第一部分：基于多组学揭示冠心病患者胸廓内动脉血管周围脂肪组织的异质 性及其与动脉粥样硬化的关联

                                   摘要

背景：血管周围脂肪组织（Perivascular adipose tissue, PVAT）与动脉粥样硬化形 成有关。然而，促进动脉粥样硬化的冠状动脉血管周围脂肪组织(Coronary artery perivascular adipose tissue , CA-PVAT)和抑制动脉粥样硬化的胸廓内动脉血管周 围脂肪组织(Internal thoracic artery perivascular adipose tissue , ITA-PVAT)的具体 差异尚不清楚。因此，我们主要研究 ITA-PVAT 相对于 CA-PVAT 是否具有不同 的表型并探讨 ITA-PVAT 抑制动脉粥样硬化的潜在机制。

 

方法：本研究采用RNA 测序和非靶向代谢组学技术，检测了 35 例接受冠状动 脉旁路移植术患者的ITA-PVAT 和 CA-PVAT 的基因表达谱和代谢物谱。

 

结果：

1. ITA-PVAT 和 CA-PVAT 之间共有 8334 个差异表达基因，与 ITA-PVAT 相比， CA-PVAT 中 3085 个基因表达上调，5249 个基因表达下调，这些差异基因主要参 与免疫调节、炎症反应、脂质代谢以及类固醇的合成等方面。其中，CA-PVAT 中与炎症反应、脂质代谢和类固醇合成相关的基因表达高于 ITA-PVAT。

 

2. CA-PVAT 与 ITA-PVAT 组间有 46 个负离子模式代谢物差异显著，与 ITA-PVAT  相比，CA-PVAT 中 27 个负离子模式代谢物上调，19 个负离子模式代谢物下调。 CA-PVAT 与 ITA-PVAT 组间有 23 个正离子模式代谢物差异显著，与 ITA-PVAT  相比，CV-PVAT 中有 10 个正离子模式代谢物上调，13 个正离子模式代谢物下调。 这些差异代谢物主要与脂质代谢，卟啉和叶绿素代谢、胆碱能突触代谢、缬氨酸、 亮氨酸和异亮氨酸的生物合成、酪氨酸代谢、类固醇激素的生物合成、甲状腺激 素合成、赖氨酸降解和胰岛素分泌等通路有关。

 

3.  转录组和代谢组联合分析发现 ITA-PVAT 与CA-PVAT 之间的差异基因和差异 代谢物共同富集到 58 条代谢通路。磷脂酰胆碱 40:5 、磷酸肌酸、谷胱甘肽和 N-乙酰神经氨酸等代谢物和甘油磷脂代谢、精氨酸和脯氨酸代谢、氨基酸和核苷 酸糖代谢、甲状腺激素合成和谷胱甘肽代谢等代谢通路是 ITA-PVAT 抑制动脉粥 样硬化的潜在代谢物和代谢通路。

 

结论：CA-PVAT 和 ITA-PVAT 之间的转录组特征和代谢组特征均存在明显差异。 PVAT  的基因表达差异和代谢物谱差异与 CA  和 ITA 在动脉粥样硬化易感性上

 

的差异相吻合。特别是与 CA-PVAT 相比，ITA-PVAT 具有较低的炎症反应相关 基因表达，较高含量的抑制动脉粥样硬化代谢产物以及较低含量的促进动脉粥样 硬化代谢产物，表明ITA-PVAT 可能具有独立于 ITA 本身的抑制动脉粥样硬化的 能力。

 

第二部分：基于多组学揭示冠心病患者大隐静脉血管周围脂肪组织的异质性及 其与动脉粥样硬化的关联

                                           摘要

背景：血管周围脂肪组织（Perivascular adipose tissue, PVAT）与动脉粥样硬化形 成有关 。研究表明 ，冠状动脉血管周围脂肪组织(Coronary artery perivascular adipose tissue , CA-PVAT)促进动脉粥样硬化形成；而大隐静脉血管周围脂肪组织 （Saphenous vein perivascular adipose tissue, SV-PVAT）具有抗动脉粥样硬化作用 并可维持大隐静脉移植物的长期通畅。然而，SV-PVAT 抵抗动脉粥样硬化的机 制尚未完全明确，特别是物质代谢方面。因此，本研究通过比较 SV-PVAT 与 CA-PVAT 来探讨 SV-PVAT 抑制动脉粥样硬化的潜在机制。

 

方法：本研究采用RNA 测序和非靶向代谢组学技术，检测了 35 例接受冠状动 脉旁路移植术患者的 CA-PVAT 以及 SV-PVAT 的基因表达谱和代谢物谱。

 

