This study by Zhang et al entitled ‘Keratin 1 attenuates hypoxic pulmonary artery hypertension through suppressing pulmonary artery media smooth muscle expansion’ is presented in a recent edition of Acta Physiologica.¹ Leading up to this research were at least three key ideas: (a) that keratin 1 (Krt1) is considered the gatekeeper of the skin's innate immune response for its important regulatory role of both local and systemic inflammation,² (b) that inflammation is central to the pathogenesis of pulmonary arterial hypertension (PAH) ³ and (c) that keratins can promote apoptosis of smooth muscle cells derived from human atherosclerotic lesions.⁴ Therefore, the authors hypothesized that Krt1 regulates abnormal pulmonary arterial smooth muscle cell (PASMC) growth that occurs in vascular remodelling indicative of PAH. These data are the first and only to inversely relate Krt1 expression and function with PAH and compel the reader to take a fresh look at the keratin family, specifically Krt1, with regard to regulation of, and as a potential treatment for, PAH.

Utilizing primary cells, including pulmonary arterial endothelial cells, smooth muscle cells (PASMC), and fibroblasts from digested rat lungs, hypoxia reduced Krt1 expression and activity most significantly in PASMC. Further investigation in PASMC revealed that Krt1 knockdown resulted in accelerated cell proliferation, migration, transformation from contractile to synthetic phenotype and activation of TLR7 and NF‐ĸB, all of which were reversed by Krt1 overexpression. In vivo experiments in two PAH rat models, hypoxia and monocrotaline (MCT), determined that Krt1 improved RV function, as measured by right ventricular systolic pressure (RVSP) and right heart hypertrophy (as measured by Fulton's index), and alleviated pulmonary vascular remodelling. Krt1 expression was negatively correlated with RVSP and Fulton's index. In both models of PH, overexpression of the Krt1 gene using AAV6 vectors administered by tracheal injection resulted in reduced RVSP and Fulton's index, improved cardiac function, decreased pulmonary vascular thickness and vascular collagen deposition. These results point to Krt1 as a key regulator of pulmonary vascular smooth muscle cell contractile activity and proliferation, inflammation and as a potential PAH therapy.

Zhang et al propose a novel mechanism for hypoxia‐induced PAH through Yin and Yang Factor 1 (YY1)‐mediated suppression in PASMC (figure).¹ YY1 protein is well‐known in cancer biology, having multiple regulatory functions in cellular development, differentiation and proliferation.⁵ Zhang et al report that hypoxia decreases transcription factor YY1 leading to Krt1 suppression in two ways that are consistent with their data¹: (a) the hypoxia‐mediated decrease in YY1 results in decreased YY1 binding/activation of the Krt1 promoter, suppressing Krt1 transcription, and (b) hypoxia‐mediated decreased YY1 leads to greater expression of methylase, DNA (cytosine‐5)‐methyltransferase 1 (Dnmt1), since YY1 inhibits Dnmt1 expression. Dnmt1 also contains Krt1 promoter binding sites, and methylation of the Krt1 promoter suppresses Krt1 transcription. In summary, hypoxia decreases YY1 leading to suppressed Krt1 transcription both directly, by decreased Krt1 promoter stimulation and indirectly through Dnmt1‐mediated Krt1 promoter methylation. These findings are reversed when YY1 is overexpressed, indicating a key role for YY1 as a regulator of Krt1 expression in PASMC.

Currently, it is not known how Krt1 suppression by hypoxia activates TLR7 and NF‐ĸB. TLR7 is part of the TLR family, important for activation of innate immunity and inflammation, with its main function to detect single stranded RNA in the process of pathogen recognition. A recent genetic bioinformatics study identified differential expression of the TLR7 gene in patients with idiopathic PAH compared to controls.⁶ In rat pulmonary vessels, Krt1 is suppressed by hypoxia, and TLR7 and NF‐ĸB expression as well as inflammatory factors S100a8 and S100a9 are all upregulated. This TLR7‐mediated innate immune inflammatory cascade promotes proliferation and migration of PASMC leading to vascular remodelling and PAH. Krt1 overexpression or pre‐incubation with TLR 7 inhibitor (IRS661) reversed the TLR7‐mediated inflammation.¹

