In the current issue of Acta Physiologica, Li and colleagues¹ demonstrate that the thromboxane‐prostanoid receptor (TP receptor) is the culprit for vascular smooth muscle cell (VSMC) transformation from a contractile (differentiated) to the synthetic (dedifferentiated) phenotype by prostacyclin when the prostacyclin receptor (IP receptor) is deficient. Through a wide array of convincing experiments, including transcriptome profiling and network analysis, as well as pharmacological studies in cultured VSMCs, they conclude that IP receptor deficiency per se results in increased expression of membrane TP receptors, which can be activated by prostacyclin with the consequent VSMC phenotypic transformation through activation of RhoA/ROCKs, MEK1/2 and JNK.

Actually, the role of TP receptor mediating the detrimental vascular effects of prostacyclin under pathological conditions, such as hyperlipidaemia and hypertension, has been reported previously. Under normal conditions, prostacyclin activates IP receptors exerting vascular beneficial effects including vasodilatation, inhibition of platelet activation and inhibition of VSMC phenotypic changes. The activation of TP receptors, leading to the opposite vascular effects, mainly occurs with thromboxane A₂, although prostacyclin when present at large amounts can also activate TP receptors (Figure 1). The presence of large amounts of prostacyclin in the intercellular space is likely the consequence of (1) upregulation of cyclooxygenase and prostacyclin synthase in endothelial cells, as demonstrated in spontaneously hypertensive rats²; and (2) impaired IP receptor‐mediated nitric oxide production, as demonstrated in hyperlipidaemic mice³ leading to a reduced negative feedback control on endothelial prostacyclin production (Figure 1). Therefore, activation of TP receptors by prostacyclin can be the result of molecular and/or functional changes in endothelial cells, accounting for the occurrence of endothelium‐dependent contractions, a characteristic of endothelial dysfunction associated with cardiovascular diseases.⁴

Clearly, prostacyclin being shifted to activate TP receptors can also be the result of smooth muscle dysfunction, associated with a reduced IP receptor responsiveness, as occurred in aortae of spontaneously hypertensive rats,⁵ or with reduced expression/function of IP receptor, as demonstrated in VSMCs with IP receptors being knocked down or mutated to a dysfunctional R212C (IP^R212C) form.¹ This phenomenon can be explained by the “yin‐yang” theory: with the favourable IP receptor‐mediated effects being impaired the harmful TP receptor signalling becomes dominated (Figure 1). By contrast, the present study by Li et al¹ and the previous one by Wilson et al⁶ elegantly demonstrate that there is a direct impact of IP receptor deficiency on membrane TP receptor expression in VSMCs. In the latter study, it is concluded that the greater level of membrane TP receptors in aortic smooth muscle cells from IP receptor‐knockout mice, compared to those from wildtype mice, is attributed to the lack of the physical association of IP and TP receptors; and that cyclic adenosine monophosphate (cAMP), the second messenger produced following IP receptor activation (Figure 1), does not play a role in regulating the levels of membrane TP receptor upregulation in VSMCs. However, Li and colleagues¹ argue that the physical association of IP and TP receptors is unlikely responsible for the increased membrane TP receptor level, based on their findings showing that (1) the level of membrane TP receptors was higher not only in VSMCs with IP receptor downregulation (by silencing RNA) but also in VSMCs transfected with IP^R212C receptors which, like the wildtype IP receptors, form heterodimer with TP receptors⁷; and that (2) IP receptor‐knockdown per se did not affect the cytosolic or the total cellular level of TP receptor protein in rat aortic VSMCs. Instead, they propose that the reduced cAMP production accounts for the reduced cytosolic TP receptor level, and hence increased membrane TP receptor level, in cells with IP receptor‐knockdown, compared with those without the knockdown, because the reduced cytosolic TP receptor level was observed only after iloprost exposure.¹ While their proposal is the logical deduction, it does not explain for the higher membrane TP receptor expression in VSMCs with IP receptor deficiency under basal condition (without iloprost stimulation and with comparable cAMP level).¹ Therefore, the mechanism underlying the redistribution of TP receptor from cytoplasm to the membrane has not been fully explored: it is possible that, in addition to the intracellular cAMP level, the ratio of functional IP to TP receptors (which is reduced by IP receptor downregulation or IP^R212C receptor overexpression, despite the latter still permitting physical association with TP receptors⁷) also determines the trafficking of the heterodimer (with TP receptor characteristic due to IP receptor deficiency) to the membrane.

