第一部分 实验性肺动脉高压大鼠模型心室结构和功能变化及右心室适应性的性别差异：心脏磁共振成像研究

 

研究目的：

肺动脉高压是一种以肺血管重塑和阻力增加为特征的严重疾病，最终可导致右心室衰竭，并影响患者生存。肺动脉高压的发病率和右心室的适应性在不同性别之间存在差异，但这些差异的机制尚未完全明确。因此，本研究旨在使用Sugen-低氧大鼠模型，结合心脏磁共振成像，深入分析肺动脉高压对心室结构和功能的影响，并进一步探讨性别差异的生理基础。

 

研究方法：

使用不同性别的Sprague Dawley大鼠建立Sugen-低氧大鼠模型，然后对正常对照组和肺动脉高压组大鼠进行右心导管检查和心脏磁共振成像评估，测量心室射血分数、心室舒张末期容积、心室质量等心脏功能和结构参数，并结合右心室收缩压分析性别对右心室适应性的影响。

 

研究结果：

肺动脉高压大鼠模型的右心室出现了显著的适应性改变，表现为右心室壁增厚、右心室扩张、射血分数下降等，提示右心室功能损伤。左心室参数与正常对照组相比无明显改变，但肺动脉高压大鼠出现了显著的室间隔移位，并表现出性别差异。雌性肺动脉高压大鼠的右心室损伤更为严重，右心室射血分数、心脏指数等参数显著低于雄性大鼠，且室间隔移位更加严重。相关分析发现，右心室收缩压显著影响右心室适应性参数，而性别本身与右心室功能的差异无显著关联。

 

研究结论：

在实验性肺动脉高压模型中，肺动脉高压导致大鼠右心室结构和功能的显著变化，雌性大鼠的右心室适应性受损更加严重，右心室适应性的性别差异主要是由于雌性大鼠更严重的肺血管病变导致更高的肺动脉压力和右室后负荷，而性别并非决定右心室适应性的重要因素。这些结果为肺动脉高压及右心室适应性的性别差异机制提供了实验依据，同时为今后基于性别差异制定肺动脉高压个体化治疗策略奠定了研究基础。

 

第二部分 骨形态发生蛋白/转化生长因子-β信号通路失衡通过驱动激活蛋白-1异常表达促进肺动脉高压的分子机制研究

 

研究目的：

肺动脉高压是一种严重的肺血管疾病，现有的治疗主要为血管扩张剂，对血管内皮细胞和平滑肌细胞的异常增殖影响有限。骨形态发生蛋白（Bone morphogenetic protein，BMP）/转化生长因子-β（Transforming growth factor-β，TGF-β）超家族信号失衡是导致肺动脉高压中血管细胞增殖失调的主要原因。本研究探讨了BMP/TGF-β信号通路的失衡如何通过驱动激活蛋白-1（Activating protein-1，AP-1）异常表达，进而促进肺动脉高压发生与发展的分子机制。

 

研究方法：

首先检测了肺动脉高压样本与正常对照中AP-1表达水平的差异，然后使用多西环素诱导的短发夹RNA稳定敲低骨形态发生蛋白II型受体（Bone morphogenetic protein receptor type 2，BMPR2）内皮集落形成细胞模型，模拟肺动脉高压的BMPR2低表达状态，通过外源性添加骨形态发生蛋白9（Bone morphogenetic protein 9，BMP9）、激活素A（Activin A）、肿瘤坏死因子-α（Tumor necrosis factor-α，TNFα）等配体，采用实时荧光定量PCR检测BMP/TGF-β信号通路经典途径下游靶基因及AP-1转录因子家族成员的mRNA表达变化。在此基础上，进一步评估了TNFα预处理的炎症环境对BMP/TGF-β信号通路配体调控作用的影响，比较了经典通路的激活与AP-1转录因子在有无TNFα预处理条件下的差异。

 

