兽研[内容请查看原文]
引言
血管内皮细胞衰老和凋亡及其导致的毛细血管稀疏（以微血管网络密度降低为特征）是年龄相关性肾脏疾病的标志（Tesfamariam and DeFelice 2007; Jourde‐Chiche et al. 2019; Li et al. 2022）。在患有慢性肾脏病（CKD）的猫患者中，通过定量免疫组织化学在受累肾脏中显示的毛细血管稀疏证据表明，其与其他组织病理学病变中的肾脏功能障碍呈正相关（Paschall et al. 2023）。临床数据提示，通过保护肾脏内皮和毛细血管网络功能来减缓肾功能丧失（Goto et al. 2014）可能延长CKD动物的寿命（Yamaguchi et al. 2013）。
贝前列素是一种化学性质稳定的前列环素合成类似物，口服给药后具有高活性和持久活性（Toda 1988; Niwano et al. 2003; Matsumoto et al. 2002）。先前的一项临床研究提示，贝前列素治疗与猫CKD患者血清肌酐水平的变化之间存在关联（Takenaka et al. 2018）；然而，贝前列素对总体生存率和特异性肾脏内皮保护的直接作用仍有待充分阐明。最近，Ito等人（2023）报道贝前列素治疗与CKD猫的总体生存率改善显著相关。贝前列素是由两对立体异构体——贝前列素-314d和贝前列素-315d及其对映体——组成的消旋混合物，总摩尔比为1:1:1:1。已发表的数据提供证据表明，贝前列素的不同异构体具有不同的药理效力（Kajikawa et al. 1989; Shen et al. 2019）；然而，尚不清楚这些效应是否具有物种特异性和/或细胞类型特异性，以及它们会在猫内皮细胞（FECs）中诱导何种效应。
对于FECs的体外实验，先前描述的永生化FEC系是使用猿猴病毒40大T抗原（SV40LT）和人端粒酶逆转录酶催化亚基（hTERT）建立的（Olyslaegers et al. 2013）。FECs已被证明在很大程度上保持原代内皮细胞的结构和功能表型，以及对肿瘤坏死因子（一种凋亡诱导剂）的免疫反应（Olyslaegers et al. 2013）。
本研究旨在建立使用各种营养饥饿条件诱导凋亡的体外FEC模型，并研究贝前列素消旋混合物及其异构体（-314d和-315d）如何对FECs提供保护性抗凋亡效应。此外，本研究旨在确定在FECs中诱导保护性（抗凋亡）效应所需的最低有效剂量贝前列素。
[内容请查看原文]
材料与方法
[内容请查看原文]
化学品与试剂
药用级贝前列素及分离的贝前列素-314d和贝前列素-315d异构体由Toray Industries, Inc.（日本东京）制造并提供。这些化学结构见图1。将其用UltraPure蒸馏水（Thermo Fisher Scientific, Waltham, MA, USA）溶解至10 mM浓度，并用0.22 µm滤器灭菌。溶液再用蒸馏水进一步稀释10倍至1 mM，并在4°C下保存直至使用。尽管本研究未进行手性HPLC分析以直接确认溶解后纯度，但这些异构体在这些特定条件下的立体化学稳定性已通过先前药理学研究的既定方案得到验证（Shen et al. 2019）。由于化合物保存在4°C的中性水溶液中，避免了已知会诱导前列腺素差向异构化的酸性条件。此外，商业供应商提供的材料数据表确认这些化合物在冷藏水溶液中可稳定数周，确保了其适用于我们的体外细胞培养实验及其稳定性。
[图：外消旋贝前列素、beraprost-314d和beraprost-315d的化学结构。beraprost-314d及其对映体]
[内容请查看原文]
细胞系与细胞培养条件
FECs购自Applied Biological Materials（加拿大列治文）。这些细胞来源于健康猫主动脉，并如前所述进行永生化（Olyslaegers et al. 2013）。按照制造商说明，细胞在Prigrow III培养基中培养，该培养基含有10%胎牛血清（FBS）、50 µg/mL内皮细胞生长补充剂（ECGS）、0.1 mg/mL硫酸庆大霉素、1 mM丙酮酸钠、1%（v/v）非必需氨基酸、10 U/mL肝素钠和1%青霉素-链霉素混合物（“完全培养基”）。完全培养基保存在4°C，使用前预热至室温（22°C）。
