzy RM Hammad GA Egila SE Elkasas AN Fouad NA Association of Tumor Necrosis factor-α (TNF-α)− 308A/G (rs1800629) Gene Polymorphism with Carotid Artery Atherosclerosis in Rheumatoid Arthritis Patients Egypt Rheumatol 2020 42 3 177 181 10.1016/j.ejr.2020.05.004 22.Zardi EM, Pipita ME, Giorgi C, Lichinchi D, Zardi DM, Afeltra A. Differences in Carotid Atherosclerosis Between Patients with Ankylosing Spondylitis Treated with Tumor Necrosis Factor-α Antagonists and Healthy Matched Controls. Medicine (Baltimore). 2018;97(27):e11250. doi: 10.1097/MD.0000000000011250. Zardi EM Pipita ME Giorgi C Lichinchi D Zardi DM Afeltra A Differences in Carotid Atherosclerosis Between Patients with Ankylosing Spondylitis Treated with Tumor Necrosis Factor-α Antagonists and Healthy Matched Controls Medicine Baltimore 2018 97 27 e11250 10.1097/MD.0000000000011250 23.Benson KK, Hu W, Weller AH, Bennett AH, Chen ER, Khetarpal SA, et al. Natural Human Genetic Variation Determines Basal and Inducible Expression of PM20D1, an Obesity-associated Gene. Proc Natl Acad Sci U S A. 2019;116(46):23232-42. doi: 10.1073/pnas.1913199116. Benson KK Hu W Weller AH Bennett AH Chen ER Khetarpal SA et al Natural Human Genetic Variation Determines Basal and Inducible Expression of PM20D1, an Obesity-associated Gene Proc Natl Acad Sci U S A 2019 116 46 23232 23242 10.1073/pnas.1913199116. 24.Li D, Liu Y, Gao W, Han J, Yuan R, Zhang M, et al. Inhibition of miR-324-5p Increases PM20D1-mediated White and Brown Adipose Loss and Reduces Body Weight in Juvenile Mice. Eur J Pharmacol. 2019;863:172708. doi: 10.1016/j.ejphar.2019.172708. Li D Liu Y Gao W Han J Yuan R Zhang M et al Inhibition of miR-324-5p Increases PM20D1-mediated White and Brown Adipose Loss and Reduces Body Weight in Juvenile Mice Eur J Pharmacol 2019 863 172708 10.1016/j.ejphar.2019.172708

Vinculação acadêmica

Não há vinculação deste estudo a programas de pós-graduação.

Fontes de financiamento: O presente estudo não teve fontes de financiamento externas.

 Original Article Clinical Significance of Peptidase M20 Domain Containing 1 Ii Patients with Carotid Atherosclerosis Huang Xincheng 1 He Peiyuan 2 0000-0001-9849-6507 Wu Linling 3 China Department of the Cardiovascular Medicine, the Fourth People’s Hospital of Chengdu, Chengdu, Sichuan – China China Health Management Center, Sichuan Provincial People’s Hospital, Chengdu, Sichuan – China China Department of the Second Ward of Acute Psychosis, the Fourth People’s Hospital of Chengdu, Chengdu, Sichuan – China Mailing Address: Linling Wu • Department of the Second Ward of Acute Psychosis, the Fourth People’s Hospital of Chengdu, n. 8, Huli West First Lane, Chengdu, Sichuan 610000, China. E-mail: wulinling_12@126.com

Author Contributions

Conception and design of the research and Critical revision of the manuscript for intellectual contente: Huang X, Wu L; Acquisition of data and Analysis and interpretation of the data: Huang X, He P; Statistical analysis: He P, Wu L; Writing of the manuscript: Huang X.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 Abstract Background

Atherosclerosis is the main cause for most cardiovascular diseases, and new biomarkers for this condition are always needed. Peptidase M20 domain containing 1 (PM20D1) is associated with both lipid metabolism and obesity. However, no study focuses on the role of PM20D1 in carotid atherosclerosis.

 Objective

The present study aimed to investigate the role of PM20D1 in carotid atherosclerosis patients.

