巨噬细胞移动抑制因子启动子区-794CATT5-8(rs5844572)多态性与汉族高原低氧性肺动脉高压(PAH)的关系※

曹学锋，刘 杰，刘延英，马 兰

(1.青海大学高原医学研究中心，青海 西宁 810016；2.青海大学附属医院老年科，青海 西宁 810001)

1.Introduction

Pulmonary arterial hypertension(PAH)is a potentially fatal vascular disease characterized by the elevation of mean pulmonary arterial pressure(mPAP)to above 25 mmHg[1].High altitude hypoxia induced PAH may lead to progressive hypoxemia and ultimately threaten the health of people living on the plateau.PAH can be initiated by hypoxia and it can also exacerbate hypoxia，thus forming a vicious circle.Hypoxia plays a pivotal role in remodeling of vascular smooth muscle cells and inflammation of small pulmonary arteries，which can have a substantial impact on the development of PAH.The circulating inflammatory markers of IL-6，IL-1 and CRP are upregulated in response to hypoxia induced inflammation at high altitude，which may also contribute to the development of PAH.Thus，both inflammatory response and pulmonary endothelial dysfunction have been implicated in the development and progression of PAH[2].

Macrophage migration inhibitory factor(MIF)is a key pleiotropic pro-inflammatory mediator and a promising therapeutic target of PAH[3]，and it can promote a wide variety of pathophysiological processes such as gene expression，cell differentiation，apoptosis and inflammatory response.Human MIF gene is located on chromosome 22q(11.2)and consists of 3 exons and 2 introns.It is reported that the protein and mRNA levels of MIF were elevated in lung tissues of PAH rats.Le Hiress et al found that the concentrations of circulating MIF were increased in the serum of PAH patients，and treatment with MIF antagonist(ISO-1)partially reversed the development of PAH and substantially reduced inflammatory cell infiltration in rats[4].Huang et al showed that MIF antagonist(MIF098)could prevent PAH by inhibiting the proliferation，migration，muscularization and fibrosis of pulmonary artery smooth muscle cells in both animal models and cell experiments[5].MIF may induce pulmonary artery smooth muscle cell proliferation by upregulating cyclinD1 expression and subsequently inducing arterial remodeling in the development of PAH in broilers[6].The promoter region of MIF has -794CATT5-8(rs5844572)microsatellite repeats，which are functionally related to the promoter activity and high expression of MIF gene and thus the migration and proliferation of endothelial cells and vascular smooth muscle cells under hypoxic conditions[7].Therefore，MIF promoter polymorphisms may be associated with an increased risk of inflammatory diseases due to increased MIF protein production.The overexpression of MIF can accelerate hypoxia induced inflammatory responses and thus the development of PAH.

Non-adaptive Han Nationality have been found to be more susceptible to PAH than others when exposed to the same altitude[8].However，it remains unclear whether MIF -794CATT5-8(rs5844572)microsatellite polymorphisms could affect the susceptibility of Han Nationality to high altitude hypoxia induced PAH.We investigated the relationship between MIF -794CATT5-8(rs5844572)polymorphisms and high altitude hypoxia induced PAH in Han Chinese，and identified MIF genes associated with the susceptibility to high altitude hypoxia induced PAH.Thus，this study may provide a genetic basis for clinical prevention and treatment of high altitude hypoxia induced PAH.

2.Participants and methods

2.1 PAH patients and healthy subjects

A total of 83 male PAH patients(age 38.3±10.0years)and 145 healthy subjects from lower altitude(age 38.7±9.6years)who ascended acutely to Yushu with an average elevation of 3 800 m a.s.l in the Qinghai-Tibet Plateau，China，were included in this study.All 83 PAH patients were incorporated according to the Qinghai diagnostic criteria of high altitude pulmonary arterial hypertension(HAPH)from the VI World Congress on Mountain Medicine and High Altitude Physiology(Xining，China；2004)[9].PAH patients were hospitalized at People′s Hospital of Yushu Prefecture.The mean pulmonary arterial pressure(mPAP)was measured by echocardiography with the same standard.The mPAP higher than 30 mmHg was included in PAH patients，while mPAP lower than 30 mmHg was included in healthy subjects.Those with a history of cardiopulmonary disease such as myocardial infarction and heart failure，and pneumonia were excluded.The two groups were matched for age，gender，weight and ethnicity，and their baseline characteristics were shown in Table 1.All participants signed the informed consent for participation，and all protocols were approved by the Ethics Committee of the Medical College of Qinghai University.

Table 1 Characteristics of study subjects

2.2 Clinical tests

Body weight，body mass index(BMI)，blood pressure and heart rate were measured by standard methods.Haematocrit(HCT)and red blood cell(RBC)count were determined from venous blood samples using the Mindray Hematology Analyzer(BC-2300，Shenzhen，China).Pulse oxygen saturation(SpO2)were recorded using a pulse oximeter(Ohmeda 3700 Pulse Oximeter，Datex-Ohmeda，Boulder，Colorado，USA).Mean pulmonary arterial pressure(mPAP)was measured by echocardiography(EchoPac，Version 6.3，GE Healthcare).

