Liver-heart cross-talk mediated by coagulation factor XI protects against heart failure
凝血因子Xi介导的肝心串扰对心力衰竭的保护作用

To identify endocrine circuits mediating liver-heart cross-talk (10), we took advantage of a recently developed bioinformatics approach that uses natural variation to identify correlations between tissues. For this, we used a panel of 100 diverse inbred mouse strains in the HMDP (6, 11). Global transcriptomic data from the heart and the liver were generated across all 100 inbred strains and used to detect the correlations between secreted proteins from the liver and their downstream effects on the heart (Fig. 1A). By assessing the strength of cross-tissue predictions, we generated a list of potential liver-heart mediators (Fig. 1B and table S1). The top-ranked candidates included Igfbp7, Lipc, Emilin1, Lgals9, St6gal1, Ghr, Crlf2, Lcat, and F11. This list revealed several previously described mediators with consistent functions. For instance, insulin-like growth factor-binding protein-7 (Igfbp7) has been reported to be correlated with diastolic function in HFrEF and HFpEF patients (12).
为了识别介导肝脏-心脏串扰的内分泌回路（10），我们利用了最近开发的生物信息学方法，该方法使用自然变异来识别组织之间的相关性。为此，我们在HMDP中使用了一组100种不同的近交系小鼠品系（6，11）。在所有100个近交系中生成来自心脏和肝脏的全局转录组数据，并用于检测来自肝脏的分泌蛋白与其对心脏的下游效应之间的相关性（图1A）。通过评估跨组织预测的强度，我们生成了潜在的肝-心介质列表（图1B和表S1）。排名靠前的候选基因包括Igfbp 7、Lipc、Emilin 1、Lgals 9、St 6 gal 1、Ghr、Crlf 2、Lcat和F11。该列表显示了几个先前描述的具有一致功能的介体。 例如，据报道胰岛素样生长因子结合蛋白-7（Igfbp 7）与HFrEF和HFpEF患者的舒张功能相关（12）。

On the basis of their specific expression in the liver, data from the literature, and functional annotation, we selected Hgfac, C8g, and F11 as candidate mediators of liver-heart communication (Fig. 1B). To determine whether these factors have clinically relevant effects on the heart, we examined them in a mouse model of HFpEF, which is characterized by diastolic dysfunction. We overexpressed these genes individually in the livers of C57BL/6J male mice with an adeno-associated virus serotype 8 (AAV8) vector carrying target genes or green fluorescent protein (GFP) control and directed by the liver-specific thyroid hormone-binding globulin promoter (fig. S1A). After AAV8 injection, mice were subjected to a “two-hit” HFpEF model induced by a combination of high-fat diet (HFD) and inhibition of nitric oxide synthase using Nω-nitrol-arginine methyl ester (l-NAME) (13), followed by assessment of cardiac functions (fig. S1B). After 7 weeks of HFD + l-NAME feeding, mice developed heart failure phenotypes that recapitulated the clinical symptoms of HFpEF, including diastolic dysfunction [increased E/A ratio, E/eʹ ratio, left ventricular (LV) mass, heart weight, and lung weight], metabolic disorders (increased body weight, fat mass, plasma lipids, and glucose intolerance), exercise intolerance (reduced running distance), and preserved LV ejection fraction (LVEF) (fig. S1, C to N).
基于它们在肝脏中的特异性表达、来自文献的数据和功能注释，我们选择Hgfac、C8 g和F11作为肝-心通讯的候选介质（图1B）。为了确定这些因素是否对心脏有临床相关影响，我们在HFpEF小鼠模型中对其进行了检查，HFpEF的特征在于舒张功能障碍。我们在C57 BL/6 J雄性小鼠的肝脏中分别过表达这些基因，使用携带靶基因的腺相关病毒血清型8（AAV 8）载体或绿色荧光蛋白（GFP）对照，并由肝脏特异性甲状腺肿结合球蛋白启动子指导（图S1 A）。在AAV 8注射后，使小鼠经受通过高脂肪饮食（HFD）和使用Nω-硝基-精氨酸甲酯（I-NAME）抑制一氧化氮合酶的组合诱导的“两次打击”HFpEF模型（13），随后评估心脏功能（图S1 B）。 在HFD + l-NAME喂养7周后，小鼠发展出心力衰竭表型，其概括了HFpEF的临床症状，包括舒张功能障碍[增加的E/A比率、E/E比值、左心室（LV）质量、心脏重量和肺重量]、代谢紊乱[增加的E/A比率、E/E比值、左心室（LV）质量、心脏重量和肺重量]、代谢紊乱[增加的E/A比率、E/E比值、左心室（LV）质量、心脏重量和肺重量]、代谢紊乱[增加的E/A比率、E/E比值、左心室（LV）质量和肺重量]。（体重、脂肪量、血脂和葡萄糖耐受不良增加）、运动不耐受（减少的跑步距离）和保留的LV射血分数（LVEF）（图S1，C至N）。