研究结果：

转录因子AP-1在肺动脉高压及相关致病因子刺激下表达上调。BMP9除了通过经典BMP信号通路调控下游靶基因外，还能诱导激活素A表达上调，进而部分介导TGF-β信号分支激活，并通过BMPR2依赖的非经典信号转导途径如AP-1成员cFOS实现下游基因转录调控。Activin A主要诱导TGF-β信号分支下游效应分子纤溶酶原激活剂抑制因子-1（Plasminogen activator inhibitor type 1，PAI-1）的表达，而BMPR2基因敲低可削弱其诱导效应。在TNFα预处理的炎症病理条件下，BMPR2表达进一步下调，导致BMP经典信号传导降低，同时BMP9对TGF-β信号分支的串扰显著增强，表现为Activin A和PAI-1的显著表达升高，此外，AP-1关键组分cFOS表达被激活，但BMPR2敲低可消除这种激活效应。

 

研究结论：

BMP9除了通过经典BMP信号分支发挥生物学效应，还可通过上调Activin A与TGF-β信号分支形成串扰，并经由BMPR2依赖的AP-1非经典途径调控下游基因的表达。在炎症条件下，BMPR2表达减少，BMP分支信号下降，BMP/TGF-β信号通路进一步失衡，BMP9进一步激活TGF-β分支及AP-1通路，加剧病理性血管重塑及内皮功能障碍。这些结果从分子层面阐释了BMP9在肺动脉高压中矛盾性作用的机制，也为基于BMP/TGF-β信号及AP-1干预的治疗策略奠定理论基础。

 

 

第三部分 组织蛋白酶B介导的激活蛋白-1活化在心肌纤维化中的作用及机制研究

 

研究目的：

心肌纤维化是多种心血管疾病进展过程中的关键病理环节，主要表现为成纤维细胞的异常活化和细胞外基质过度沉积，最终导致心脏结构与功能的显著受损。转录因子激活蛋白-1（Activating protein-1，AP-1）在多种病理性纤维化中具有重要作用，但其在心肌纤维化中的具体调控机制仍不清楚。本研究旨在探讨组织蛋白酶B（Cathepsin B，Ctsb）介导的AP-1激活在心肌纤维化中的作用及机制，为心肌纤维化干预策略提供新的思路与靶点。

 

研究方法：

首先建立多柔比星诱导的小鼠心肌纤维化模型，然后利用单细胞转录组测序技术检测模型组与对照组心脏组织的非心肌细胞，通过细胞分型与基因表达分析，重点研究参与心肌纤维化和产生细胞外基质的细胞类型。使用亚群分析描述成纤维细胞不同亚群的特征，然后通过RNA速率和拟时序分析揭示成纤维细胞的激活过程及相关基因表达变化。最后，通过免疫荧光染色与和实时荧光定量PCR进一步验证Ctsb介导的AP-1激活在心肌纤维化中的作用。

 

研究结果：

多柔比星处理后的小鼠出现明显的心肌损伤与纤维化，病理学分析可见细胞外基质的显著增生和胶原沉积。单细胞测序结果显示，心肌纤维化过程中细胞外基质的主要来源为成纤维细胞，拟时序分析揭示了成纤维细胞从静止状态向肌成纤维细胞的分化轨迹。Ctsb和多个AP-1转录因子家族成员在多柔比星处理组的成纤维细胞中表达上调，Ctsb可能通过调控AP-1相关信号通路，促进细胞外基质过度沉积与纤维化进程，在多柔比星诱导的心肌纤维化中发挥关键作用。

 

研究结论：

通过单细胞转录组测序的方法，本研究系统地构建了多柔比星诱导的心肌纤维化的非心肌细胞基因表达图谱，深入解析了细胞外基质的来源及成纤维细胞在纤维化过程中的分化轨迹。Ctsb在基质纤维细胞和肌成纤维细胞中显著高表达，且可能通过调控AP-1转录因子促进心肌纤维化的发生与发展。这些发现不仅深化了对心肌纤维化细胞与分子机制的认识，也为心肌纤维化的临床研究提供了重要的分子基础和潜在治疗靶点。

 

 

Part One: Sex Differences in Ventricular Structural and Functional Changes and Right Ventricular Adaptation in an Experimental Pulmonary Arterial Hypertension Rat Model: A Cardiac Magnetic Resonance Imaging Study

 

Aims:

Pulmonary arterial hypertension is a severe disease characterized by pulmonary vascular remodeling and increased resistance, ultimately leading to right ventricular failure and affecting patient survival. The incidence of pulmonary arterial hypertension and right ventricular adaptation differs between sexes, but the mechanisms underlying these differences remain unclear. Therefore, this study aims to utilize the Sugen-hypoxia rat model in combination with cardiac magnetic resonance imaging to comprehensively analyze the effects of pulmonary arterial hypertension on ventricular structure and function and further explore the physiological basis of sex differences.