 Methods

The present prospective observational study contained a total of 231 carotid atherosclerosis patients, who went to our department between July 2018 and December 2019. Blood samples and medical characteristics were also obtained from 231 healthy individuals with the same body mass index distribution of carotid atherosclerosis patients. Serum PM20D1 was determined using enzyme-linked immunosorbent assay. Clinical and demographic characteristics of all patients were collected, including age, sex, body mass index and medical history. Levels of C-reactive protein, tumor necrosis factor, homocysteine, as well as total cholesterol, triglyceride, high-density leptin cholesterol and low-density leptin cholesterol were recorded. Statistical analysis was conducted using the SPSS software, with p<0.05 as statistically different.

 Results

Serum PM20D1 levels were markedly lower in carotid atherosclerosis patients when compared to the healthy control, which were significantly lower in severe carotid atherosclerosis patients and carotid atherosclerosis/stroke patients. Patients with unstable plaques showed markedly lower PM20D1 when compared to patients with stable plaques. No significant difference was found among carotid atherosclerosis patients with different body mass index. Patients with higher PM20D1 levels showed significantly lower expression of C-reactive protein, tumor necrosis factor, homocysteine, triglyceride, total cholesterol and low-density leptin cholesterol. PM20D1 was negatively correlated with C-reactive protein, tumor necrosis factor, homocysteine, total cholesterol and low-density leptin cholesterol in carotid atherosclerosis patients, and could be used as a biomarker for severe carotid atherosclerosis patients or carotid atherosclerosis patients with stroke. Sex, tumor necrosis factor, homocysteine and PM20D1 were risk factors for carotid atherosclerosis.

 Conclusion

PM20D1 was decreased in carotid atherosclerosis patients and was associated with severity, plaque stability, and levels of C-reactive protein, tumor necrosis factor, homocysteine, triglyceride, total cholesterol and low-density leptin cholesterol in carotid atherosclerosis patients.

 Carotid Artery Diseases Lipids Body Mass Index Introduction

As the main cause of most cardiovascular diseases, atherosclerosis (AS) can occur early in lifetime and remain asymptomatic for long periods before onset.1,2 Among kinds of atherosclerosis, carotid atherosclerosis (CAS) is thought to be a predictor for ischemic stroke.3 It is reported that carotid plaque is a risk factor for ischemic stroke, which is also associated with the cadmium, and increased carotid intima-media thickness and presence of carotid plaque are associated with increased risk of ischemic stroke in individuals with atrial fibrillation.4,5 Many risk factors are reported to be associated with CAS, including dysfunction of lipid metabolism,6 hypertension,7 diabetes,8 age,9 smoking10 etc. However, deeper insights are still needed for CAS onset and clinical outcomes.

Peptidase M20 domain containing 1 (PM20D1) is a newly identified secreted enzyme enriched in uncoupling protein 1 (UCP1+), versus UCP1- adipocytes.11 A recent study found PM20D1 is associated with lipid metabolism and might be associated with obesity,12 which are all risk factors for CAS. It was also found that PM20D1 could regulate lipidated amino acid uncouplers of mitochondria and increased PM20D1 augmented energy expenditure.13 However, up to now, no study focuses on the role of PM20D1 in CAS.

The present research performed a prospective observational study in order to investigate the clinical significance of PM20D1 in CAS patients. This research might provide clinical evidence for the role of PM20D1 in CAS.

 Methods and materials Patients

The present prospective observational study enrolled a total of 231 CAS patients, who went to our department between July 2018 and December 2019. The inclusion criteria were: 1) all patients were diagnosed as CAS according to color Doppler ultrasound of neck blood vessels and aortic arch intracranial computed tomography angiography (CTA); 2) CAS patients with stroke were admitted within 72 hours after onset, and the diagnosis of stroke was confirmed by magnetic resonance imaging (MRI) and computed tomography (CT) scan; 3) patients agreed to participate in the observational research. The following patients were excluded: 1) patients with cerebral aneurysm, arteriovenous malformation, dissection, arteritis or moyamoya disease; 2) patients with cardiogenic cerebral embolism; 3) patients with other severe system diseases, including cancer and heart, renal or liver dysfunction. The severity of CAS was defined as: 1) mild/moderate CAS group, who showed thickening of the intima-media, with intima-media thickness (IMT) >1.0 mm, or plaque information with arterial stenosis <70% and without stroke; 2) severe CAS group, who showed one or more plaques with arterial stenosis ≥70% and without stroke; 3) CAS combined with stroke group, who showed CAS combined with large artery atherosclerotic ischemic stroke and cerebral vascular stenosis >50%. The plaques were divided into stable and unstable plaques as widely accepted in clinic: 1) IMT≥1.2 mm was considered as plaque; 2) the stable plaques are plaques with uniform strong echo or medium echo; 3) the unstable plaques are soft plaque or ulcerative plaque with mixed echo or low echo.14 The measurement of IMT was conducted using a LOGIQ C9 Color Doppler ultrasound diagnostic instrument (General Electric, United States of America) with probe frequency of 7~14 MHz. The IMT of bilateral common carotid artery, proximal, distal and 1 cm from the bifurcation of common carotid artery were measured. All measurement was conducted at least three times and the mean value was considered as the final IMT value.