We observed that when overexpressed, liver-derived hepatocyte growth factor activator (HGFAC) increased LV mass and complement C8 gamma chain (C8G) decreased heart weight in the model of HFpEF (figs. S2 and S3). However, we focused on the other top candidate, FXI, because it had additional effects on several HFpEF traits, including diastolic function. FXI acts downstream of FXII (14, 15) and triggers the middle phase of the intrinsic pathway of blood coagulation by activating FIX. Like HGFAC and C8G, FXI is also exclusively expressed in the liver (Fig. 1C and fig. S4, A and B). Furthermore, on the basis of associations with heart transcript levels in the HMDP, FXI was predicted to be strongly correlated with critical pathways in the heart and a number of clinical traits related to HFpEF (Fig. 1D and fig. S4C). In addition, human genome-wide association studies (GWAS) revealed that genetic loci encompassing the F11 gene were associated with the total cholesterol and BMP7 levels (Fig. 1E and tables S2 and S3) (16). These data suggested a potential role of FXI in heart failure.
我们观察到，在HFpEF模型中，当过表达时，肝源性肝细胞生长因子激活剂（HGFAC）增加LV质量，补体C8 γ链（C8G）降低心脏重量（图1A和1B）。S2和S3）。然而，我们专注于另一个最佳候选药物FXI，因为它对几个HFpEF特征有额外的影响，包括舒张功能。FXI作用于FXII的下游（14，15），并通过激活FIX触发凝血内源性途径的中间阶段。与HGFAC和C8 G一样，FXI也仅在肝脏中表达（图1C和图S4，A和B）。此外，基于与HMDP中心脏转录物水平的相关性，预测FXI与心脏中的关键途径和与HFpEF相关的许多临床特征强烈相关（图1D和图S4C）。此外，人类全基因组关联研究（GWAS）显示，包含F11基因的遗传基因座与总胆固醇和BMP 7水平相关（图1）。 表1E和表S2和S3）（16）。这些数据表明FXI在心力衰竭中的潜在作用。

We observed reduced plasma FXI in the mice on HFD + l-NAME relative to those given a chow diet (fig. S4, D and E). We then induced the HFpEF model in 30 genetically diverse, inbred strains of mice, a subset of HMDP, to examine the association between plasma FXI and diastolic dysfunction in the context of naturally occurring variation. We found that FXI levels were inversely correlated with diastolic dysfunction after feeding the HFpEF diet (Fig. 2A). Taken together, these results suggest that FXI may protect against diastolic dysfunction.
我们观察到相对于给予普通食物的小鼠，HFD + l-NAME的小鼠中血浆FXI减少（图S4、D和E）。然后，我们在30个遗传多样的近交系小鼠（HMDP的一个子集）中诱导HFpEF模型，以检查血浆FXI和舒张功能障碍之间的相关性。我们发现，喂食HFpEF饮食后，FXI水平与舒张功能障碍呈负相关（图2A）。综上所述，这些结果表明，FXI可预防舒张功能障碍。

We then directly validated the function of FXI in the HFpEF model using overexpression. C57BL/6J male mice injected with AAV8-GFP or AAV8-F11 were subjected to chow diet or HFD + l-NAME for 7 weeks (fig. S1B). After AAV8 injection, F11 expression was elevated in the liver and FXI protein was increased in the plasma (Fig. 2, B and C, and fig. S5, A and B). We injected sufficient virus to increase FXI protein levels only modestly (about 1.4-fold) to avoid nonphysiological artifacts. Plasma alanine transaminase levels were not significantly changed (P = 0.29) by FXI overexpression, suggesting no deleterious effects on the liver from overexpression (fig. S5C). FXI protein was not detected in the heart, confirming the specificity of AAV8 to target the liver and supporting the concept that FXI is an endocrine factor produced by the liver that affects the heart (fig. S5D). Mice receiving AAV8-F11 had lower body weight and fat mass after HFpEF compared with those receiving AAV8-GFP (fig. S5E). Blood pressure was not affected by FXI (fig. S5F). Consistent with our genetic results in the HMDP, FXI overexpression decreased E/A ratio, E/eʹ ratio, heart weight, and lung weight in the HFpEF model while preserving LVEF, indicating an improvement in diastolic function (Fig. 2, D to I, and fig. S5G). Running distance was also improved by FXI overexpression, indicating that FXI ameliorates exercise intolerance in HFpEF (Fig. 2J). FXI overexpression also had beneficial metabolic effects on fat mass and plasma lipid levels (fig. S6, A to D) but not on glucose tolerance (fig. S6, E to G).
然后，我们使用过表达直接验证了FXI在HFpEF模型中的功能。注射AAV 8-GFP或AAV 8-F11的C57 BL/6 J雄性小鼠经受普通饮食或HFD +1-NAME 7周（图S1 B）。AAV 8注射后，肝脏中F11表达升高，血浆中FXI蛋白增加（图2，B和C，以及图S5，A和B）。我们注射了足够的病毒，仅适度增加FXI蛋白水平（约1.4倍），以避免非生理性伪影。FXI过表达未显著改变血浆丙氨酸转氨酶水平（P = 0.29），表明过表达对肝脏无有害影响（图S5 C）。在心脏中未检测到FXI蛋白，证实了AAV 8靶向肝脏的特异性，并支持FXI是由肝脏产生的影响心脏的内分泌因子的概念（图S5 D）。与接受AAV 8-GFP的小鼠相比，接受AAV 8-F11的小鼠在HFpEF后具有较低的体重和脂肪量（图S5 E）。 血压不受FXI影响（图S5 F）。与我们在HMDP中的遗传结果一致，在HFpEF模型中，FXI过表达降低了E/A比、E/E比值、心脏重量和肺重量，同时保留了LVEF，表明舒张功能改善（图2，D至I和图S5 G）。FXI过表达也改善了跑步距离，表明FXI改善了HFpEF中的运动不耐受（图2 J）。FXI过表达还对脂肪量和血浆脂质水平具有有益的代谢作用（图S6，A至D），但对葡萄糖耐量无作用（图S6，E至G）。

