Quantification of N-acetyl-seryl-aspartyl-lysyl-proline in hemodialysis patients administered angiotensin-converting enzyme inhibitors by stable isotope dilution liquid chromatography-tandem mass spectrometry

We developed a sensitive, selective and accurate method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to determine N-terminal thymosin-β peptides of Ac-SDKP and Ac-ADKP in human plasma samples. Quantification of Ac-SDKP and Ac-ADKP was performed using solid phase extraction (SPE) based on C(18), reversed phase LC separation, and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) and Ac-ADKP-d(7) were synthesized for the internal standards. These MRM monitoring ions were m/z 488→129 (quantitative ion)/226 for Ac-SDKP, m/z 496→137 for Ac-SDKP-(13)C(6), (15)N(2), m/z 472→129 (quantitative ion)/226 for Ac-ADKP, and m/z 479→129 for Ac-ADKP-d(7), respectively. Lower limit of quantitation (LLOQ) of Ac-SDKP and Ac-ADKP was 0.1ng/mL in human plasma. Recovery values were ranged from 94.7% to 106.3% for inter- (RSD: 0.6-3.5%) and intra- (RSD: 0.4-4.9%) day assays. Plasma Ac-SDKP levels were significantly higher in hemodialyzed subjects treated with angiotensin-converting enzyme inhibitors of enalapril (27.3±24.6ng/mL, n=10) and trandolapril (12.3±16.9ng/mL, n=18) than healthy (0.4±0.2ng/mL, n=7) and hemodialyzed subjects (0.6±0.2ng/mL, n=34). This analytical method would be useful to measure N-terminal thymosin-β peptides in human plasma for the clinical study.     

Ac-SDKP在博莱霉素诱导的肺纤维化小鼠中作用机制的探讨

目的 探讨小鼠肺间质纤维化模型中,Ac SDKP通过抑制内质网应激进而干预上皮-间质转化过程而延缓肺纤维化发展的机制.方法 将24只9周龄健康雄性C57BL/6小鼠[体质量(25±2)g]随机分为3组:对照组(N组)、肺纤维化模型组(M组)、肺纤维化+Ac-SDKP组(P组),每组8只.选取其中16只小鼠,采用气管内注入博莱霉素(5 mg/kg)法建立小鼠肺纤维化模型,21d后随机选取8只纤维化小鼠,腹腔内埋入Ac-SDKP微量注射泵,持续干预21 d.于各组造模21 d后处死小鼠,取肺组织,观察肺组织病理学变化;测定羟脯氨酸含量,判断造模是否成功并评估肺组织纤维化严重程度;Western blot、免疫组化法检测内质网应激相关蛋白GRP78、CHOP,间质组织标志物α-SMA、Vementin,上皮组织标志物E-cadherin蛋白表达水平.结果 ①肉眼观察:N组双肺表面及切面光滑,未见结节形成;M组双肺表面及切面均可见散在结节性病变;P组肺组织表面也可见小结节,但较M组稀少.②HE及Masson染色:M组出现明显的纤维化改变;P组与M组相比,肺泡炎及肺纤维化的程度均有所减轻(P＜0.05).③羟脯氨酸含量测定:M组较N组含量升高;P组较M组含量降低,P组较N组含量仍有所升高(P值均＜0.05).④免疫组化结果:N组肺组织中αSMA、Vimentin、CHOP及GRP78少量表达,M组表达较N组明显增加,P组较M组表达减少,但仍多于N组(P值均＜0.05);N组肺组织中E-cadherin表达较多,M组表达较N组减少,P组较M组表达增加,但少于N组(P值均＜0.05).⑤Western blot结果:与N组比较,M组CHOP、GRP78、Vimentin蛋白相对表达量均升高,E-cadherin蛋白相对表达量降低;而P组各蛋白相对表达量均介于N组和M组之间(P值均＜0.05).结论 在肺纤维化发病过程中,Ae-SDKP可能通过抑制内质网应激减缓上皮-间质转化,从而发挥其抗肺间质纤维化的作用.     

The role of thymosin-β4 in kidney disease

Therapies that modulate inflammation and fibrosis have the potential to reduce the morbidity and mortality associated with chronic kidney disease (CKD). A promising avenue may be manipulating thymosin-β4, a naturally occurring peptide, which is the major G-actin sequestering protein in mammalian cells and a regulator of inflammation and fibrosis. Thymosin-β4 is already being tested in clinical trials for heart disease and wound healing. This editorial outlines the evidence that thymosin-β4 may also have therapeutic benefit in CKD.     