Besides the mechanism underlying the relationship between vascular expressions of IP and TP receptors, there remains a great deal to be elucidated regarding the impact of the relationship on the vascular function. TP receptors are detected also in endothelial cells. Would endothelial IP receptor deficiency also result in increased membrane TP receptors in endothelial cells? If so, what would the consequence of endothelial TP receptor upregulation be? If a reduced cAMP production, as a result of IP receptor deficiency, accounts for the increased membrane TP receptors in VSMCs, would an increase cAMP production increase the internalization of TP receptors? Recently it was reported that the responsiveness of IP receptors is enhanced by the genetic deletion of apolipoprotein E (apoE).³ The increased IP receptor responsiveness in aortae of male apoE‐deficient mice fed a high fat diet, compared with the wild type counterparts (with same age, sex and diet), is manifested by an increased cAMP production upon exposure to IP receptor agonists; this is associated with a reduced level of membrane TP receptors in aortae with endothelium and a reduced TP receptor protein level in aortae without endotelium.³ The findings, therefore, support an inverse relationship between cAMP production and membrane TP receptor level. Despite the lower expression, contractions to the TP receptor agonist U46619 in aortae without endothelium are greater in apoE‐deficient mice fed with high fat diet than in those of the wild type counterparts.³ The dissociation between membrane expression and responsiveness of TP receptors highlights the importance in conducting functional study in addition to assays on protein expressions in order to determine the vascular impact of IP‐TP receptor interaction.

In an effort to examine the contractile activity in cultured VSMCs, Li and colleagues conducted the collagen contraction gel assay.¹ To their disappointment, they found out that, even using the well‐established contracting agents [including receptor‐dependent (phenylephrine and angiotensin II causing inositol phosphate production to trigger calcium release from intracellular stores) and ‐independent vasoconstrictors (potassium chloride causing depolarization to trigger calcium influx)], a sensitive measurement of contractile activity could not be detected. It was reported that phenotypic changes from quiescent (contractile) to proliferative (synthetic) state occurs with cell passage and with increasing time following cell seeding during the culturing of VSMCs. The “pro‐synthetic” status of cultured VSMCs may account for the unsuccessful detection of contractile activity using the collagen contraction gel assay. Nevertheless, Li and colleagues¹ clearly demonstrate that only in VSMCs with deficient/dysfunctional IP receptors the IP receptor agonist iloprost increased cell proliferation, a response resulting from the transformation of VSMCs to the synthetic phenotype, thereby substantiating the results based on changes in the levels of contractile proteins (smooth muscle myosin heavy chain) and synthetic proteins (osteopontin and proliferation cell nuclear antigen). Moreover these iloprost‐induced effects in VSMCs with deficient/dysfunctional IP receptors were associated with RhoA activation and were prevented with genetic/pharmacological inhibition of TP receptor, thus confirming that the deficiency/dysfunction of IP receptors results in IP receptor agonist activating TP receptor to induce/enhance the conversion of VSMCs to the synthetic phenotype.

The findings of Li and colleagues¹ provide an explanation for the clinical observation of an accelerated atherothrombosis in patients with a dysfunctional IP^R212C receptor⁸ and the association of the presence of IP^R212C mutation with intimal hyperplasia or thrombotic episodes.⁹ Therefore, they provide scientific evidence for the use of TP receptor antagonist to retard the progression of atherothrombotic diseases, especially in patients with genetic variant (R212C mutant) of the IP receptor. Their findings also raise a concern regarding the clinical use of IP receptor agonists, which are the therapy of choice for the management of pulmonary arterial hypertension, a progressive disease with high morbidity and mortality characterized by pulmonary vasoconstriction and vascular remodelling.¹⁰ If the increased expression of membrane TP receptors by IP receptor deficiency/dysfunction also occurs in pulmonary vascular smooth muscle cell, not only would IP receptor agonists be ineffective but they, by acting on TP receptors promoting contractile to synthetic phenotype transformation in smooth muscle cells, would also exacerbate the progression of pulmonary arterial hypertension in patients with dysfunctional (for example, the R212C mutant variant) IP receptors. Would TP receptor antagonist be particularly useful in this group of patients? Should genotyping be required before the initiation of IP receptor agonists in patients with pulmonary arterial hypertension? The study of Li and colleagues, by highlighting the interactions between IP and TP receptors and the consequent impact on vascular remodelling, thus has implications not only in the progression of atherothrombotic diseases but also in the clinical use of IP receptor agonists in the management of pulmonary arterial hypertension.