To test whether FXI overexpression affects blood coagulation, we measured blood thrombin-antithrombin complexes in mice with GFP or FXI overexpression and found that they were not significantly changed (P = 0.79) in mice receiving AAV8-F11 versus AAV8-GFP (Fig. 2K), suggesting that the coagulation system was not affected by FXI overexpression. It has been found that mean platelet volume, reflecting the size and activity of platelets, is increased in decompensated heart failure patients and correlates with disease severity, serving as an independent predictor of 6-month mortality after decompensation (17). We observed a small but significant increase of mean platelet volume upon HFpEF development (P < 0.05), and FXI overexpression reversed it (P < 0.01) (fig. S6H). FXI overexpression significantly reduced circulating inflammatory cells (P < 0.05) and cytokine levels [interleukin β (IL-β) and IL-6, P < 0.05; interferon γ, P < 0.01] in the HFpEF model (fig. S6, I and J). Moreover, the expression of inflammatory genes in the heart was also reduced by FXI overexpression (Fig. 2L and fig. S6K). When mice were maintained on a chow diet, the number of blood immune cells was not changed by FXI overexpression (fig. S6L).
为了测试FXI过表达是否影响血液凝固，我们测量了GFP或FXI过表达的小鼠中的血液凝血酶-抗凝血酶复合物，发现它们在接受AAV 8-F11的小鼠中相对于AAV 8-GFP没有显著变化（P = 0.79）（图2K），表明凝血系统不受FXI过表达的影响。研究发现，反映血小板大小和活性的平均血小板体积在失代偿性心力衰竭患者中增加，并与疾病严重程度相关，可作为失代偿后6个月死亡率的独立预测因素（17）。我们观察到HFpEF发展后平均血小板体积的小但显著的增加（P < 0.05），并且FXI过表达逆转了它（P < 0.01）（图S6 H）。在HFpEF模型中，FXI过表达显著降低了循环炎性细胞（P < 0.05）和细胞因子水平[白细胞介素β（IL-β）和IL-6，P < 0.05;干扰素γ，P < 0.01]（图S6，I和J）。 此外，心脏中炎性基因的表达也通过FXI过表达而降低（图2L和图S6K）。当小鼠维持食物饮食时，血液免疫细胞的数量不因FXI过表达而改变（图S6L）。

To further test whether the cardiac infiltration of inflammatory cells was attenuated by FXI, we performed multiplex immunohistochemistry using antibodies against macrophages (F4/80), T cells (CD3), monocytes (Ly6C), and granulocytes (Ly6G). We observed significantly decreased inflammatory cells (F4/80, P < 0.0001; CD3, P < 0.05; Ly6C and Ly6G, P < 0.01) in heart tissue from FXI-overexpressing mice versus GFP-overexpressing mice (Fig. 2, M and N), suggesting that FXI overexpression reduced inflammation in heart tissue in the HFpEF model. In addition, FXI overexpression also decreased fibrosis in the heart (Fig. 2, O and P).
为了进一步测试FXI是否减弱了炎性细胞的心脏浸润，我们使用抗巨噬细胞（F4/80）、T细胞（CD 3）、单核细胞（Ly 6C）和粒细胞（Ly 6 G）的抗体进行了多重免疫组织化学。我们观察到与GFP过表达小鼠相比，FXI过表达小鼠的心脏组织中的炎性细胞显著减少（F4/80，P < 0.0001; CD 3，P < 0.05; Ly 6C和Ly 6 G，P < 0.01）（图2，M和N），表明FXI过表达减少了HFpEF模型中心脏组织的炎症。此外，FXI过表达也减少了心脏中的纤维化（图2，O和P）。