Keratins were first characterized by x‐ray diffraction in horse hair, distinguishing α‐ and β‐keratins by the α‐helical or β‐sheet molecular structure of the rod domain.⁷ Currently, it is known mainly as a stable cytoskeletal element and for its role in skin diseases. Zhang et al are the first to specifically link Krt1, inflammation, pulmonary vascular remodelling, and PAH.¹ Focusing on the abnormal activation of pulmonary vascular smooth muscle cells, these studies support a role for keratins in vascular remodelling.

Overall, this is a very interesting set of comprehensive experiments looking at the mechanism in vitro and in vivo of how Krt1 protects against PAH, opening up a new field of research to further explore keratins as potential PAH therapy. Future studies will certainly focus on (a) mechanism of Krt1 activation of the TLR7‐NF‐ĸB pathway, (b) mechanism of Krt1 suppression by hypoxia, including potential post‐translational modifications of Krt1 such as formation of disulphide bonds, phosphorylation and glycosylation, (c) mechanism by which hypoxia regulates the increase in promoter methylation to inhibit Krt1 expression and to (d) explore the hypoxia‐driven Krt1 suppression in fibroblasts. G proteins, PI3K kinase, MAP kinase and Src kinase are all needed for cytokeratin expression.⁴ Transcriptional factors that are important in PAH development should be tested including STAT3, FOXM1, FOXO, NOTCH and NFAT.¹ Studies to determine if Krt1 increases endogenous pulmonary vascular endothelial nitric oxide production would be useful, since it is known that depletion of nitric oxide is correlated with an increase in Krt1 transcript.⁸ In conclusion, Krt1 is an exciting novel potential therapeutic target for PAH and these studies provide the first insight into the hypoxia‐mediated Krt1 suppression and subsequent pulmonary arterial smooth muscle cell expansion, indicative of pulmonary vascular remodelling in PAH.

张等人的这项研究题为“角蛋白 1 通过抑制肺动脉中层平滑肌扩张来减轻缺氧性肺动脉高压”，发表在最新一期的《生理学报》上。¹这项研究至少提出了三个关键观点：(a) 角蛋白 1 (Krt1) 因其对局部和全身炎症的重要调节作用而被认为是皮肤先天免疫反应的看门人，2 (b ⁾炎症角蛋白是肺动脉高压 (PAH) 发病机制的核心³，并且 (c) 角蛋白可以促进源自人类动脉粥样硬化病变的平滑肌细胞的凋亡。⁴因此，作者假设 Krt1 调节异常肺动脉平滑肌细胞 (PASMC) 生长，这种生长发生在表明 PAH 的血管重塑中。这些数据是第一个也是唯一一个将 Krt1 表达和功能与 PAH 反向相关的数据，并迫使读者重新审视角蛋白家族，特别是 Krt1，关于 PAH 的调节和潜在治疗。

利用原代细胞，包括肺动脉内皮细胞、平滑肌细胞 (PASMC) 和来自消化的大鼠肺部的成纤维细胞，缺氧可最显着地降低 PASMC 中的 Krt1 表达和活性。对 PASMC 的进一步研究表明，Krt1 敲低导致细胞增殖、迁移、从收缩表型向合成表型的转化加速以及 TLR7 和 NF-ĸB 的激活，所有这些都可以通过 Krt1 过表达来逆转。在缺氧和野百合碱 (MCT) 两种 PAH 大鼠模型中进行的体内实验确定，Krt1 可改善右心室收缩压 (RVSP) 测量的右心室功能和富尔顿指数测量的右心肥大，并减轻肺血管重塑。Krt1表达与RVSP和Fulton指数呈负相关。在两种 PH 模型中，通过气管注射施用 AAV6 载体过度表达 Krt1 基因，导致 RVSP 和 Fulton 指数降低，改善心脏功能，减少肺血管厚度和血管胶原沉积。这些结果表明 Krt1 是肺血管平滑肌细胞收缩活性和增殖、炎症的关键调节因子，并且是一种潜在的 PAH 治疗方法。