在本期《生理学报》中，Li及其同事¹证明了血栓烷-前列腺素受体（TP受体）是前列环素从收缩（分化）表型向合成（去分化）表型转化为血管平滑肌细胞（VSMC）的罪魁祸首。当前列环素受体（IP受体）不足时。通过一系列令人信服的实验，包括转录组谱分析和网络分析，以及在培养的VSMC中进行的药理研究，他们得出结论，IP受体本身的缺乏会导致膜TP受体的表达增加，其可以被前列环素激活，因此通过激活RhoA / ROCKs，MEK1 / 2和JNK进行VSMC表型转化。

实际上，先前已经报道了TP受体在诸如高脂血症和高血压的病理条件下介导前列环素的有害血管作用的作用。在正常情况下，前列环素激活IP受体，发挥血管有益作用，包括血管舒张，抑制血小板活化和抑制VSMC表型改变。TP受体的激活导致相反的血管作用，主要发生在血栓烷A _2中，尽管前列环素大量存在时也会激活TP受体（图1）。自发性高血压大鼠²在内皮细胞中存在大量前列环素可能是由于以下因素的结果：（1）内皮细胞中环氧合酶和前列环素合酶的上调²；（2）IP受体介导的一氧化氮产生受损，如高脂血症小鼠^3所示导致减少对内皮前列环素产生的负反馈控制（图1）。因此，前列环素对TP受体的激活可能是内皮细胞分子和/或功能改变的结果，这说明了内皮依赖性收缩的发生，这是与心血管疾病有关的内皮功能障碍的特征。⁴

显然，前列环素转移到激活TP受体也可能是平滑肌功能障碍的结果，与自发性高血压大鼠主动脉⁵发生的IP受体反应性降低有关，或IP受体的表达/功能降低，如具有IP受体的VSMC被敲低或突变为功能异常的R212C（IP ^R212C）形式。¹这种现象可以用“阴阳”理论来解释：随着良好的IP受体介导的作用受损，有害的TP受体信号转导成为主导（图1）。相比之下，Li等人^1的本研究，Wilson等人⁶的前一项研究优雅地证明了IP受体缺乏对VSMC中膜TP受体表达有直接影响。在后者的研究中，得出的结论是，与野生型小鼠相比，IP受体敲除小鼠的主动脉平滑肌细胞中的膜TP受体水平更高，这归因于IP和TP受体缺乏物理联系。环磷酸腺苷（cAMP）是IP受体激活后产生的第二个信使（图1），在调节VSMC中膜TP受体上调的水平中不起作用。但是，李和同事¹他们的发现表明，IP和TP受体的物理联系不太可能导致膜TP受体水平的升高（1）不仅在IP受体下调的VSMC中，膜TP受体的水平更高（通过沉默RNA） ），也可以在转染了IP ^R212C受体的VSMC中使用，该受体像野生型IP受体一样与TP受体形成异二聚体⁷; （2）IP受体的敲除本身并不影响大鼠主动脉VSMC的胞质或TP受体蛋白的总细胞水平。相反，他们提出，与没有敲除的细胞相比，具有IP受体敲低的细胞中cAMP产量的减少是由于胞浆TP受体水平的降低，因此膜TP受体水平的增加是因为观察到了胞浆TP受体水平的降低。仅在服用伊洛前列素后。¹尽管他们的建议是合理的推论，但它不能解释在基础条件下（无伊洛前列素刺激且cAMP水平可比）具有IP受体缺乏的VSMC中更高的膜TP受体表达。^1个因此，尚未完全探讨TP受体从细胞质到膜的重新分布的潜在机制：除了细胞内cAMP水平外，功能性IP与TP受体的比例可能会降低（由于IP受体下调而降低）或IP ^R212C受体过表达，尽管后者仍允许与TP受体发生物理缔合⁷）也决定了异二聚体（由于IP受体缺乏而具有TP受体特征）向膜的运输。