To investigate the molecular mechanism underlying the impact of FXI on the heart, we performed RNA sequencing (RNA-Seq) of the mice with FXI versus GFP overexpression in heart and adipose. Compared with GFP controls, 124 genes in the heart were significantly changed (adjusted P < 0.05) by FXI overexpression (fig. S7, A to C). Differentially expressed genes were enriched in pathways related to circadian rhythm, cardiac muscle contraction, inflammation, focal adhesion, the phosphatidylinositol 3-kinase (PI3K)–Akt pathway, and insulin signaling (fig. S7D). In contrast to the heart, only six genes in white adipose tissue were significantly changed (adjusted P < 0.05) by FXI overexpression (fig. S7E). Tcap (Titin-cap), and Lrrc10 (Leucine-rich repeat-containing 10), two genes involved in cardiac myofibril assembly and cardiac muscle tissue morphogenesis (2), were increased in the heart tissue of FXI-overexpressing mice (fig. S7, F and G).
为了研究FXI对心脏影响的分子机制，我们对心脏和脂肪中FXI与GFP过表达的小鼠进行了RNA测序（RNA-Seq）。与GFP对照相比，心脏中的124个基因被FXI过表达显著改变（调整的P < 0.05）（图S7，A至C）。差异表达的基因在与昼夜节律、心肌收缩、炎症、粘着斑、磷脂酰肌醇3-激酶（PI 3 K）-Akt途径和胰岛素信号传导相关的途径中富集（图S7 D）。与心脏相反，白色脂肪组织中只有6个基因被FXI过表达显著改变（调整的P < 0.05）（图S7 E）。Tcap（Titin-cap）和Lrrc 10（富含亮氨酸的重复序列10），这两个基因参与心脏肌原纤维组装和心肌组织形态发生（2），在FXI过表达小鼠的心脏组织中增加（图S7、F和G）。

To identify pathways perturbed by FXI, we again turned to the HMDP. Because we had performed global transcriptomics in the heart as well as the liver in all 100 strains, we could identify heart genes in which expression was correlated with the expression of FXI in the liver. On the basis of this, we examined the protein or RNA levels of the predicted pathways (Fig. 1E) and RNA-Seq (fig. S7), including the PI3K-Akt, nuclear factor κB, SMAD, and tumor necrosis factor-α (TNF-α) pathways (Fig. 3A and fig. S8A). We observed that members of the BMP pathway were correlated with FXI expression, and the link to BMP was supported by data from human GWAS (discussed below). Consistent with this, our overexpression studies showed that FXI induced an increase in SMAD1/5 phosphorylation and a decrease in TNF-α in the heart but not in other tissues (Fig. 3A and fig. S8, A to H), suggesting activation of the BMP-SMAD1/5 pathway and a decrease of inflammation in the heart. To test whether nuclear p-SMAD1/5 was also increased, we isolated the nuclear fraction from the same heart tissue and observed that it was significantly induced in the FXI overexpression group relative to GFP controls (P < 0.0001) (fig. S8I). We injected C57BL/6J male mice with saline control or mouse FXI protein and, after 2 hours, observed the phosphorylation of SMAD1/5 in the heart but not in other tissues, supporting the tissue-specific activation of the BMP-SMAD1/5 pathway by FXI (fig. S8, J to M). Plasminogen activator inhibitor-1 (PAI-1) was comparable in the hearts receiving AAV8-F11 relative to those receiving AAV8-GFP (fig. S8A). However, FXI overexpression reversed the expression of the fibrotic and inflammatory genes Col5a1, Col5a3, Adam19, IL1β, IL6, and Tnf in the heart but not in other tissues examined, consistent with the observed decrease in fibrosis and inflammation in the heart (Fig. 3B and fig. S8, N and O).
为了确定FXI干扰的途径，我们再次转向HMDP。因为我们已经在所有100个菌株的心脏和肝脏中进行了全局转录组学，所以我们可以鉴定心脏基因，其中表达与肝脏中FXI的表达相关。在此基础上，我们检查了预测途径（图1 E）和RNA-Seq（图S7）的蛋白质或RNA水平，包括PI 3 K-Akt、核因子κB、SMAD和肿瘤坏死因子-α（TNF-α）途径（图3A和图S8 A）。我们观察到BMP途径的成员与FXI表达相关，并且与BMP的联系得到了来自人GWAS的数据的支持（下文讨论）。与此一致，我们的过表达研究表明，FXI诱导心脏中SMAD 1/5磷酸化增加和TNF-α降低，但在其他组织中未诱导（图3A和图S8，A至H），表明BMP-SMAD 1/5通路激活和心脏炎症减少。 为了测试核p-SMAD 1/5是否也增加，我们从相同的心脏组织中分离核级分，并观察到相对于GFP对照，它在FXI过表达组中被显著诱导（P < 0.0001）（图S8 I）。我们向C57 BL/6 J雄性小鼠注射生理盐水对照或小鼠FXI蛋白，2小时后，观察到心脏中SMAD 1/5的磷酸化，但其他组织中没有，支持FXI对BMP-SMAD 1/5途径的组织特异性激活（图S8，J至M）。相对于接受AAV 8-GFP的心脏，接受AAV 8-F11的心脏中的纤溶酶原激活物抑制剂-1（派-1）相当（图S8 A）。然而，FXI过表达逆转了心脏中纤维化和炎症基因Col 5a 1、Col 5a 3、Adam 19、IL 1 β、IL 6和Tnf的表达，但未逆转检查的其他组织中的表达，这与观察到的心脏纤维化和炎症减少一致（图3B和图S8，N和O）。