将FECs置于25 cm²培养瓶（Corning Inc.，Corning，NY，USA）中，在37°C、5% CO₂加湿培养箱（Thermo Fisher Scientific）中培养，直至达到对数生长期亚融合状态。吸弃培养基后，用Dulbecco磷酸盐缓冲盐水（D-PBS；5 mL）洗涤细胞两次，用0.25%（w/v）胰蛋白酶-1 mM乙二胺四乙酸（0.3 mL）消化 detachment，并重悬于完全培养基（3 mL）中以终止胰酶消化。收集细胞悬液，计数细胞并按1：3至1：4比例分瓶接种于25 cm²培养瓶中进行传代。培养3天后，将第3代（P3；群体倍增水平[PDL] 5.2）细胞冻存于Cellbanker（Takara Bio Inc.，Shiga，Japan）中，于–80°C保存备用。为进一步分析，将冻存细胞解冻并培养4天。随后，将细胞传代并以每孔4 × 10⁵个细胞的密度接种于24孔培养板（TPP，Trasadingen，Switzerland）中的完全培养基中，取P5、PDL10.1，每孔0.5 mL。
[内容请查看原文]
不同饥饿培养条件下FECs中Caspase 3/7活性的测定
为建立饥饿诱导凋亡的最佳培养条件，制备了六种含不同浓度FBS（0.5%、0.1%或0%）的饥饿培养基，每种均含或不含50 µg/mL ECGS。将已在完全培养基中培养43–44小时的FECs用D-PBS（0.5 mL）洗涤一次，然后在24孔板中向FECs加入完全培养基或六种饥饿培养基之一（每种条件三复孔，0.5 mL），孵育4小时，随后测定caspase-3/7活性，这是凋亡的关键指标（Taylor et al. 2008）。
孵育4小时后，移除培养基，替换为含20 mM 4-(2-羟乙基)-1-哌嗪乙磺酸、250 mM蔗糖、50 mM氯化钾、2.5 mM氯化镁和1 mM二硫苏糖醇的冰冷细胞裂解缓冲液（0.2 mL/孔）。随后将板在–80°C冷冻，然后在室温（22°C）下解冻一次。使用超声破碎仪UR-21P（Tomy，Tokyo，Japan）以最低功率水平（0–1）脉冲10–20秒，在冰上对细胞裂解物进行超声处理。使用Apo-ONE Homogeneous Caspase-3/7 Assay（Promega，Madison，WI，USA）按照制造商方案在96孔板（Corning）中测定Caspase-3/7活性。使用SpectraMax M3酶标仪（Molecular Devices，San Jose，CA，USA）获取485和530 nm处的荧光强度，结果以Bradford法（Bio-Rad，Hercules，CA，USA）定量的蛋白含量进行归一化。完全培养基组作为对照（相对caspase-3/7活性，以完全培养基对照的百分比表示）。
[内容请查看原文]
饥饿FECs中及贝前列素处理后的Caspase-3/7活性测定
按上述方法培养FECs并转移至24孔板。细胞分别与0.5 mL完全培养基（不含贝前列素）、不含贝前列素的饥饿培养基（0.1% FBS，不含ECGS）或含四种不同浓度贝前列素、beraprost-314d或beraprost-315d之一的饥饿培养基孵育4小时，浓度依据既往研究选择（Kajikawa et al. 1989；Kainoh et al. 1992）。每种条件共加入9个孔，形成三次独立的三复孔实验。孵育后，按上述方法测定caspase-3/7活性。
[内容请查看原文]
饥饿FECs中及贝前列素处理后Bax和Bcl2表达的定量
在单独的六孔培养板中制备细胞用于RNA提取。在此之前，将FECs与2.0 mL完全培养基、饥饿培养基或含四种浓度（0.1、0.5、1.0或5.0 nM）贝前列素（外消旋混合物）之一的饥饿培养基孵育4小时。孵育后，小心移除培养基，替换为NucleoSpin RNA（Macherey-Nagel，Düren，Germany）提供的350 µL裂解缓冲液。按照制造商说明提取和纯化RNA。使用ReverTra Ace™ qPCR RT Master Mix（Toyobo Co.，Ltd.，Osaka，Japan）对提取的RNA进行逆转录。互补DNA（cDNA）于–20°C保存备用。
使用THUNDERBIRD Next SYBR™ qPCR Mix（Toyobo Co.，Ltd.）在Applied Biosystems 7500 Real-Time PCR System（Thermo Fisher Scientific）上定量相对基因表达。用于扩增目标基因的寡核苷酸引物如下：β-actin（Jursza et al. 2014）（F：ATCAAGGAGAAGCTGTGCTACGT；R：CGTTGCCGATGGTGATCA）、Bax（Zhao et al. 2008）（F：CCGATGGCAACTTCAACTGGG；R：GTCAGCACTCCCGCCACAAAG）和Bcl2（Zhao et al. 2008）（F：GGGATGGCGTGAACTGG；R：CAGGAGAAATCAAACAGAGGC）。所有实验均进行三次，mRNA相对表达量采用2-ΔΔCt法计算。β-actin用作mRNA的内参基因。