张等人提出了一种通过 PASMC 中阴阳因子 1 (YY1) 介导的抑制来抑制缺氧诱导 PAH 的新机制（图）。¹ YY1 蛋白在癌症生物学中众所周知，在细胞发育、分化和增殖中具有多种调节功能。⁵张等人报道，缺氧会降低转录因子 YY1，从而以两种方式抑制 Krt1，这与他们的数据¹一致：(a) 缺氧介导的 YY1 减少导致 YY1 与 Krt1 启动子的结合/激活减少，从而抑制 Krt1 (b) 缺氧介导的 YY1 减少导致甲基化酶、DNA (胞嘧啶-5)-甲基转移酶 1 (Dnmt1) 的表达增加，因为 YY1 抑制 Dnmt1 表达。Dnmt1 还包含 Krt1 启动子结合位点，Krt1 启动子的甲基化会抑制 Krt1 转录。总之，缺氧会降低 YY1，从而通过减少 Krt1 启动子刺激直接抑制 Krt1 转录，并通过 Dnmt1 介导的 Krt1 启动子甲基化间接抑制 Krt1 转录。当 YY1 过表达时，这些发现发生逆转，表明 YY1 作为 PASMC 中 Krt1 表达的调节因子发挥着关键作用。

目前，尚不清楚缺氧抑制 Krt1 如何激活 TLR7 和 NF-ĸB。TLR7是TLR家族的一部分，对于先天免疫和炎症的激活很重要，其主要功能是在病原体识别过程中检测单链RNA。最近的一项遗传生物信息学研究发现，与对照组相比，特发性 PAH 患者的 TLR7 基因表达存在差异。⁶在大鼠肺血管中，Krt1 因缺氧而受到抑制，TLR7 和 NF-ĸB 表达以及炎症因子 S100a8 和 S100a9 均上调。这种 TLR7 介导的先天免疫炎症级联反应促进 PASMC 的增殖和迁移，导致血管重塑和 PAH。Krt1 过表达或与 TLR 7 抑制剂 (IRS661) 预孵育可逆转 TLR7 介导的炎症。¹

角蛋白首先通过马毛中的 X 射线衍射进行表征，通过杆结构域的 α 螺旋或 β 片层分子结构区分 α 和 β 角蛋白。⁷目前，它主要被认为是一种稳定的细胞骨架元素及其在皮肤病中的作用。张等人是第一个将 Krt1、炎症、肺血管重塑和 PAH 明确联系起来的人。¹这些研究重点关注肺血管平滑肌细胞的异常激活，支持角蛋白在血管重塑中的作用。

总的来说，这是一组非常有趣的综合实验，着眼于 Krt1 如何预防 PAH 的体外和体内机制，开辟了一个新的研究领域，以进一步探索角蛋白作为潜在的 PAH 疗法。未来的研究肯定会集中在（a）Krt1激活TLR7-NF-ĸB通路的机制，（b）缺氧抑制Krt1的机制，包括Krt1潜在的翻译后修饰，例如二硫键的形成、磷酸化和糖基化，（c）缺氧调节启动子甲基化增加以抑制 Krt1 表达的机制，（d）探索成纤维细胞中缺氧驱动的 Krt1 抑制。G 蛋白、PI3K 激酶、MAP 激酶和 Src 激酶都是细胞角蛋白表达所必需的。⁴应测试在 PAH 发生过程中重要的转录因子，包括 STAT3、FOXM1、FOXO、NOTCH 和 NFAT。¹确定 Krt1 是否增加内源性肺血管内皮一氧化氮生成的研究将是有用的，因为已知一氧化氮的消耗与 Krt1 转录物的增加相关。⁸总之，Krt1 是 PAH 的一个令人兴奋的新型潜在治疗靶点，这些研究首次深入了解缺氧介导的 Krt1 抑制和随后的肺动脉平滑肌细胞扩张，表明 PAH 中的肺血管重塑。