目前国际上共有六大类30多种药物 （包括复合制剂），分别为核苷类反转录酶抑制剂（NRTIs）、非核苷类反转录酶抑制剂（NNRTIs）、蛋白酶抑制剂（PIs）、整合酶抑制剂（INSTIs）、融合抑制剂（FIs）及CCR5抑制剂。国内的抗反转录病毒治疗（ARV）药物有 NRTIs、NNRTIs、PIs、INSTIs 以及 FIs 五大类（包含复合制剂），见表3。

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2.3 Reagents and instruments

The characteristics of study subjects，including sample size，age range，sex，body weight(Kg)，body mass index (BMI)，HCT(%)，RBC(×1012)，SpO2(%)and mean pulmonary artery pressure(mPAP)(mmHg)，are described in Table 1.PAH patients have significantly higher mPAP(34～85mmHg)，but lower SpO2than Non PAH healthy subjects.

2.4 Genomic DNA extraction and genotyping of MIF promoter microsatellite polymorphisms

Genomic DNA was extracted from peripheral blood leukocytes using a GentraPuregene Blood Kit.MIF promoter polymorphism sequences were amplified by polymerase chain reaction(PCR)(ABI 9700 thermocycler)using the following FAM-labeled primers synthesized by the Bioassay Laboratory of CapitalBio Corporation：Forward primer，5′-CCTTGTCCTCTTCCTGCTATGT-3′；Reverse primer，5′-TCTGGGCAACTTCAGCTCCT-3′.

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The differences in genotype and allele frequency between the two groups were tested by using SPSS statistical software(version 27.0，SPSS，Inc，Chicago，IL).Measurement data were expressed as mean standard deviation，and t-test was used for comparison between groups.Fisher′s exact probability method for counting data and Hardy Weinberg test were used to test the level α=0.05.

Target fragmentswere amplified as follows：pre-denaturation at 94 ℃ for 5 min，30 cycles of 94 ℃ for 30 s，60 ℃ for 30 s and 72 ℃ for 20 s，and a final extension at 72 ℃ for 60 min.Amplified fragments were resolved on 1.5% agarose gels.The amplification length was 230 bp.Short Tandem Repeat(STR)was detected based on standard capillary electrophoresis using an ABI 3730 XL sequencer(Applied Biosystems，USA).Then，9 μL of the mixture(8.5μL hi-DI+0.5μL Genescan Marker ROX500)and diluted STR were added successively.The sequencing peak map was imported into GeneMapper4.1 software(ABI)for identification.

2.5 Statistical method

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3.Results

3.1 Characteristics of subjects

The reagents and instruments used in this study included PCR thermocycler(ABI 9700)，sequencer(ABI 3730XL)，low speed centrifuge(Eppendorf Centrifuge 5415R，Germany)，ultralow temperature freezer(Forma 725)，gel scanning imaging system(Labworks，UVP，USA)，UV spectrophotometer(Beckman DU800，USA)，steady flow electrophoresis apparatus(DYY-6B，Beijing Sixty-one Instrument Factory，China)，refrigerated centrifuge，small desktop centrifuge，electrophoresis tank，GentraPuregene Blood Kit(158389 Qiagen，Germany)，D2000 DNA-Marker(Tiangen，Beijing)，agarose gel powder(Formatas)，gene amplification reagents(Capitalbio Technology，Beijing)，sequencing reagents(ABI)，and GeneMapper 4.1 software(ABI).

3.2 MIF-794CATT5-8(rs5844572)polymorphisms

通过有限元计算的裂尖应力场和应变场来计算J积分，得到在临界载荷时沿厚度分布的J积分,如图12所示。由图可见，从试样中心面到表面的J积分基本呈现变小趋势，其中最大值为381.3kJ/m2，最小值为137.4kJ/m2，该范围包含了按照GB/T 21143标准计算的J积分的起裂断裂韧度值(351.4 kJ/m2)。另外，有限元计算的J积分平均值为293.1kJ/m2，记为JiFem，与光测试验结果JiDic(293.4kJ/m2)接近。

Table 2 MIF-794-CATT5-8(rs5844572)polymorphisms of study subjects

PAH is an early pathophysiological manifestation of acute ascent to high altitude，which can lead to severe systemic hypoxemia，right ventricular failure and even death.Elevated circulating MIF levels have been shown to correlate with PAH[10-12]，and the polymorphisms in the promoter region of MIF gene can affect gene transcription and are associated with the pathogenesis of rheumatoid arthritis and several other inflammatory diseases[13].MIF-794CATT5-8(rs5844572)polymorphisms are tetranucleotide repeat polymorphisms located at -794 bp upstream of mRNA transcriptional start site and a higher number of repeats is associated with increased expression of MIF in some inflammatory diseases[14].