[内容请查看原文]
通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记进行原位细胞死亡检测
对于转移酶dUTP缺口末端标记（TUNEL）染色，将FECs接种于4孔培养载玻片（Corning）上直至达到融合。按照上述基因表达实验中相同的饥饿/处理方案，细胞孵育4小时后，将培养基替换为冰冷的4%多聚甲醛（Fujifilm Wako Pure Chemical Corp.，大阪，日本）进行细胞固定。载玻片于4°C孵育25分钟，随后使用0.2% Triton X-100（Sigma-Aldrich，圣路易斯，MO，美国）在室温下透化5分钟。按照制造商推荐，使用DeadEnd荧光TUNEL系统（Promega）常规进行TUNEL染色。随后使用DAPI Fluoromount-G封片剂（Southern Biotechnology Associates，伯明翰，AL，美国）盖上干净的盖玻片（24 × 24 mm；Matsunami，大阪，日本），以对细胞核进行复染。根据Akbar和Kim（2002）的改良方法，通过计数绿色荧光细胞的数量来确定凋亡细胞数。简言之，每个重复使用Keyence一体化荧光显微镜BZ-X810（Keyence Ltd.，大阪，日本）在四个随机视野中至少计数150个细胞。TUNEL阳性细胞的百分比基于每个重复计数的总细胞数确定。
[内容请查看原文]
统计分析
所有实验均进行生物学三重复。每个重复包含三个技术重复，在统计分析前取平均值。采用单因素方差分析比较多组均值。饥饿条件间的差异采用双侧Dunnett检验，将各处理浓度与饥饿对照进行比较。三个处理组间效应的差异采用双尾Holm–Bonferroni检验。显著性水平设定为p < 0.05。
[内容请查看原文]
结果
[内容请查看原文]
建立FECs饥饿条件以诱导凋亡相关事件
为建立激活caspase-3/7的适宜培养基条件，将FECs在不同量FBS（含或不含ECGS补充）中孵育。完全培养基组作为对照（相对caspase-3/7活性，以完全培养基对照的百分比表示）。饥饿组中caspase-3/7平均活性范围为134.2%至215.1%（表S1）。尽管由于本评估的预实验性质和有限效能而省略了正式统计分析，但在减少或去除FBS/ECGS补充后，FECs中观察到caspase-3/7激活的明显趋势（图S1）。
基于这些结果，选择含0.1% FBS且不含ECGS作为后续药理学实验的最适饥饿条件。选择含0.1%血清（FBS）的条件是为了应对完全无血清条件下可能破坏内源性前列环素功能的风险，正如在人内皮细胞中已有报道（Spector等，1985）。
[内容请查看原文]
贝前列素及其组分异构体贝前列素-314d和贝前列素-315d对FECs中caspase-3/7活性的抑制作用
饥饿诱导的caspase-3/7激活被0.5 nM或更高浓度的贝前列素或贝前列素-314d处理显著抑制。在贝前列素-315d组中，在5 nM浓度下观察到抑制作用（p < 0.025）（图2A）。贝前列素组的caspase-3/7活性水平与贝前列素-314d组几乎相同（图2B），而贝前列素-315d组的活性水平显著更高。更多细节见表S2。
[图：贝前列素对饥饿诱导的FECs中caspase-3/7活化的影响。(A) 数据以散点图和平均值±]
[内容请查看原文]
贝前列素处理饥饿FECs中Bax/Bcl2的表达
饥饿条件下FECs中促凋亡Bax的平均相对基因表达与贝前列素浓度呈负相关（图3A）。与未处理组相比，1.0 nM或更高浓度贝前列素处理后，饥饿FECs中Bax表达显著下调（p < 0.05）。即使在高浓度下，贝前列素或其异构体处理也未引起抗凋亡Bcl2表达的显著变化。
[图：贝前列素对FECs中凋亡相关事件的影响。(A) Bax和Bcl2的相对表达，凋亡相]
[内容请查看原文]
贝前列素处理饥饿FECs中TUNEL法检测的DNA片段化