To determine the localization of p-SMAD1/5, we stained p-SMAD1/5 and the markers of cardiomyocytes (troponin I), fibroblasts (vimentin), macrophages (CD68), and endothelial cells (CD31) in the heart after FXI overexpression. p-SMAD1/5 was colocalized with troponin I, but not with other markers (fig. S9), suggesting that p-SMAD1/5 was mainly activated in cardiomyocytes. To directly test whether FXI protein activates the BMP-SMAD1/5 pathway in cardiomyocytes, we incubated neonatal rat ventricular myocytes (NRVMs), human embryonic stem cell–induced cardiomyocytes, and other cell lines with control medium or medium containing human activated FXI protein (FXIa) in the presence of phenylephrine for 24 hours. We observed that FXIa increased the phosphorylation of SMAD1/5 and decreased the expression of Nppa, Nppb, Col5a3, and Adam19 in NRVMs and human embryonic stem cell–induced cardiomyocytes but not in other cell types (Fig. 3, C and D, and fig. S10).
为了确定p-SMAD 1/5的定位，我们对FXI过表达后心脏中的p-SMAD 1/5和心肌细胞（肌钙蛋白I）、成纤维细胞（波形蛋白）、巨噬细胞（CD 68）和内皮细胞（CD 31）的标志物进行染色。p-SMAD 1/5与肌钙蛋白I共定位，但不与其他标志物共定位（图S9），表明p-SMAD 1/5主要在心肌细胞中活化。为了直接测试FXI蛋白是否激活心肌细胞中的BMP-SMAD 1/5通路，我们在苯丙氨酸存在下将新生大鼠心室肌细胞（NRVM）、人胚胎干细胞诱导的心肌细胞和其他细胞系与对照培养基或含有人活化FXI蛋白（FXIa）的培养基孵育24小时。我们观察到FXIa增加了SMAD 1/5的磷酸化，并降低了NRVM和人胚胎干细胞诱导的心肌细胞中Nppa、Nppb、Col 5a 3和Adam 19的表达，但在其他细胞类型中未观察到（图3，C和D，以及图S10）。

The above experiments were performed with male mice and we were interested in determining whether the results were similar with females. C57BL/6J female mice injected with AAV8-GFP or AAV8-F11 were subjected to HFD + l-NAME for 7 weeks (fig. S11A). After AAV8 injection, F11 expression was increased in the liver (fig. S11B). We observed similar effects of FXI in female mice, including decreased body mass, decreased inflammatory cells in the blood, reduced plasma lipids, increased SMAD1/5 phosphorylation, improved diastolic function, and reduced heart weight and lung weight (fig. S11, C to N). By contrast, blood pressure was comparable between the FXI and GFP groups (fig. S11O).
上述实验是用雄性小鼠进行的，我们有兴趣确定结果是否与雌性小鼠相似。注射AAV 8-GFP或AAV 8-F11的C57 BL/6 J雌性小鼠经受HFD +1-NAME 7周（图S11 A）。AAV 8注射后，肝脏中F11表达增加（图S11 B）。我们在雌性小鼠中观察到FXI的相似作用，包括体重减轻、血液中炎性细胞减少、血脂降低、SMAD 1/5磷酸化增加、舒张功能改善以及心脏重量和肺重量减轻（图S11，C至N）。相比之下，FXI和GFP组之间的血压相当（图S11 O）。