Additionally, patients were further divided into different body mass index (BMI) groups: the normal group, with BMI<24 kg/m2; the overweight group, with 28 kg/m2>BMI≥24 kg/m2; and the obesity group, with BMI>28 kg/m2.15 Blood samples and medical characteristics were also obtained from 231 healthy individuals, who came to routine physical examination with the same BMI distribution of CAS patients. All patients signed the informed consent. The present study was approved by the ethical committee of the Fourth People’s Hospital of Chengdu (CDH-2018-057).

 Measurement of PM20D1

The blood samples of all cases were collected within 24 hours of admission or coming to outpatient. Briefly, 5 ml of blood was collected into tubes without any anticoagulant. After 1 hour, centrifugation was conducted at 2,000 g for 15 minutes, in room temperature, and the serum samples were obtained. The serum PM20D1 levels were measured by enzyme-linked immunosorbent assay (ELISA), using a PM20D1 kit (MYBioSource, cat. no. MBS280518), strictly according to the manufacturer’s instruction.

 Data collection and measurement

Clinical and demographic characteristics of all patients were collected, including age, sex, BMI and medical history. Routing whole blood test was performed using an automatic biochemical analyzer (Hitachi 7600, Hitachi Corporation, Japan), and the levels of C-reactive protein (CRP), tumor necrosis factor (TNF-α), homocysteine (Hcy), as well as total cholesterol (TC), triglyceride (TG), high-density leptin cholesterol (HDL-ch) and low-density leptin cholesterol (LDL-ch), were recorded.

 Statistical analysis

Data distributed normally was expressed by mean ± standard deviation (SD), and non-normal distributed data was expressed as median (interquartile range). Categorical variables were shown as number (rates). The distribution of the data was analyzed by Kolmogorov-Smirnov method. For normally distributed data, comparison between two groups was conducted by unpaired t-test and one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, which was used for comparison among three or more groups. For non-normal distributed data, Mann-Whitney test was used for comparison between two groups, and Kruskal-Wallis test was used for comparison among three or more groups, following with Dunn’s post hoc test. Rates were analyzed using chi-squared test. Correlation among PM20D1, Hcy, lipid metabolism and inflammatory factors was analyzed using the Pearson’s correlation. A receiver operating characteristic (ROC) curve was used for analysis of PM20D1 in CAS patients. Logistic regression was used for analysis of risk of CAS. p<0.05 was considered as statistically different. All calculation was performed using SPSS 25.0 (SPSS Inc., Chicago, United States of America) or GraphPad 6.0 (GraphPad Software, San Diego, California, United States of America).

 Results Basic clinical characteristics of all patients

This research included a total of 231 CAS patients, with 152 cases of mild/moderate CAS, 50 cases of severe CAS and 29 cases of CAS with stroke. The ratio of the sum of overweight and obesity patients was significantly higher in severe CAS patients when compared to the mild/moderate patients (Table 1). The ratio of unstable plaque was significantly higher in severe CAS and CAS/stroke patients. The levels of CRP, TNF-α and Hcy were markedly higher in severe CAS patients and CAS/stroke patients when compared to the mild/moderate patients. Only TG, TC and LDL-ch were found to be remarkably higher in severe CAS patients and CAS/stroke patients when compared to the mild/moderate cases. Besides, the healthy control showed