Effects of thymosin β4 and its N-terminal fragment Ac-SDKP on TGF-β-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis

Thymosin β4 (Tβ4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tβ4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis.

Objectives: In this study, the putative anti-proliferative and anti-fibrogenic effects of Tβ4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tβ4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis.

Methods: We utilized both control and TGF-β-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tβ4 were assessed in CD1 mice treated with bleomycin.

Results: In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-β-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-β. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tβ4 failed to prevent fibrosis induced by the drug at 14 and 21 days.

Conclusion: We conclude that, compared to Tβ4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.     

Prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner

BACKGROUND AND PURPOSE A serine protease, prolyl oligopeptidase (POP) has been reported to be involved in the release of the pro-angiogenic tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Ac-SDKP) from its precursor, 43-mer thymosin β4 (Tβ4). Recently, it was shown that both POP activity and the levels of Ac-SDKP are increased in malignant tumours. The aim of this study was to clarify the release of Ac-SDKP, and test if POP and a POP inhibitor, 4-phenyl-butanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047), can affect angiogenesis. EXPERIMENTAL APPROACH We used HPLC for bioanalytical and an enzyme immunoassay for pharmacological analysis. Angiogenesis of human umbilical vein endothelial cells was assessed in vitro using a 'tube formation' assay and in vivo using a Matrigel plug assay (BD Biosciences, San Jose, CA, USA) in adult male rats. Moreover, co-localization of POP and blood vessels was studied. KEY RESULTS We showed the sequential hydrolysis of Tβ4: the first-step hydrolysis by proteases to <30-mer peptides is followed by an action of POP. Unexpectedly, POP inhibited the first hydrolysis step, revealing a novel regulation system. POP with Tβ4 significantly induced, while KYP-2047 effectively prevented, angiogenesis in both models compared with Tβ4 addition itself. POP and endothelial cells were abundantly co-localized in vivo. CONCLUSIONS AND IMPLICATIONS We have now revealed that POP is a second-step enzyme in the release of Ac-SDKP from Tβ4, and it has novel autoregulatory effect in the first step. Our results also advocate a role for Ac-SDKP in angiogenesis, and suggest that POP has a pro-angiogenic role via the release of Ac-SDKP from its precursor Tβ4 and POP inhibitors can block this action.     

An Ac-SDKP analogue resistant to angiotensin converting enzyme exhibits anti-fibrosis effects and improves heart function in mice after myocardial infarction

To explore the effect of an Ac-SDKP analog on left ventricular remodeling after myocardial infarction, we synthesized the analog Ac-SD_DK_DP by replacing Asp and Lys with their D isomers. The biological activities of Ac-SD_DK_DP were confirmed using flow cytometry, qRT-PCR, Western blots and fluorescence microscopy. The protective effects of Ac-SD_DK_DP on infarcted hearts were assessed in mice with myocardial infarction (MI). The half-life of Ac-SD_DK_DP was prolonged to over 2 h from a few minutes that Ac-SDKP has. Compared with Ac-SDKP, the analog exhibited stronger inhibition on the differentiation of macrophages, expression of arginase I (ARG I) and TGF-β1 in mature macrophages, proliferation and secretion of collagen type I in cardiac fibroblasts. In MI mice mode, Ac-SD_DK_DP decreased collagen deposition and TGF-β1 expression in myocardium, thus improvingthe FS (%) to 23.0±7.8 compared with 11.2±6.2 in untreated mice and 11.7±5.3 in Ac-SDKP treated mice (P<0.05). This work shows that the Ac-SDKP analogue is potentially useful for protective treatment for heart failure post-MI. In addition, the anti-fibrosis mechanism of Ac-SDKP was correlated with the alternative activation (M2) of macrophages by assessing ARG I and TGF-β1, two important fibrosis-related molecules secreted in M2 macrophages.     