除了IP和TP受体的血管表达之间关系的潜在机制外，关于该关系对血管功能的影响还有很多要阐明的地方。在内皮细胞中也检测到TP受体。内皮细胞IP受体缺乏还会导致内皮细胞膜TP受体增加吗？如果是这样，内皮TP受体上调的后果将是什么？如果由于IP受体缺乏而导致cAMP产量减少导致VSMC中膜TP受体增加，那么cAMP产量的增加是否会增加TP受体的内在化？最近有报道说，载脂蛋白E（apoE）的基因缺失增强了IP受体的反应能力。³与野生型（年龄，性别和饮食相同）相比，高脂饮食的雄性apoE缺陷型雄性小鼠主动脉IP受体反应性增强，这通过暴露于IP受体激动剂后cAMP产生得以体现。这与具有内皮的主动脉中的膜TP受体水平降低和不具有内皮的主动脉中的TP受体蛋白水平降低有关。³因此，这些发现支持了cAMP产生与膜TP受体水平之间的反比关系。尽管表达较低，但是在饲喂高脂饮食的apoE缺陷型小鼠中，无内皮的主动脉中TP受体激动剂U46619的收缩比野生型小鼠更大。³ 膜表达与TP受体反应性之间的分离突出了进行功能研究的重要性，此外还进行了蛋白表达测定以确定IP-TP受体相互作用对血管的影响。

为了检查培养的VSMC中的收缩活性，Li及其同事进行了胶原蛋白收缩凝胶测定。^1个令他们失望的是，他们发现，即使使用成熟的收缩剂（包括依赖受体的（苯肾上腺素和血管紧张素II引起肌醇磷酸的产生触发从细胞内存储中释放钙）和独立于血管收缩剂（氯化钾引起去极化触发钙涌入）]，则无法检测到敏感的收缩活动测量值。据报道，在VSMC培养过程中，随着细胞的传代和细胞接种后时间的增加，表型从静止（收缩）状态向增殖（合成）状态变化。培养的VSMC的“合成前”状态可能说明使用胶原蛋白收缩凝胶测定法未能成功检测到收缩活性。不过，李和同事¹清楚地表明，只有在IP受体功能不足/功能失调的VSMC中，IP受体激动剂iloprost才能增加细胞增殖，这是VSMC转化为合成表型所引起的反应，从而基于收缩蛋白（平滑肌）水平的变化证实了结果肌球蛋白重链）和合成蛋白（骨桥蛋白和增殖细胞核抗原）。此外，这些伊洛前列素在具有不足/功能失调的IP受体的VSMC中诱导的作用与RhoA激活相关，并被TP受体的遗传/药理学抑制所阻止，从而证实IP受体的缺乏/功能失调导致IP受体激动剂激活TP受体。诱导/增强VSMC向合成表型的转化。

Li等[ ^1]的发现为IP ^R212C受体功能障碍^8的患者加速动脉粥样硬化以及IP ^R212C突变与内膜增生或血栓形成的关联提供了解释。⁹因此，它们为使用TP受体拮抗剂来延缓动脉粥样硬化性疾病的进展提供了科学依据，特别是在IP受体具有遗传变异（R212C突变）的患者中。他们的发现也引起人们对IP受体激动剂临床应用的关注，IP受体激动剂是治疗肺动脉高压的一种选择疗法，这是一种以肺血管收缩和血管重塑为特征的高发病率和高死亡率的进行性疾病。¹⁰如果在肺血管平滑肌细胞中还发生了IP受体缺乏/功能障碍引起的膜TP受体表达增加，那么IP受体激动剂不仅无效，而且它们通过作用于TP受体而在平滑肌细胞中促进从收缩到合成表型转化，也将加重IP受体功能异常（例如R212C突变体变体）患者的肺动脉高压的进展。TP受体拮抗剂在这类患者中是否特别有用？肺动脉高压患者在启动IP受体激动剂之前是否应该进行基因分型？Li和同事的研究通过强调IP和TP受体之间的相互作用及其对血管重塑的影响，