To determine whether the effects of FXI that we observed were specific to the HFpEF model, we examined a “multi-hit” HFpEF model induced by the combination of aging, HFD, and angiotensin II (18). Aged C57BL/6J male mice were injected with AAV8-GFP or AAV8-F11 and then fed a HFD for 12 weeks. After 8 weeks of HFD, mice were infused with angiotensin II for 4 weeks (fig. S12A). F11 mRNA was increased in the liver by AAV8-F11 compared with AAV8-GFP controls (fig. S12B). Similar to the beneficial effects in the “two-hit” HFpEF model, we observed significant improvement in diastolic function and related traits in FXI-overexpressing mice relative to GFP-overexpressing mice, including reduced body mass (P < 0.05) (fig. S12C) and improved diastolic function as measured by lower E/A ratio (P < 0.05), lower E/eʹ ratio (P < 0.001), and lower LV mass (P < 0.05) (fig. S12, D to H). In addition, FXI-overexpressing mice exhibited reduced heart weight and plasma total cholesterol, as well as increased p-SMAD1/5 relative to GFP controls (fig. S12, I to K).
为了确定我们观察到的FXI效应是否对HFpEF模型具有特异性，我们检查了由衰老、HFD和血管紧张素II联合诱导的“多次打击”HFpEF模型（18）。用AAV 8-GFP或AAV 8-F11注射老年C57 BL/6 J雄性小鼠，然后喂食HFD 12周。HFD 8周后，向小鼠输注血管紧张素II 4周（图S12 A）。与AAV 8-GFP对照相比，通过AAV 8-F11在肝脏中增加F11 mRNA（图S12 B）。与“两次打击”HFpEF模型中的有益作用相似，我们观察到相对于GFP过表达小鼠，FXI过表达小鼠的舒张功能和相关性状显著改善，包括体重减轻（P < 0.05）。（图S12 C）和通过较低的E/A比测量的改善的舒张功能（P < 0.05），较低的E/E比值（P < 0.001），和较低的LV质量（P < 0.05）（图S12，D至H）。 此外，相对于GFP对照，过表达FXI的小鼠表现出心脏重量和血浆总胆固醇降低，以及p-SMAD 1/5增加（图S12，I至K）。

To confirm that FXI overexpression activates BMP signaling to protect against diastolic dysfunction, we blocked the BMP receptor with the dorsomorphin homolog 1 (DMH1) (19). DMH1 is a selective inhibitor of activin receptor-like kinase 3, a type 1 BMP receptor. In NRVMs, DMH1 treatment suppressed SMAD1/5 phosphorylation induction by FXIa (fig. S13A). We also examined the effect of DMH1 in vivo. C57BL/6J mice were injected with AAV8-F11 and fed with HFD + l-NAME for 7 weeks. Injection of DMH1 every other day to block SMAD1/5 phosphorylation (20) suppressed the change of body mass induced by FXI (fig. S13B), as well as the effect of FXI on p-SMAD1/5 levels, diastolic function, adipose weight, blood cell numbers, and plasma cholesterol (Fig. 3, E to H, and fig. S13, C to G). These results confirmed that FXI protects against HFpEF by activating the BMP pathway.
为了证实FXI过表达激活BMP信号传导以防止舒张功能障碍，我们用dorsomorphin同系物1（DMH 1）阻断BMP受体（19）。DMH 1是激活素受体样激酶3（一种1型BMP受体）的选择性抑制剂。在NRVM中，DMH 1处理抑制了FXIa对SMAD 1/5磷酸化的诱导（图S13 A）。我们还研究了DMH 1在体内的作用。C57 BL/6 J小鼠注射AAV 8-F11，并用HFD + l-NAME喂养7周。每隔一天注射DMH 1以阻断SMAD 1/5磷酸化（20）可抑制FXI诱导的体重变化（图S13 B），以及FXI对p-SMAD 1/5水平、舒张功能、脂肪重量、血细胞数量和血浆胆固醇的影响（图3，E至H，图S13，C至G）。这些结果证实，FXI通过激活BMP通路保护免受HFpEF。