结果：

1. SV-PVAT 和 CA-PVAT 之间共有 10355 个差异表达基因，与 CA-PVAT 相比， SV-PVAT 中 4710 个基因表达上调，5625 个基因表达下调。这些差异基因主要参 与器官组织发育、免疫调节、炎症反应及脂质代谢等方面，其中，CA-PVAT 中 与免疫调节、炎症反应、心肌细胞分化、心肌收缩及脂质代谢相关的基因表达高 于 SV-PVAT。

2. SV-PVAT 与 CA-PVAT 中发现有 71 个负离子模式代谢物差异显著，与 CA-PVAT  相比，SV-PVAT 中 31 个负离子模式代谢物上调，40 个负离子模式代谢物下调； SV-PVAT 与 CA-PVAT 中有 36 个正离子模式代谢物差异显著，与 CA-PVAT 相比， SV-PVAT 中有 21 个正离子模式代谢物上调，15 个正离子模式代谢物下调。这些 差异代谢物主要与氨基酸糖和核苷酸糖代谢、牛磺酸和低牛磺酸代谢、丁酸盐代 谢、谷胱甘肽代谢和α-亚麻酸代谢有关。CA-PVAT 和 SV-PVAT 中均有多种代谢 物参与甘油磷脂代谢通路。

3.  转录组学和代谢组学联合分析发现 79 条共同富集通路。13(S)-羟基十八碳三 烯酸、癸酸、棕榈油酸、磷脂酰肌醇 36:4 、磷脂酰乙胺醇 38:4 、溶血磷脂酰胆 碱 15:0、琥珀酸和鞘磷脂 1-磷酸等代谢物和甘油磷脂代谢、脂肪酸的生物合成、 α-亚麻酸代谢、鞘磷脂信号通路、氧化磷酸化、苯丙氨酸代谢与丙氨酸、天冬氨 酸和谷氨酸代谢等代谢通路是 SV-PVAT 抑制动脉粥样硬化的潜在代谢物和代谢 通路。

 

结论： SV-PVAT 与CA-PVAT 在基因表达模式和代谢物谱中存在显著异质性。

 

特别是 SV-PVAT  和 CA-PVAT  在冠心病患者中与炎症、脂质代谢和心肌过程有 关的基因表达的异质性以及与氨基酸代谢、脂质代谢和能量代谢有关的代谢物的 异质性表明 SV-PVAT 抑制动脉粥样硬化的潜在机制与炎症反应、脂质代谢、氨 基酸代谢和能量代谢有关。

 

 

第三部分：冠心病患者不同部位血管周围脂肪组织神经酰胺组成及其与心血管 疾病危险因素的关联

                                            摘要

背景：血管周围脂肪组织可以通过分泌生物活性物质来调节血管功能和结构。研 究表明，胸部脂肪组织可分泌神经酰胺，后者会导致血管的氧化应激损伤，并与 肥胖患者的不良预后相关。冠状动脉血管周围脂肪组织与冠状动脉粥样硬化、血 管炎症和细胞因子失衡有关。临床研究表明，在冠状动脉旁路移植术中，保留大 隐静脉血管周围脂肪组织可以增加大隐静脉移植物的通畅率。我们推测冠状动脉 血管周围脂肪组织和大隐静脉血管周围脂肪组织对血管的不同影响可能是神经 酰胺组成差异引起的。因此，本研究旨在比较大隐静脉血管周围脂肪组织与其他 血管周围脂肪组织在神经酰胺组成上是否存在差异。

 

方法：本研究采用靶向代谢组学技术，检测了30 例接受冠状动脉旁路移植术患 者的胸部皮下脂肪组织、大隐静脉血管周围脂肪组织以及冠状动脉血管周围脂肪 组织样本的神经酰胺谱。采用线性回归模型评价脂肪组织神经酰胺与心血管疾病 危险因素的相关性。

 

结果：在胸部皮下脂肪组织、大隐静脉血管周围脂肪组织以及冠状动脉血管周围 脂肪组织中均可检测出 15 种神经酰胺。与冠状动脉血管周围脂肪组织相比，大 隐静脉血管周围脂肪组织有 12 种神经酰胺含量明显降低。与皮下脂肪相比，大 隐静脉血管周围脂肪组织有 5 种神经酰胺含量降低，2 种神经酰胺含量升高。冠 状动脉血管周围脂肪组织多种神经酰胺与心血管疾病危险因素显著相关。

 

结论：本研究中发现冠心病患者不同部位血管周围脂肪组织神经酰胺组成有显著 差异，有助于解释动脉粥样硬化斑块的易感性差异。改善血管周围脂肪组织神经 酰胺谱的治疗策略可能有益于冠心病患者。

 

 

                                                Part I 

                                             Abstract

BACKGROUND:    Perivascular    adipose    tissue    (PVAT)    is    associated    with atherogenesis. However, the specific differences between coronary artery perivascular adipose  tissue  (CA-PVAT),  which  promotes  atherosclerosis,  and  internal  thoracic artery perivascular adipose tissue  (ITA-PVAT), which inhibits atherosclerosis,  are unknown. Therefore, we focused on whether ITA-PVAT has a different phenotype relative to CA-PVAT and explored the potential mechanisms by which ITA-PVAT inhibits atherosclerosis.