根据TUNEL阳性细胞核百分比确认，未用贝前列素处理的饥饿FECs中存在凋亡相关DNA片段化（图3B）。与未用贝前列素处理的饥饿FECs相比，贝前列素处理饥饿FECs显著减少了TUNEL阳性凋亡细胞数。在低至0.1 nM的浓度下，TUNEL阳性细胞数显著减少（p < 0.01）；然而，在贝前列素浓度高于1.0 nM时，TUNEL阳性细胞未进一步减少。
[内容请查看原文]
讨论
本研究的目的是建立一种体外模型，用于评估贝前列素及其立体异构体在饥饿条件下对猫内皮细胞的药理作用，并利用该模型评估贝前列素在这些细胞中抗凋亡作用的疗效及作用方式。通过测定不同营养剥夺细胞培养条件下猫内皮细胞中caspase 3/7活性，确定了猫内皮细胞发生凋亡相关事件的最佳饥饿条件。在研究贝前列素对饥饿诱导的猫内皮细胞死亡的保护性抗凋亡药理作用时，贝前列素的外消旋混合物及其异构体beraprost-314d表现出优于贝前列素异构体315d的caspase抑制效果。通过TUNEL染色法证实了贝前列素在该猫内皮细胞模型中的抗凋亡作用。此外，Bax相对表达的定量分析显示，贝前列素处理可剂量依赖性地下调Bax表达。
永生化细胞系通常比新鲜分离的细胞具有更高的存活能力和抗凋亡能力，且已成功尝试在永生化内皮来源细胞系（如人EA.hy926细胞）中诱导凋亡（Jia et al. 2012）。然而，内皮细胞因其来源不同而表现出不同的生物学行为（Thorin and Shreeve 1998）。通过异位表达SV40LT抗原和hTERT的相对稳健的永生化方案所建立的猫内皮细胞系能否在这些培养条件下用于诱导凋亡，尚未被探索。ECGS和FBS含有维持猫内皮细胞体外增殖所必需的营养物质，如蛋白质和生长因子。本研究结果表明，剥夺或减少ECGS和FBS可激活caspase-3/7并诱导猫内皮细胞发生凋亡相关事件。尽管仅使用0.1% FBS的饥饿条件被选为贝前列素药理学研究的最适条件，但所呈现的所有条件均可应用于未来有关猫内皮功能的各种药理学研究。
贝前列素是四种对映体的外消旋体，各单一异构体已被证明具有不同的亲和力和药效学特性（Kajikawa et al. 1989; Shen et al. 2019）。先前关于beraprost-314d与贝前列素药效学的比较研究表明：（1）在大鼠肺动脉中涉及内皮源性NO产生的舒张反应的估计半数最大有效浓度（EC50）在两组间相似（贝前列素4.6 µM vs. beraprost-314d 4.7 µM）；（2）在人肺动脉平滑肌细胞中升高cAMP的EC50，beraprost-314d比贝前列素低4.6倍。因此，有研究提出将贝前列素进行异构体分离以获得beraprost-314d可显著影响其药理学特性（Shen et al. 2019）。然而，在人医领域，一项随机、双盲、安慰剂对照的3期试验对口服beraprost-314d联合吸入曲前列尼尔进行了研究，以考察从基线至临床恶化的时间，但结果未能达到统计学显著性（Oudiz et al. 2019）。在本研究中，beraprost-314d对猫内皮细胞中饥饿诱导的caspase-3/7激活的抑制作用与贝前列素几乎一致，这与先前所述的大鼠肺动脉内皮源性NO产生的结果相符（Shen et al. 2019）。基于一项啮齿动物研究（Goto et al. 2014），贝前列素在猫慢性肾病中的药理靶点被认为主要是内皮而非血管平滑肌，这一观点得到以下证据的支持：临床剂量的贝前列素在健康猫中未显示血流动力学参数的变化（Takenaka et al. 2018）。因此，尽管需要进一步的体内验证，我们目前的结果引出以下假设：对映体分离未必能在改善贝前列素对猫慢性肾病患者临床疗效方面提供实质性优势。
然而，本研究并未探讨贝前列素诱导其抗凋亡效应的具体机制。贝前列素参与体外内皮细胞对抗氧化应激的维持（Kainoh等，1992），并在CKD和糖尿病啮齿动物模型的体内研究中发挥作用（Goto等，2014；Matsumoto等，2002）。贝前列素还被证明可通过蛋白激酶A和环磷酸腺苷（cAMP）信号通路刺激人和牛内皮细胞中的前列环素信号传导及一氧化氮（NO）产生（Niwano等，2003）。前列环素由内皮释放，与内皮细胞的细胞膜及核受体结合，作为自分泌因子调控细胞存活（Narumiya等，1999；Lim和Dey，2002；Cines等，1998；Hoffmann等，2001）。CKD常见于老年猫的诊断中（Sparkes等，2016）。内皮细胞衰老因NO合成减少而导致凋亡诱导增强，这被认为会进一步引起内皮细胞衰老，形成恶性循环（Hoffmann等，2001）。鉴于上述内容及本研究的发现，我们假设贝前列素对凋亡相关事件的减弱可能代表了一种保护猫CKD肾脏毛细血管网的潜在机制。然而，这是否涉及体内通过NO或前列环素信号通路对Bax和caspase-3通路的调控，目前仍不明确，需要进一步研究。