 

Methods:

Male and female Sprague Dawley rats were used to establish the Sugen-hypoxia pulmonary arterial hypertension model. Right heart catheterization and cardiac magnetic resonance imaging were performed on both normal control and pulmonary arterial hypertension groups. Parameters such as ventricular ejection fraction, end-diastolic volume, and ventricular mass were measured to assess cardiac function and structural changes. Additionally, right ventricular systolic pressure was analyzed to determine the impact of sex on right ventricular adaptation.

 

Results:

The pulmonary arterial hypertension rat model exhibited significant adaptive changes in the right ventricle, including right ventricular wall thickening, right ventricular dilation, and a decrease in ejection fraction, indicating right ventricle functional impairment. Left ventricular parameters showed no significant changes compared to the control group. However, pulmonary arterial hypertension rats exhibited notable septal displacement with significant sex differences. Female pulmonary arterial hypertension rats experienced more severe right ventricular damage, with significantly lower right ventricular ejection fraction and cardiac index compared to male rats, along with more significant septal displacement. Correlation analysis revealed that right ventricular systolic pressure had a significant impact on RV adaptation parameters, whereas sex itself was not directly associated with RV functional differences.

 

Conclusion:

In the experimental pulmonary arterial hypertension model, pulmonary arterial hypertension leads to significant changes in right ventricular structure and function. Female rats exhibit more severe impairment in right ventricular adaptation, primarily due to more severe pulmonary vascular lesions, resulting in higher pulmonary arterial pressure and increased right ventricle afterload. However, sex itself is not a decisive factor in right ventricular adaptation. These findings provide experimental evidence for the mechanisms underlying sex differences in pulmonary arterial hypertension and right ventricular adaptation, laying the foundation for future research on individualized pulmonary arterial hypertension treatment strategies based on sex differences.

 

Part Two: Molecular Mechanism of Bone Morphogenetic Protein/ Transforming Growth Factor-β Signaling Imbalance Driving Activator Protein-1 Dysregulation Leading to Pulmonary Arterial Hypertension

 

Aims:

Pulmonary arterial hypertension is a severe pulmonary vascular disease. Current treatments mainly focus on vasodilators, which have limited effects on the abnormal proliferation of endothelial and smooth muscle cells. The imbalance of the bone morphogenetic protein (BMP)/transforming growth factor-β (TGF-β) superfamily signaling pathway is a key driver of vascular cell proliferation dysregulation in PAH. This study investigates how the imbalance in BMP/TGF-β signaling promotes the development and progression of pulmonary arterial hypertension through aberrant expression of activating protein-1 (AP-1).

 

Methods:

First, the expression levels of AP-1 were analyzed in pulmonary arterial hypertension samples and normal controls. A doxycycline-inducible short hairpin RNA stable knockdown model targeting bone morphogenetic protein receptor type 2 (BMPR2) in endothelial colony-forming cells was established to mimic the low BMPR2 expression state in pulmonary arterial hypertension. Exogenous ligands, including bone morphogenetic protein 9 (BMP9), activin A, and tumor necrosis factor-α (TNFα), were used to stimulate cells. Real-time quantitative PCR was employed to assess changes in the mRNA expression of downstream target genes in the BMP/TGF-β signaling canonical pathway and members of the AP-1 transcription factor family. Furthermore, the effect of TNFα-induced inflammatory conditions on the regulatory role of BMP/TGF-β pathway ligands was evaluated. The differences in canonical pathway activation and AP-1 transcription factor expression were compared with and without TNFα pre-treatment.