METHODS: In this study, we examined the gene expression profiles and metabolite profiles of ITA-PVAT and CA-PVAT in 35 patients who underwent coronary artery bypass grafting using RNA sequencing and untargeted metabolomics.

Results:

1. A total of 8334 differentially expressed genes were found between ITA-PVAT and CA-PVAT. 3085 genes were up-regulated and 5249 genes were down-regulated in CA-PVAT compared to ITA-PVAT, and these differentially expressed genes were mainly involved in immune regulation, inflammatory response, lipid metabolism, and steroid  synthesis.  Among  them,  the  expression  of  genes  related  to  inflammatory response, lipid metabolism and steroid synthesis was higher in CA-PVAT than in ITA-PVAT.

2. There were 46 negative ion pattern metabolites that differed significantly between the CA-PVAT and ITA-PVAT groups, and 27 negative ion pattern metabolites were up-regulated  and   19   negative   ion   pattern  were   down-regulated   in   CA-PVAT compared  with  ITA-PVAT.   Twenty-three  positive   ion-mode   metabolites  were significantly different between the CA-PVAT and ITA-PVAT groups, and 10 positive ion-mode metabolites were up-regulated and 13 positive ion-mode metabolites were down-regulated   in   CV-PVAT    compared   with    ITA-PVAT.   These    differential metabolites were mainly associated with lipid metabolism, porphyrin and chlorophyll

metabolism,   cholinergic    synaptic   metabolism,   valine,    leucine   and    isoleucine biosynthesis, tyrosine metabolism,  steroid  hormone  biosynthesis, thyroid hormone synthesis, lysine degradation and insulin secretion pathways.

3. Combined transcriptome and metabolome analyses revealed that differential genes and differential metabolites between ITA-PVAT and CA-PVAT were co-enriched in 58    metabolic     pathways.     Metabolites    such     as     phosphatidylcholine     40:5, phosphocreatine, glutathione and N-acetylneuraminic acid and metabolic pathways such  as  glycerophospholipid  metabolism,  arginine  and  proline  metabolism,  amino acid and nucleotide glucose metabolism, thyroid hormone synthesis and glutathione metabolism were the potential metabolites and metabolic pathways for ITA-PVAT to inhibit atherosclerosis.

CONCLUSIONS:   Significant   differences   in   both   transcriptomic   profiles   and metabolomic  profiles  were  observed  between  CA-PVAT  and  ITA-PVAT.  The differences in gene expression and metabolite profiles of PVAT coincide with the differences  in  atherosclerosis   susceptibility  between  CA  and  ITA.  In  particular, ITA-PVAT demonstrated lower expression of genes associated with inflammatory responses, higher levels of metabolites that inhibit atherosclerosis, and lower levels of metabolites  that  promote   atherosclerosis,  when   compared  with   CA-PVAT.  This suggests   that   ITA-PVAT    may   have    the   capacity    to   inhibit    atherosclerosis independently of ITA itself.

 

 

                                            Part II

                                          Abstract
BACKGROUND:    Perivascular    adipose    tissue    (PVAT)     is    associated    with atherosclerosis  formation.  Studies  have   shown  that   coronary  artery  perivascular adipose   tissue    (CA-PVAT)   promotes    atherogenesis,   whereas    saphenous   vein perivascular adipose tissue (SV-PVAT) has anti-atherosclerotic effects and maintains long-term  patency  of  saphenous  vein  grafts.  However,  the  mechanism  by  which SV-PVAT resists atherosclerosis has not been fully clarified, especially in terms of substance metabolism. Therefore, the present study was conducted to investigate the potential   mechanism   of   SV-PVAT   in   inhibiting   atherosclerosis   by   comparing SV-PVAT with CA-PVAT.

METHODS: In this study, the gene expression profiles and metabolite profiles of CA-PVAT as well as SV-PVAT were examined in 35 patients who underwent coronary artery bypass grafting using RNA sequencing and untargeted metabolomics.