本研究存在若干局限性。本研究使用的体外饥饿模型不能完全再现猫CKD肾脏的复杂微环境，该微环境涉及尿毒症毒素、血流动力学改变以及复杂的细胞间相互作用。虽然使用了多种互补方法（caspase-3/7活性、TUNEL染色和Bax表达）来评估细胞死亡，但这些检测方法存在局限性，可能无法明确排除严重饥饿应激引起的其他形式的细胞死亡或DNA损伤。虽然我们的数据清楚地表明贝前列素可减弱培养FECs中这些凋亡相关事件，但尚待确定这种抑制作用是否直接有助于预防毛细血管稀疏或减缓CKD猫的肾功能障碍。我们目前的发现应被视为初步结果。需要进一步研究，包括体内研究和利用原代猫肾内皮细胞的实验，以验证这些发现与猫CKD的直接临床相关性。
综上所述，这些发现表明贝前列素处理即使在低浓度下也能抑制培养FECs中的凋亡相关事件。这些发现有助于理解贝前列素在抗凋亡和内皮保护作用方面的作用方式，这可能与CKD猫相关。未来，为进一步探索本研究的临床相关性，有必要使用与内皮损伤和NO失调相关的临床病理学标志物（如人类中所提出的那样（Jourde-Chiche等，2019））以及病理活检，在猫CKD患者中确认目前的发现。
[内容请查看原文]
结论
细胞培养基中的营养剥夺可诱导FECs中caspase-3/7活性并增加凋亡相关事件。贝前列素剂量依赖性地抑制caspase-3/7活化。异构体beraprost-314d的最低有效剂量与贝前列素一致，而异构体beraprost-315d的最低有效剂量更高。贝前列素即使在低浓度下也能抑制caspase-3/7活性，下调Bax表达，并抑制FEC死亡。
作者贡献
T.M.参与了数据管理、形式分析、研究、方法学、验证、可视化及写作——原始草稿。C.N.B.B.M.参与了数据管理、形式分析、研究、可视化及写作——原始草稿。T.Y.参与了概念化、数据管理、形式分析、资金获取、监督、验证、可视化及写作——审阅与编辑。
资金
作者无需报告任何内容。
利益冲突
T.M.是Toray Industries, Inc.的雇员。其余作者声明无利益冲突。
伦理声明
由于本研究仅使用已建立的猫细胞系进行，未涉及任何使用活体动物或原代组织采集的程序，因此无需伦理批准。
支持信息
[内容请查看原文]
[内容请查看原文]
查看英文原文▾
[内容请查看原文]
Introduction
Vascular endothelial cell senescence and apoptosis and the resulting capillary rarefaction, characterized by reduced density of microvascular networks, are hallmarks of age‐related kidney disorders (Tesfamariam and DeFelice 2007; Jourde‐Chiche et al. 2019; Li et al. 2022). In feline patients with chronic kidney disease (CKD), evidence of capillary rarefaction using quantitative immunohistochemistry in the affected kidneys shows a positive correlation with kidney dysfunction among other histopathological lesions (Paschall et al. 2023). Clinical data suggest that slowing the loss of kidney function by preserving the renal endothelium and capillary network functions (Goto et al. 2014) may prolong the lifespan of animals with CKD (Yamaguchi et al. 2013).