 

Results:

AP-1 transcription factor expression was upregulated in pulmonary arterial hypertension and under stimulation by related pathogenic factors. In addition to regulating downstream target genes via the canonical BMP signaling pathway, BMP9 induced the upregulation of activin A, partially mediating the activation of the TGF-β signaling branch. This process involved BMPR2-dependent non-canonical signaling pathways, such as AP-1 member cFOS, which mediated downstream gene transcription. Activin A primarily induced the expression of the TGF-β downstream effector molecule plasminogen activator inhibitor type 1 (PAI-1), while BMPR2 knockdown weakened this induction effect. Under inflammatory conditions with TNFα pre-treatment, BMPR2 expression was further downregulated, leading to a decrease in canonical BMP signaling. Meanwhile, BMP9-mediated crosstalk with the TGF-β signaling branch was significantly enhanced, as evidenced by the elevated expression of activin A and PAI-1. Additionally, the key AP-1 component cFOS was activated, but its activation was abolished by BMPR2 knockdown.

 

Conclusion:

In addition to its role in the classical BMP signaling branch, BMP9 exerts biological effects by upregulating activin A, thereby enhancing crosstalk with the TGF-β signaling branch. Through BMPR2-dependent non-classical AP-1 pathways, BMP9 regulates downstream gene expression. Under inflammatory conditions, reduced BMPR2 expression leads to further BMP/TGF-β signaling imbalance, decreased BMP branch signaling, and enhanced activation of the TGF-β and AP-1 pathways by BMP9. This exacerbates pathological vascular remodeling and endothelial dysfunction. These findings elucidate the molecular mechanisms underlying the paradoxical role of BMP9 in pulmonary arterial hypertension and provide a theoretical foundation for therapeutic strategies targeting BMP/TGF-β signaling and AP-1.

 

 

Part Three: Mechanism of Cathepsin B Mediated Activator Protein-1 Activation in Myocardial Fibrosis

 

Aims:

Myocardial fibrosis is a critical pathological process in the progression of various cardiovascular diseases, characterized by abnormal activation of fibroblasts and excessive extracellular matrix deposition, ultimately leading to significant structural and functional impairment of the heart. The transcription factor activating protein-1 (AP-1) plays an important role in pathological fibrosis; however, its specific regulatory mechanisms in myocardial fibrosis remain unclear. This study aims to investigate the role and mechanism of cathepsin B (Ctsb)-mediated AP-1 activation in myocardial fibrosis, providing new insights and potential therapeutic targets for fibrosis intervention.

 

Methods:

A doxorubicin-induced mouse model of myocardial fibrosis was established. Single-cell RNA sequencing was used to analyze non-cardiomyocyte populations in cardiac tissue from both the model and control groups. Cell classification and gene expression profiling were performed to identify key cell types involved in myocardial fibrosis and extracellular matrix production. Subcluster analysis was conducted to characterize different fibroblast subclusters, followed by RNA velocity and pseudotime trajectory analysis to reveal fibroblast activation processes and associated gene expression changes. Finally, immunofluorescence staining and real-time quantitative PCR were used to validate the role of Ctsb-mediated AP-1 activation in myocardial fibrosis.

 

Results:

Doxorubicin-treated mice exhibited significant myocardial injury and fibrosis, as evidenced by histological analysis showing substantial extracellular matrix proliferation and collagen deposition. Single-cell sequencing revealed that fibroblasts were the primary source of extracellular matrix in myocardial fibrosis. Pseudotime analysis delineated the differentiation trajectory of fibroblasts from a quiescent state to activated myofibroblasts. Ctsb and multiple AP-1 transcription factor family members were upregulated in fibroblasts from the doxorubicin-treated group. Ctsb is likely to promote excessive extracellular matrix deposition and fibrosis progression by regulating AP-1-associated signaling pathways, playing a critical role in doxorubicin-induced myocardial fibrosis.

 

Conclusion:

Using single-cell RNA sequencing, this study systematically mapped the non-cardiomyocyte gene expression landscape in doxorubicin-induced myocardial fibrosis, providing a comprehensive analysis of extracellular matrix sources and fibroblast differentiation trajectories. Ctsb was significantly upregulated in matrix fibroblasts and myofibroblasts, potentially promoting myocardial fibrosis by regulating AP-1 transcription factors. These findings not only enhance the understanding of cellular and molecular mechanisms underlying myocardial fibrosis but also provide crucial molecular insights and potential therapeutic targets for clinical research on myocardial fibrosis.