– Basic clinical characteristics of all patients
Variables All CAS, n=231 Mild/moderate CAS, n=152 Severe CAS, n=50 CAS/stroke, n=29 Healthy, n=231 P1* P2#
Age, y 57.85±9.04 58.17±9.35 58.32±8.90 55.37±7.37 58.36±8.97 0.543 0.289
Sex, female (%) 105 (45.45) 64 (42.11) 25 (50.00) 16 (55.17) 108 (46.75) 0.854 0.177
BMI, n (%) 0.968 0.176
<24 kg/m2 98 (42.42) 65 (42.76) 15 (30.00) 8 (27.59) 102 (44.16)
28 kg/m2>BMI≥24 kg/m2 76 (32.90) 48 (31.58) 20 (40.00) 11 (37.93) 73 (31.60)
>28 kg/m2 57 (24.68) 39 (25.66) 15 (30.00) 10 (34.48) 56 (24.24)
Complications, n (%) - 0.993
Diabetes 62 (26.84) 39 (25.66) 15 (30.00) 8 (27.59) - 0.790
Hypertension 114 (49.35) 75 (49.34) 24 (48.00) 15 (51.72) - 0.868
History of coronary heart disease 41 (17.75) 27 (17.76) 9 (18.00) 5 (17.24) - 0.990
History of stroke 12 (5.19) 7 (4.61) 3 (6.00) 2 (6.90) - 0.804
Current smoker 129 (55.84) 87 (57.24) 24 (48.00) 18 (62.07) 97 (41.99) 0.090 0.126
Plaque, n (%) - - <0.001
Stable 103 (44.59) 81 (53.29) 17 (34.00) 5 (17.24) -
Unstable 128 (55.41) 71 (46.71) 33 (66.00) 24 (82.76) -
CRP, mg/L 6.91 (0.52~34.87) 5.13 (0.52~9.99) 17.01 (2.92~34.87) 13.40 (3.01~33.64) 4.23 (0.57~10.01) <0.001 <0.001
TNF-α, pg/ml 24.18±12.99 17.45±4.40 37.01±14.70 37.34±13.59 14.81±2.81 <0.001 <0.001
Hcy, μmol/L 12.28±3.22 10.78±2.34 15.08±2.72 15.33±2.61 7.46±1.45 <0.001 <0.001
TC, mmol/L 4.26±0.78 4.00±0.66 4.80±0.72 4.66±0.84 3.96±0.68 <0.001 <0.001
TG, mmol/L 1.48±0.51 1.37±0.54 1.65±0.40 1.77±0.36 1.28±0.45 <0.001 <0.001
LDL-ch, mmol/L 3.01±0.55 2.87±0.44 3.25±0.61 3.33±0.66 2.74±0.41 <0.001 <0.001
HDL-ch, mmol/L 1.15±0.08 1.15±0.07 1.16±0.08 1.15±0.09 1.16±0.08 0.473 0.735

*P1 comparison between all carotid atherosclerosis (CAS) and the healthy control. #P2 comparison among mild/moderate, severe and CAS/stroke patients. One-way analysis of variance followed by Tukey’s post hoc test was used for normally distributed data. For non-normal distributed data, Kruskal-Wallis test was used for comparison among mild/moderate, severe and CAS/stroke patients, following with Dunn’s post hoc test. Rates were analyzed using chi-squared test. BMI: body mass index; CRP: C-reactive protein; TNF-α: tumor necrosis factor; Hcy: homocysteine; TC: total cholesterol; TG: triglyceride; LDL-ch: low-density leptin cholesterol; HDL-ch: high-density leptin cholesterol.

significantly lower levels of CRP, TNF-α and Hcy, as well as TG, TC and LDL-ch than all CAS patients. No other significant difference was found.

 PM20D1 was associated with severity and plaque stability of CAS patients

Then, the expression of serum PM20D1 in CAS patients was determined. It was found that serum PM20D1 levels were markedly lower in CAS patients when compared to the healthy control (Figure 1). Severe CAS patients and CAS/stroke patients showed significantly lower PM20D1 levels when compared to the mild/moderate patients. However, no significant difference was found between severe and CAS/stroke patients. Additionally, patients with unstable plaques showed markedly lower PM20D1 expression than patients with stable plaques.

– Serum levels of PM20D1 in carotid atherosclerosis (CAS) patients with different severity and plaque stability. ***p<0.001, **p<0.01.

PM20D1 was not associated with carotid atherosclerosis patients with different body mass index

To further investigate the role of PM20D1 in CAS patients, we also evaluated the levels of PM20D1 in CAS patients with different BMI distribution. No significant difference was observed among patients with different BMI (Figure 2).