The FXI protein is conserved in human, mouse, rat, and other species and consists of four apple domains and one catalytic domain (fig. S14A). It is present in that plasma as a zymogen, which exists as a homodimer consisting of two identical polypeptide chains linked by disulfide bonds (fig. S14B) (21). During FXI activation, an internal peptide bond is cleaved by FXIIa (or XII) in each of the two chains, resulting in activated FXIa, a serine protease composed of two heavy and two light chains held together by disulfide bonds (fig. S14B). To test whether the catalytic domain is required for the function of FXI on the heart, we introduced two missense mutations in human and mouse FXI catalytic domains (fig. S14, A to E). These mutations were predicted to be exposed at the surface of the FXI molecule and to cause functional defects (type II mutation) (22). Next, we tested their function in vitro using a co-culture system. Huh7 human liver cells and AML12 mouse liver cells were transfected with respective human or mouse plasmids containing GFP control, wild-type (WT) F11, or F11 with mutations. Then, cells were placed in co-cultures with NRVMs or 3T3-L1 adipocytes (fig. S15A). Twenty-four hours after transfection, FXI was highly induced in both Huh7 cells and AML12 cells (fig. S15B). In NRVMs, phosphorylation of SMAD1/5 was induced by WT FXI overexpression from both human and mouse liver cells, whereas mutant FXI did not exhibit a comparable effect (fig. S15C). By contrast, SMAD1/5 phosphorylation was not significantly (P = 0.84) induced by FXI in 3T3-L1 adipocytes, suggesting a heart-specific effect (fig. S15D). Consistent with phosphorylated SMAD1/5, Col5a3 was decreased by WT FXI but not mutant FXI in NRVMs, indicating that the catalytic activity is required for its effect (fig. S15E).
FXI蛋白在人、小鼠、大鼠和其他物种中是保守的，由四个苹果结构域和一个催化结构域组成（图S14 A）。它作为酶原存在于血浆中，作为由通过二硫键连接的两条相同多肽链组成的同源二聚体存在（图S14 B）（21）。在FXI活化过程中，两条链中的每条链的内部肽键被FXIIa（或XII）切割，产生活化的FXIa，这是一种丝氨酸蛋白酶，由两条重链和两条轻链通过二硫键连接在一起（图S14 B）。为了测试催化结构域是否是心脏上FXI功能所必需的，我们在人和小鼠FXI催化结构域中引入了两个错义突变（图S14，A至E）。预测这些突变暴露于FXI分子表面并导致功能缺陷（II型突变）（22）。接下来，我们使用共培养系统在体外测试它们的功能。 Huh 7人肝细胞和AML 12小鼠肝细胞用含有GFP对照、野生型（WT）F11或具有突变的F11的相应人或小鼠质粒转染。然后，将细胞置于与NRVM或3 T3-L1脂肪细胞的共培养物中（图S15 A）。转染后24小时，FXI在Huh 7细胞和AML 12细胞中均被高度诱导（图S15 B）。在NRVM中，SMAD 15的磷酸化由来自人和小鼠肝细胞的WT FXI过表达诱导，而突变体FXI未表现出相当的作用（图S15 C）。相比之下，FXI在3 T3-L1脂肪细胞中未显著诱导SMAD 15磷酸化（P = 0.84），表明心脏特异性效应（图S15 D）。与磷酸化SMAD 15一致，在NRVM中WT FXI降低了Col 5a 3，但突变体FXI没有降低，表明其作用需要催化活性（图S15 E）。

To test the effect of missense mutation in vivo, we produced AAV8 with the mouse WT and mutant F11 coding sequences. AAV8 containing GFP control, WT F11, and mutant F11 (mF11-Mut2) was injected into C57BL/6J male mice followed by HFD + l-NAME for 7 weeks, after which plasma FXI was increased in FXI group and was comparable to the mutant FXI group (Fig. 3I and fig. S15F). Body weight and fat mass were decreased by WT FXI overexpression, but there was no significant difference (P > 0.05) between groups of mutant FXI and GFP controls (fig. S15, G to I), suggesting functional defects of mutant FXI. Consistently, the effects of FXI on p-SMAD1/5, heart weight, E/A ratio, E/eʹ ratio, adipose weight, plasma cholesterol, and blood immune cells were not observed in mice carrying mutant FXI, demonstrating that catalytic activity is essential for the function of FXI in protecting against deleterious phenotypes in HFpEF (Fig. 3, J to M, and fig. S15, J to P).
为了测试体内错义突变的效果，我们用小鼠WT和突变体F11编码序列产生AAV 8。将含有GFP对照、WT F11和突变体F11（mF 11-Mut 2）的AAV 8注射到C57 BL/6 J雄性小鼠中，随后注射HFD + l-NAME 7周，之后FXI组中血浆FXI增加并且与突变体FXI组相当（图3 I和图S15 F）。WT FXI过表达降低了体重和脂肪量，但在突变体FXI组和GFP对照组之间没有显著差异（P > 0.05）（图S15，G至I），表明突变体FXI的功能缺陷。同样，在携带突变FXI的小鼠中未观察到FXI对p-SMAD 15、心脏重量、E/A比、E/E比值、脂肪重量、血浆胆固醇和血液免疫细胞的影响，表明催化活性对于FXI在保护HFpEF免受有害表型影响方面的功能至关重要（图3，J至M和图S15，J至P）。

As a serine protease, FXIa catalyzes the proteolysis of its substrates. BMP7 is synthesized as a large precursor molecule (inactive) that is cleaved to growth factor dimer or monomer (active) by proteolytic enzymes (23). We found that the cleavage site of the full-length BMP7 protein, at an arginine, is a common FXIa cleavage site (fig. S16, A and B) (24). We therefore hypothesized that BMP7 is a substrate of FXIa that mediates SMAD1/5 activation. FXI overexpression increased BMP7 growth factor dimer and monomer in the heart (Fig. 3N). Moreover, incubation of FXIa with BMP7 protein resulted in the cleavage of BMP7 (fig. S16C). Knocking down BMP7 or treatment with BMP7 antibody in NRVMs greatly reduced the activation of SMAD1/5 by FXIa (Fig. 3, O and P). The BMP7 protein is considerably enriched in heart tissue and cardiomyocytes (fig. S17), which may explain the preferential effect of FXI on the heart. The prodomain of BMP7 appears to bind to the extracellular matrix (23), suggesting that FXI cleaves the precursor BMP7 bound to the extracellular matrix in the heart; this then releases the dimer and monomer growth factors from the matrix to bind to the BMP receptor and activate SMAD1/5.
作为丝氨酸蛋白酶，FXIa催化其底物的蛋白水解。BMP 7作为大的前体分子（无活性）合成，其被蛋白水解酶切割成生长因子二聚体或单体（活性）（23）。我们发现，全长BMP 7蛋白在精氨酸处的切割位点是常见的FXIa切割位点（图S16，A和B）（24）。因此，我们假设BMP 7是FXIa介导SMAD 1/5激活的底物。FXI过表达增加了心脏中的BMP 7生长因子二聚体和单体（图3 N）。此外，FXIa与BMP 7蛋白孵育导致BMP 7裂解（图S16 C）。在NRVM中敲低BMP 7或用BMP 7抗体处理大大降低了FXIa对SMAD 1/5的激活（图3，O和P）。BMP 7蛋白在心脏组织和心肌细胞中显著富集（图S17），这可以解释FXI对心脏的优先作用。 BMP 7的前结构域似乎与细胞外基质结合（23），表明FXI切割与心脏细胞外基质结合的前体BMP 7;然后从基质中释放二聚体和单体生长因子，与BMP受体结合并激活SMAD 1/5。