Ac-SDKP通过抑制内质网应激对肺纤维化细胞凋亡的影响

目的探讨体内、外肺间质纤维化模型中Ac-SDKP延缓肺纤维化发展的可能机制。 方法体内实验：将24只健康雄性C57BL/6小鼠随机分为对照组（8只）、肺纤维化模型组（8只）、肺纤维化+Ac-SDKP治疗组（8只）。采用气管内注入博来霉素（5 mg/kg）法建立小鼠肺纤维化模型,对照组为气管内注入等体积0.9%氯化钠溶液。造模后肺纤维化+Ac-SDKP治疗组小鼠腹腔内埋入Ac-SDKP微量注射泵（Ac-SDKP 800μg·kg^-1·d^-1,冰乙酸0.1 mol/L,卡托普利100 mg·kg^-1·d^-1）,持续干预21 d。对照组及肺纤维化模型组小鼠均于造模完成后第21天处死,肺纤维化+Ac-SDKP治疗组小鼠于干预21 d后处死,取肺组织观察其病理学变化;按试剂盒说明测定羟脯氨酸含量测定;TUNEL法测定肺泡上皮细胞凋亡率;Western-blot、免疫组化法检测GRP78、CHOP蛋白及mRNA的表达水平。体外实验：培养A549细胞,经TGF-β干预24 h后收集细胞,RT-PCR检测GRP78、CHOP mRNA的表达水平。正态分布计量资料多组间比较采用方差分析,两两比较采用SNK-q检验;非正态分布计量资料多组间比较采用kruskal-WallisH检验。 结果（1）一般观察：对照组小鼠双肺表面及切面光滑,未见结节形成;肺纤维化模型组肺组织表面及切面可见散在小结节;肺纤维化+Ac-SDKP治疗组肺组织表面也可见小结节,但较肺纤维化模型组稀少。（2）HE及Masson染色：肺纤维化模型组出现明显的纤维化改变,肺纤维化+Ac-SDKP治疗组肺纤维化程度有所减轻。（3）羟脯氨酸含量：3组间羟脯氨酸含量差异有统计学意义（H=20.490,P<0.01）,其中肺纤维化模型组、肺纤维化+Ac-SDKP治疗组明显高于对照组（q=0.517、0.167,P<0.05）,肺纤维化+Ac-SDKP治疗组明显低于肺纤维化模型组（q=0.351,P<0.05）。（4）肺泡上皮细胞凋亡指数：3组间差异有统计学意义（F=57.720,P<0.01）,其中肺纤维化模型组、肺纤维化+Ac-SDKP组明显高于对照组（q=8.471、3.639,均P<0.01）,肺纤维化+Ac-SDKP组明显低于肺纤维化模型组（q=4.832,P<0.01）。（5）CHOP、GRP78蛋白水平表达：肺纤维化模型组、肺纤维化+Ac-SDKP治疗组较对照组明显下降（q=22.630、8.921,138.812、41.233;P<0.01）,肺纤维化+Ac-SDKP治疗组较肺纤维化组明显升高（q=13.711、97.583,P<0.01）。（6）GRP78、CHOP mRNA表达量：TGF-β干预组较对照组明显升高（q=8.791、7.782,P<0.05）;TGF-β+Ac-SDKP干预组较单纯Ac-SDKP干预组明显升高（q=4.399、5.561,P<0.05)。 结论Ac-SDKP可能通过抑制内质网应激减缓肺泡上皮细胞凋亡,从而发挥其抗肺间质纤维化的作用。     

Regeneration of chronic myocardial infarction by injectable hydrogels containing stem cell homing factor SDF-1 and angiogenic peptide Ac-SDKP

Regeneration of chronic myocardial infarction (CMI) is one of the challenging issues due to its limited regeneration activity compared to acute or sub-acute stage. In this study, we examined whether combination of stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) injected with biomimetic hydrogels promote regeneration of cardiac function in a CMI model. We evaluated the regeneration of chronically infarcted myocardium using injectable biomimetic hydrogels containing two therapeutic factors; stromal-derived factor-1 (SDF-1) and Ac-SDKP for stem cell homing and angiogenesis, respectively. Injection of the two therapeutic factors into the infarct region of the left ventricle showed that the biomimetic hydrogels containing two therapeutic factor exhibited significantly improved left ventricle function, increased angiogenesis, decreased infarct size and greatest wall thickness within the infarct region at 4 weeks post-treatment. From these results, it is clear that hydrogels containing two therapeutic factors showed synergistic effects on regeneration in the chronic heart failure model. In conclusion, these results suggest that combination of stem cell homing factor with angiogenic peptides recruit stem cells to the microenvironments, increase the expression of angiogenic genes, enhance the matured vessel formation and improve the cardiac function in chronic MI.