Beraprost is a chemically stable synthetic analogue of prostacyclin with high and long‐lasting activity after oral administration (Toda 1988; Niwano et al. 2003; Matsumoto et al. 2002). A previous clinical study suggested an association between beraprost treatment and changes in serum creatinine levels in feline patients with CKD (Takenaka et al. 2018); however, the direct effects of beraprost on overall survival and specific renal endothelial protection remain to be fully elucidated. Recently, Ito et al. (2023) reported that beraprost therapy is significantly associated with improved overall survival in cats with CKD. Beraprost is a racemic mixture consisting of two pairs of stereoisomers, beraprost‐314d and beraprost‐315d, and their enantiomers, in a total molar ratio of 1:1:1:1. Published data provide evidence that different isomers of beraprost possess different pharmacological potencies (Kajikawa et al. 1989; Shen et al. 2019); however, it is unknown whether these effects are species‐ and/or cell type‐specific, and what kind of effects they would induce in feline endothelial cells (FECs).
For in vitro experiments with FECs, the previously described immortalized FEC line was established using the simian virus 40 large T antigen (SV40LT) and the catalytic subunit of human telomerase reverse transcriptase (hTERT) (Olyslaegers et al. 2013). FECs have been shown to largely maintain the structural and functional phenotypes of primary endothelial cells, as well as an immunologic response to tumour necrosis factor (Olyslaegers et al. 2013), an inducer of apoptosis.
This study aimed to establish an in vitro FEC model using various nutritional starvation conditions to induce apoptosis and investigate how the racemic mixture of beraprost and its isomers (−314d and −315d) can provide a protective, anti‐apoptotic effect on FECs. Additionally, this study aimed to determine the minimum effective dose of beraprost required to induce protective (anti‐apoptotic) effects in FECs.
[内容请查看原文]
Materials and Methods
[内容请查看原文]
Chemicals and Reagents
Pharmaceutical‐grade beraprost and separated beraprost‐314d and beraprost‐315d isomers were manufactured and provided by Toray Industries, Inc. (Tokyo, Japan). These chemical structures are shown in Figure 1. These were dissolved with UltraPure distilled water (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 10 mM and sterilized with a 0.22‐µm filter. The solution was further diluted 10 times with the distilled water to 1 mM and kept at 4°C until use. Although chiral HPLC analysis was not performed in the present study to directly confirm post‐dissolution purity, the stereochemical stability of these isomers under these specific conditions has been validated by established protocols from previous pharmacological investigations (Shen et al. 2019). Because the compounds were maintained in a neutral aqueous solution at 4°C, the acidic conditions known to induce epimerization of prostaglandins were avoided. Furthermore, material data sheets provided by commercial suppliers confirm that these compounds remain stable in aqueous solutions for several weeks under refrigeration, ensuring their suitability and stability for our in vitro cell culture experiments.
[图：The chemical structures of the racemic beraprost, beraprost‐314d, and beraprost‐315d. Beraprost‐314d and its enantiomer ]
[内容请查看原文]
Cell Line and Cell Culture Conditions
The FECs were purchased from Applied Biological Materials (Richmond, Canada). These were derived from healthy feline aortas and immortalized as previously described (Olyslaegers et al. 2013). Following the manufacturer's instruction, the cells were cultured in Prigrow III Medium containing 10% fetal bovine serum (FBS), 50 µg/mL endothelial cell growth supplement (ECGS), 0.1 mg/mL gentamycin sulphate, 1 mM sodium pyruvate, 1% (v/v) non‐essential amino acids, 10 U/mL heparin sodium, and 1% penicillin‐streptomycin mixture (“complete medium”). The complete medium was stored at 4°C and was warmed to room temperature (22°C) before use.