We sought to further examine the cardioprotective effect of FXI using FXI knockout male mice in which the F11 gene was disrupted by a PGK-neo cassette (25). F11 transcripts in the liver of heterozygous null mice (F11-Het) were reduced by ~50% compared with WT littermates (Fig. 4A). FXI was either absent or barely detectable in other tissues (Fig. 4A and fig. S4, A and B). Adult WT and F11-Het mice were then subjected to HFD + l-NAME for 7 weeks to induce HFpEF phenotypes. Compared with WT littermates on the HFpEF diet, p-SMAD1/5 was reduced in the hearts of F11-Het mice (Fig. 4B). Consistent with reduced p-SMAD1/5, F11-Het mice exhibited more severe diastolic dysfunction, as evidenced by the increased E/A ratio, E/eʹ ratio, and LV mass but preserved ejection fraction (Fig. 4, C to G). Moreover, heart weight and lung weight were higher in F11-Het mice relative to WT controls, suggesting cardiac hypertrophy and lung congestion in FXI-deficient mice (Fig. 4, H and I). Exercise tolerance was also decreased in F11-Het mice compared with WT mice (Fig. 4J). By contrast, blood pressure was not significantly changed (P > 0.05) by FXI deficiency (fig. S18), indicating that FXI does not influence heart function through effects on blood pressure. These results collectively demonstrated the increased severity of diastolic dysfunction in FXI-deficient mice. We observed consistent effects of FXI in female mice with FXI heterozygous knockout (fig. S19).
我们试图使用FXI基因敲除雄性小鼠进一步检查FXI的心脏保护作用，其中F11基因被PGK-neo盒破坏（25）。与WT同窝出生小鼠相比，杂合无效小鼠（F11-Het）肝脏中的F11转录物减少约50%（图4A）。在其他组织中，FXI不存在或几乎检测不到（图4A和图S4，A和B）。然后使成年WT和F11-Het小鼠经受HFD +1-NAME 7周以诱导HFpEF表型。与使用HFpEF饮食的WT同窝仔相比，F11-Het小鼠心脏中的p-SMAD 1/5减少（图4 B）。与p-SMAD 1/5降低一致，F11-Het小鼠表现出更严重的舒张功能障碍，如E/A比、E/E比值和LV质量增加但射血分数保持所证明的（图4，C至G）。此外，相对于WT对照，F11-Het小鼠的心脏重量和肺重量更高，表明FXI缺陷小鼠的心脏肥大和肺充血（图4，H和I）。 与WT小鼠相比，F11-Het小鼠的运动耐量也降低（图4J）。相比之下，FXI缺乏没有显著改变血压（P > 0.05）（图S18），表明FXI不会通过影响血压影响心脏功能。这些结果共同证明了FXI缺陷小鼠中舒张功能障碍的严重程度增加。我们在FXI杂合敲除雌性小鼠中观察到FXI的一致效应（图S19）。

To determine the clinical relevance of FXI, we quantified plasma FXI in human patients with HFpEF and in normal participants. Plasma FXI protein was not significantly different (P = 0.91) between non–heart failure controls and HFpEF patients (Fig. 4K). However, plasma FXI was inversely correlated with E/eʹ ratio in all participants (Fig. 4L), including HFpEF patients (Fig. 4M), supporting the conclusion that FXI protects against diastolic dysfunction in HFpEF.
为了确定FXI的临床相关性，我们定量了HFpEF患者和正常受试者的血浆FXI。血浆FXI蛋白在非心力衰竭对照和HFpEF患者之间没有显著差异（P = 0.91）（图4K）。然而，在所有参与者（图4L）中，包括HFpEF患者（图4 M），血浆FXI与E/E比值呈负相关，支持FXI可预防HFpEF舒张功能障碍的结论。