RESULTS:
1. There were  10355 differentially expressed genes between SV-PVAT and CA-PVAT, and 4710 genes were up-regulated and 5625 genes were down-regulated in SV-PVAT compared with CA-PVAT. These differentially expressed genes were mainly involved in organ tissue development, immune regulation, inflammatory response and lipid metabolism, among which, the  expression of genes related to immune regulation, inflammatory  response,  cardiomyocyte  differentiation,  myocardial  contraction  and lipid metabolism was higher in CA-PVAT than in SV-PVAT.
2.  Significant  differences  were  found  in  71  negative  ion  pattern  metabolites  in SV-PVAT versus CA-PVAT, with 31 negative ion pattern metabolites up-regulated and
40  negative  ion  pattern  metabolites  down-regulated  in  SV-PVAT  compared  to CA-PVAT.  36  positive  ion  pattern  metabolites  were   significantly   different   in SV-PVAT versus CA-PVAT, with 21 positive ion pattern metabolites up-regulated and
15  positive  ion  pattern  metabolites  down-regulated  in   SV-PVAT  compared  to CA-PVAT. These differential metabolites were mainly related to amino acid sugar and nucleotide   sugar   metabolism,    taurine   and    low   taurine    metabolism,   butyrate metabolism,  glutathione  metabolism,  and  α-linolenic  acid  metabolism.  Multiple metabolites  are  involved  in  the  glycerophospholipid  metabolic  pathway  in  both CA-PVAT and SV-PVAT.

3.  Combined  transcriptomic  and  metabolomic  analyses  identified  79  co-enriched pathways. The metabolites such as 13(S)-hydroxyoctadecatrienoic acid, caprylic acid, palmitoleic      acid,       phosphatidylinositol       36:4,      phosphatidylglycol       38:4, lysophosphatidylcholine  15:0,  succinic  acid,  and  sphingosine  1-phosphate  and  the metabolic pathways such as glycerophospholipid metabolism, fatty acid biosynthesis, alpha-linolenic acid metabolism, sphingosine signalling, oxidative phosphorylation, phenylalanine  metabolism  and  alanine,  aspartate  and  glutamate  metabolism  are potential metabolites and metabolic pathways of SV-PVAT to inhibit atherosclerosis, respectively.

CONCLUSIONS: SV-PVAT and CA-PVAT are significantly heterogeneous in gene expression patterns and metabolite profiles. In particular, the heterogeneity of gene expression related to inflammation, lipid metabolism, and myocardial processes as well as metabolites related to amino acid metabolism, lipid metabolism, and energy metabolism between SV-PVAT and CA-PVAT in patients with coronary artery disease suggests that the potential mechanisms of atherosclerosis inhibition by SV-PVAT are related to  inflammatory responses,  lipid metabolism,  amino  acid  metabolism,  and energy metabolism.

 

                                        Part III

                                      Abstract

BACKGROUND:  Perivascular  adipose  tissue  can  regulate  vascular  function  and structure by secreting bioactive substances. Studies have shown that thoracic adipose tissue secretes ceramides, the latter of which cause oxidative stress damage to the vasculature  and  are  associated  with  poor  prognosis  in  obese  patients.  Coronary perivascular  adipose  tissue  is  associated  with   coronary  atherosclerosis,  vascular inflammation and cytokine imbalance. Clinical studies have shown that preservation of saphenous vein perivascular adipose tissue increases the patency of saphenous vein grafts in coronary artery bypass grafting. We hypothesised that the different effects of coronary perivascular adipose tissue and saphenous vein perivascular adipose tissue on the vasculature may be caused by differences in ceramide composition. Therefore, the aim of this study was to compare whether saphenous vein perivascular adipose tissue differs from other perivascular adipose tissue in terms of ceramide composition.

METHODS: In this study, the ceramide profiles of chest subcutaneous adipose tissue, saphenous vein perivascular adipose tissue, and coronary artery perivascular adipose  tissue samples from 30 patients who underwent coronary artery bypass grafting were  examined  using  targeted  metabolomics.  A  linear  regression  model  was  used  to  evaluate the correlation between adipose tissue ceramides and cardiovascular disease  risk factors.

RESULTS: Fifteen ceramides were detected in chest subcutaneous adipose tissue, perivascular  adipose  tissue  of the  great  saphenous  vein,  and  perivascular  adipose tissue  of  the   coronary  arteries.  Twelve  ceramides  were  significantly  lower  in saphenous   vein   perivascular    adipose    tissue   compared    with   coronary    artery perivascular adipose tissue. Compared with subcutaneous adipose tissue, saphenous vein perivascular adipose tissue had decreased levels of 5 ceramides and increased levels of 2 ceramides. Multiple ceramides in coronary perivascular adipose tissue were significantly associated with cardiovascular disease risk factors.

CONCLUSION: Significant differences in the ceramide composition of perivascular adipose tissue at different sites in patients  with coronary artery disease were found in this  study,  which  helps  to  explain  differences  in  susceptibility  to  atherosclerotic plaques.  Therapeutic   strategies  to  improve  perivascular   adipose  tissue   ceramide profiles may be beneficial for patients with coronary artery disease.