FECs were grown in 25‐cm2 culture flasks (Corning Inc., Corning, NY, USA) at 37°C in a humidified 5% CO2 incubator (Thermo Fisher Scientific) until sub‐confluency in the logarithmic growth phase was reached. After aspirating the medium, the cells were washed twice with Dulbecco's phosphate‐buffered saline (D‐PBS; 5 mL), detached with 0.25% (w/v) trypsin‐1 mM ethylenediaminetetraacetic acid (0.3 mL), and resuspended in complete medium (3 mL) to stop trypsinization. Cell suspensions were collected, and the cells were counted and split 1:3 to 1:4 in 25‐cm2 culture flasks for passage. After 3 days of culture, the cells at passage 3 (P3;population doubling level [PDL] 5.2) were cryopreserved in Cellbanker (Takara Bio Inc., Shiga, Japan) at –80°C until use. For further analysis, the frozen stored cells were thawed and cultured for 4 days. Subsequently, the cells were passaged and seeded in complete medium in 24‐well culture plates (TPP, Trasadingen, Switzerland) at a density of 4 × 105 cells per well at P5, PDL10.1 per well (0.5 mL/well).
[内容请查看原文]
Determination of Caspase 3/7 Activity in FECs Under Various Starvation Culture Conditions
To establish the optimum culture condition for starvation‐induced apoptosis, six starvation media were prepared with varying concentrations of FBS (0.5%, 0.1%, or 0%), each with or without 50 µg/mL ECGS. FECs, which had been cultured for 43–44 h in complete medium, were washed once with D‐PBS (0.5 mL), and then either complete medium or one of six starvation media were added to the FECs in a 24‐well plate (triplicates per condition, 0.5 mL), and incubated for 4 h, before measuring the caspase‐3/7 activity, a key indicator of apoptosis (Taylor et al. 2008).
After 4 h of incubation, the medium was removed and replaced by ice‐chilled cell lysis buffer containing 20 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid, 250 mM sucrose, 50 mM potassium chloride, 2.5 mM magnesium chloride, and 1 mM dithiothreitol (0.2 mL/well). The plates were then frozen at –80°C followed by thawing at room temperature (22°C) once. The cell lysates were sonicated on ice using an ultrasonic disruptor UR‐21P (Tomy, Tokyo, Japan) at a minimum power level (0–1) for 10‐ to 20‐s pulse. Caspase‐3/7 activity was measured in 96‐well plates (Corning) using the Apo‐ONE Homogeneous Caspase‐3/7 Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. The fluorescence intensities at 485 and 530 nm were obtained using a SpectraMax M3 plate reader (Molecular Devices, San Jose, CA, USA), and the results were normalized to the protein content quantified using the Bradford assay (Bio‐Rad, Hercules, CA, USA). The complete medium group served as a control (relative caspase‐3/7 activity, % of complete‐medium control).
[内容请查看原文]
Determination of Caspase‐3/7 Activity in Starved FECs and Upon Treatment With Beraprost
FECs were cultured as described above and transferred to 24‐well plates. The cells were incubated for 4 h with either 0.5 mL of complete medium (without beraprost), starvation medium (0.1% FBS without ECGS) without beraprost, or starvation medium with one of four different concentrations of beraprost, beraprost‐314d, or beraprost‐315d, which were selected based on previous studies (Kajikawa et al. 1989; Kainoh et al. 1992). Each condition was added to a total of nine wells, resulting in three independent triplicate experiments. After incubation, the caspase‐3/7 activity was measured as described above.
[内容请查看原文]
Quantification of Bax and Bcl2 Expression in Starved FECs and Upon Treatment With Beraprost
The cells were prepared for RNA extraction in separate six‐well culture plates. Prior to this, FECs were incubated for 4 h with either 2.0 mL of complete medium, starvation medium, or starvation medium with one of four concentrations (0.1, 0.5, 1.0, or 5.0 nM) of beraprost (racemic mixture). After incubation, the medium was carefully removed and replaced by 350 µL of the lysis buffer provided by NucleoSpin RNA (Macherey‐Nagel, Düren, Germany). RNA was extracted and purified according to the manufacturer's instructions. The extracted RNA was reverse‐transcribed using the ReverTra AceTM qPCR RT Master Mix (Toyobo Co., Ltd., Osaka, Japan). The complementary DNA (cDNA) was stored at –20°C until use.