Alleles were classified as 5，6，7 and 8 repeats as described in previous studies.The frequency of 55(two short alleles)，56(one 5 allele and one 6 allele)，57(one 5 allele and one 7 allele)，58(one 5 allele and one 8 allele)，66(two 6 alleles)，67(one 6 allele and one 7 allele)，68(one 6 allele and one 8 allele) and 77(two 7 alleles)genotypes is 13.25%，22.89%，13.25%，1.20%，26.51%，20.48%，0%，and 2.41% in PAH patients；and 14.48%，33.79%，12.41%，0%，16.55%，17.93%，1.38% and 3.45% in Non-PAH healthy subjects，respectively.The frequency of 5，6，7 and 8 alleles is 31.93%，48.19%，19.28% and 0.60% in PAH patients；and 37.58%，43.10%，18.62% and 0.69% in Non-PAH healthy subjects，respectively.There was no significant difference in the frequency of 55，56，57，58，66，67，68 and 77 genotypes between the two groups (P=0.352).There was no significant difference in the frequency of alleles (P=0.652) as well.The comparison showed that there was no significant difference in the distribution genotypes and alleles frequency of MIF-794CATT5-8(rs5844572)polymorphisms between the two groups(P>0.05).

The genotype and allele frequencies of MIF-794CATT5-8(rs5844572)polymorphisms of PAH patients and healthy subjects were presented in Table 2.

The sequencing peak diagram was imported into GeneMapper4.1 software(ABI)to identify the sequencing results.Some of the results are shown in Figure 1，including heterozygotes and homozygotes.

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Figure 1 Capillary electrophoretic peaks of MIF-794CATT5-8(rs5844572)polymorphisms

4.Discussion

Abbreviations：PAH，pulmonary arterial hypertension；Non-PAH，non-pulmonary arterial hypertension

This is the first study to investigate the relationship between MIF-794CATT5-8(rs5844572)polymorphisms and high altitude hypoxia induced PAH in Han Chinese.We found that the frequency of alleles was not associated with the susceptibility to high altitude hypoxia induced PAH in Han Chinese.Meanwhile，there was no significant difference in MIF-794CATT5-8(rs5844572)genotype frequency between PAH patients and Non-PAH healthy subjects(P>0.05).Note that all PAH patients were males in this study，and previous human studies have also shown that gender and supplementary estrogen intake have a protective effect against PAH[15].Further，MIF-794CATT5-8(rs5844572)genotype and allele frequency polymorphism did not correlate with PAH.MIF is an important upstream regulator in the pro-inflammatory cascade and a potential biomarker in the assessment of PAH[15].Anti-MIFs have been implicated as a new therapeutic approach for hypoxia induced PAH and vascular remodeling in rodents.MIF antagonist(MIF098)inhibits the pathological progression of PAH，mainly by inhibiting the proliferation，migration，muscularization and fibrosis of pulmonary artery smooth muscle cells[5].MIF can also increase the proliferation of endothelial cells and smooth muscle cells.However，its role in the pathogenesis of high altitude hypoxia induced PAH remains poorly understood.Up to now，MIF is considered a vascular protective factor in humans due to its anti-inflammatory and pro-vasodilatory properties.It has been reported that the concentrations of circulating MIF were increased in the serum of PAH patients compared to healthy controls，and the treatments with MIF antagonist(ISO-1)or anti-CD74 neutralizing antibodies partially reversed the development of PAH in rats[4].It has also been reported that the MIF gene polymorphisms in the promoter region MIF-794CATT5-8(rs5844572)are closely related to the transcription activity of MIF in inflammatory diseases[16].

In this study，there is no significant difference in the genotype and allele frequency between PAH patients and Non-PAH subjects(P>0.05).Therefore，it can be concluded that MIF-794CATT5-8(rs5844572)polymorphisms may not have special effect on the occurrence and development of PAH in Han Chinese.And that high altitude hypoxia induced PAH is a multifactorial disease that is related to various environmental and genetic factors，and its important risk factors include gender，coldness，vigorous exercise，and poor physical health and so on.Furthermore，the exact molecular mechanism of high altitude hypoxia induced PAH remains unclear.Our results suggest that MIF-794CATT5-8(rs5844572)polymorphisms may not be a potential genetic risk factor of high altitude hypoxia induced PAH.

5.Limitations

Our study has several limitations that should be acknowledged.The subjects included in this study are largely young men enrolled from a single district.There is also a lack of information on the systemic oxidative stress and inflammation markers，as well as the inflammatory status such as MIF protein levels and molecules associated with the oxidative stress.Biochemical measures would contribute to better understanding MIF promoter polymorphisms associated with high altitude hypoxia induced PAH.Last but not least sampling bias and size should also be noted in order to obtain more accurate results.

6.Conclusion

In conclusion，MIF-794CATT5-8(rs5844572)polymorphisms are not associated with high altitude hypoxia induced PAH in Han Chinese，and MIF may not be a genetic risk factor of this disease.However，further studies are needed to elucidate the molecular mechanism of MIF in the pathogenesis and development of high altitude hypoxia induced PAH.

Conflictofinterest

The authors declare that there is no conflict of interest.