– Serum levels of PM20D1 in carotid atherosclerosis (CAS) patients with different body mass index (BMI).

Correlation between PM20D1, lipid metabolism and inflammatory factors

Then, patients were divided into PM20D1 high expression group and low expression group, according to the mean level of PM20D1 (4.53 ng/ml). It was observed that patients with higher PM20D1 levels showed significantly lower expression of CRP, TNF-α, Hcy, TG, TC and LDL-ch (Table 2). Pearson’s correlation was then conducted and results showed that PM20D1 was negatively correlated with CRP, TNF-α, Hcy, TC and LDL-ch in CAS patients (Table 3).

– Expression of lipid metabolism and inflammatory factors in patients with different PM20D1
Variables PM20D1 high, n=117 PM20D1 low, n=114 p*
CRP, mg/L 5.27 (0.52~33.64) 15.16 (0.68~34.87) <0.001
TNF-α, pg/ml 18.78±7.32 29.72±15.08 <0.001
Hcy, μmol/L 11.26±2.66 13.34±3.41 <0.001
TC, mmol/L 4.03±0.70 4.49±0.80 <0.001
TG, mmol/L 1.41±0.54 1.56±0.47 0.031
LDL-ch, mmol/L 2.90±0.48 3.12±0.59 0.003
HDL-ch, mmol/L 1.14±0.08 1.16±0.08 0.269

*Student’s t-test was used for comparison among mild/moderate, severe and CAS/stroke patients for normally distributed data. For non-normal distributed data, Mann-Whitney test was used for comparison between two groups. Rates were analyzed using chi-squared test. CRP: C-reactive protein; TNF-α: tumor necrosis factor; Hcy: homocysteine; TC: total cholesterol; TG: triglyceride; LDL-ch: low-density leptin cholesterol; HDL-ch: high-density leptin cholesterol.

– Correlation between PM20D1, homocysteine (Hcy), lipid metabolism and inflammatory factors
Variables Pearson’s correlation p value
CRP, mg/L -0.514 <0.001
TNF-α, pg/ml -0.585 <0.001
Hcy, μmol/L -0.598 <0.001
TC, mmol/L -0.254 <0.001
TG, mmol/L -0.059 0.198
LDL-ch, mmol/L -0.071 0.126
HDL-ch, mmol/L 0.022 0.622

CRP: C-reactive protein; TNF-α: tumor necrosis factor; Hcy: homocysteine; TC: total cholesterol; TG: triglyceride; LDL-ch: low-density leptin cholesterol; HDL-ch: high-density leptin cholesterol.

Diagnostic value of PM20D1 in carotid atherosclerosis

At last, a ROC curve was drawn in order to see the diagnostic value of PM20D1 for CAS, as well as severe CAS or CAS/stroke. It was found PM20D1 could be a potential diagnostic biomarker of CAS with cutoff value of 5.94 ng/ml, area under the ROC curve (AUC) 0.876, 95%CI (0.845~0.906), sensitivity 80.1% and specificity 73.6% (Figure 3). PM20D1 could also be used as a biomarker for severe CAS patients or CAS patients with stroke, with cutoff value of 3.99 ng/ml, AUC 0.917, 95%CI (0.883~0.951), sensitivity 81.6%, specificity 77.2%. Further binary logistic regression which included all factors of age, BMI, CRP, TNF-α, Hcy, TC, TG, LDL-ch, HDL-ch,PM20D1, as well as sex, diabetes, hypertension, history of coronary heart disease, history of stroke and current smoker, showed sex, TNF-α, Hcy and PM20D1 were risk factors for CAS (Table 4).

– Receiver operating characteristic (ROC) curves for diagnostic value of PM20D1 of carotid atherosclerosis (CAS), severe CAS or CAS/stroke.

– Binary logistic regression for risk factors of carotid atherosclerosis
Variables Wald Odds ratio 95%CI p
Age 0.011 1.003 0.935~1.078 0.914
Sex 4.210 0.208 1.073~21.401 0.040
BMI 0.524 1.057 0.909~1.231 0.469
Diabetes <0.001 <0.001 0.000 0.995
Hypertension <0.001 <0.001 0.000 0.993
History of coronary heart disease <0.001 <0.001 0.000 0.995
History of stroke <0.001 0.001 0.000 0.999
Current smoker <0.001 0.844 0.232~3.075 0.798
CRP 0.004 1.008 0.777~1.310 0.947
TNF-α 11.820 0.664 0.526~0.839 0.001
Hcy 22.852 0.155 0.073~0.334 <0.001
TC 0.462 0.697 0.248~1.967 0.496
TG 1.403 0.411 0.095~1.787 0.236
LDL-ch 1.559 0.340 0.063~1.847 0.212
HDL-ch 1.216 0.008 1.797E-6~40.532 0.270
PM20D1 18.152 8.485 3.173~22.693 <0.001

BMI: body mass index; CRP: C-reactive protein; TNF-α: tumor necrosis factor; Hcy: homocysteine; TC: total cholesterol; TG: triglyceride; LDL-ch: low-density leptin cholesterol; HDL-ch: high-density leptin cholesterol.

Discussion

Early diagnosis and prediction of CAS can greatly improve the treatment efficacy and prognosis. Thus, biomarkers for diagnosis and prognosis of CAS are of great significance. In the present study, we demonstrated that PM20D1 was down-regulated in CAS patients and was associated with the severity and plaque condition of CAS patients. Besides, PM20D1 was negatively correlated with CRP, TNF-α, Hcy, TC and LDL-ch in CAS patients.

Lipid metabolism and obesity are both associated with development of CAS. It was found that levels of TG, TC and LDL-ch were significantly higher in patients with carotid artery plaque.16 Another research found that treatment of atorvastatin or ezetimibe obviously decreased the levels of TC, TG and LDL-ch in CAS patients.17 In a recent study, Pan et al. showed that, in low-income rural residents in China, LDL-ch and TC were risk factors for early-stage atherosclerosis and carotid plaque risk increased by 24% and 62% for each 1-mmol/L increase in TC and LDL-ch.18 In our research, we also found that TG, TC and LDL-ch were highly expressed in CAS patients, especially in severe CAS cases, which was consistent with the aforementioned researches. Besides, we also observed that inflammatory factors such as CRP, TNF-α and expression of Hcy were elevated in CAS patients, which was also demonstrated in several researches.19-22

PM20D1 is a factor associated with both lipid metabolism and obesity. It was found that mice with increased circulating PM20D1 showed increased breathing and increased serum N-acyl amino acids, which improved glucose homeostasis and increased energy consumption, and thus might regulate obesity.13 Benson et al. demonstrated that decreased PM20D1 was associated with the genetic relationship of obesity and neurodegenerative diseases in humans.23 Li et al. found that inhibition of miR-324-5p increased the oxygen consumption of primary white and brown adipose tissue cells, increased fat consumption, and thus reduced the weight of mice by enhancing the levels of PM20D1.24 Long et al. observed that, in PM20D1 deficiency mice, knockdown of PM20D1 decreased the N-acetil aspartato hydrolase/synthetase activity in serum and tissues, as well as a variety of metabolic and pain phenotypes, including insulin resistance, cold temperature changes and antinociceptive behavior, which are associated with obesity, diabetes and other diseases.12 Thus, we can speculate that higher PM20D1 levels might be beneficial in the improvement of obesity and the reduction of lipid. Although there is no study demonstrating the role of PM20D1 in CAS, the down-regulation of PM20D1 in CAS patients in our study might be partly due to the dysfunction of lipid metabolism and obesity of CAS patients. Since CAS patients showed lower PM20D1 levels than healthy control who had the same BMI distribution, and CAS patients with different BMI showed no significant difference of PM20D1, this result indicated that BMI might not be associated with the level of PM20D1 in CAS patients. The negative correlation between PM20D1 and Hcy, TC and LDL-ch might be one of the reasons for the abnormal expression of PM20D1 in CAS patients. However, to substantiate all these speculations, more studies are needed in order to obtain further evidence.

The present study has some limitations. First, this is an observational study from a single center, with only 231 cases. Secondly, molecular mechanism for how PM20D1 influences CAS development is still unclear.

 Conclusion

In conclusion, this observational study demonstrated that decreased PM20D1 was associated with the severity, plaque condition, and expression of CRP, TNF-α, Hcy, TG, TC and LDL-ch in CAS patients. This study might provide novel research target for PM20D1 in CAS.

Study Association

This study is not associated with any thesis or dissertation work.

Sources of Funding: There were no external funding sources for this study.