Relative gene expression was quantified using THUNDERBIRD Next SYBRTM qPCR Mix (Toyobo Co., Ltd.) on an Applied Biosystems 7500 Real‐Time PCR System (Thermo Fisher Scientific). The oligonucleotide primers used for the amplification of the target genes were as follows: β‐actin (Jursza et al. 2014) (F: ATCAAGGAGAAGCTGTGCTACGT; R: CGTTGCCGATGGTGATCA), Bax (Zhao et al. 2008) (F: CCGATGGCAACTTCAACTGGG; R: GTCAGCACTCCCGCCACAAAG), and Bcl2 (Zhao et al. 2008) (F: GGGATGGCGTGAACTGG; R: CAGGAGAAATCAAACAGAGGC). All assays were performed three times, and the relative expression of mRNA was calculated using the 2‐ΔΔCt method. β‐actin was used as the reference gene for mRNA.
[内容请查看原文]
In Situ Cell Death Detection via Terminal Deoxynucleotidyl Transferase‐Mediated dUTP Nick End Labelling
For transferase dUTP nick end labelling (TUNEL) staining, FECs were seeded onto 4‐well culture slides (Corning) until they reached confluence. Following the same starvation/treatment protocol as in the gene expression assay described above, the cells were incubated for 4 h before replacing the culture medium with ice‐cold 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) for cell fixation. The slides were incubated at 4°C for 25 min and were subsequently permeabilized using 0.2% Triton X‐100 (Sigma‐Aldrich, St. Lois, MO, USA) for 5 min at room temperature. TUNEL staining was performed routinely using the DeadEnd Fluorometric TUNEL System (Promega), as recommended by the manufacturer. The stained culture slides were then covered with a clean cover glass (24 × 24 mm; Matsunami, Osaka, Japan) using DAPI Fluoromount‐G mounting medium (Southern Biotechnology Associates, Birmingham, AL, USA) to counterstain the cell nuclei. The number of apoptotic cells was determined by counting the number of green fluorescent cells following a modified method of Akbar and Kim (2002). Briefly, at least 150 cells were counted in four random fields per replicate using a Keyence All‐in‐One Fluorescence microscope BZ‐X810 (Keyence Ltd., Osaka, Japan). The percentage of TUNEL‐positive cells was determined based on the total number of cells counted per replicate.
[内容请查看原文]
Statistical Analysis
All experiments were done in biological triplicate. Each consisted of three technical replicates, which were averaged prior to statistical analysis. One‐way analysis of variance was used to compare the means of multiple groups. The differences between starvation conditions were tested by a two‐sided Dunnett's test to compare each treatment concentration with the starvation control. The differences in effects among the three treatment groups were tested by a two‐tailed Holm–Bonferroni test. The significance levels were set at p < 0.05.
[内容请查看原文]
Results
[内容请查看原文]
Establishment of Starvation Conditions in FECs to Induce Apoptosis‐Associated Events
To establish the appropriate culture medium conditions for activating caspase‐3/7, FECs were incubated with different amounts of FBS with or without ECGS supplementation. The complete medium group served as a control (relative caspase‐3/7 activity, % of complete‐medium control). Mean caspase‐3/7 activity in the starvation group ranged from 134.2% to 215.1% (Table S1). Although formal statistical analysis was omitted due to the pilot nature and limited power of this evaluation, a clear trend of caspase‐3/7 activation was observed in FECs following the reduction or removal of FBS/ECGS supplementation (Figure S1).
Based on these results, 0.1% FBS without ECGS was selected as the most suitable starvation condition for the subsequent pharmacological experiments. This condition containing 0.1% serum (FBS) was chosen to address the risk of disrupting endogenous prostacyclin function, which can occur under completely serum‐free conditions, as has been reported in human endothelial cells (Spector et al. 1985).
[内容请查看原文]
Inhibitory Effects of Beraprost, and Its Component Isomers Beraprost‐314d, and Beraprost‐315d on Caspase‐3/7 Activity in FECs
Starvation‐induced activation of caspase‐3/7 was significantly inhibited by treatment with beraprost or beraprost‐314d at concentrations of 0.5 nM or higher. In the beraprost‐315d group, inhibitory effects were observed at a concentration of 5 nM (p < 0.025) (Figure 2A). Caspase 3/7 activity levels in the beraprost group were nearly identical to those in the beraprost‐314d group (Figure 2B), whereas activity levels were significantly higher in the beraprost‐315d group. Additional details are provided in Table S2.
[图：Effects of beraprost on starvation‐induced caspase‐3/7 activation in FECs. (A) The data represent dot plots and means ± ]
[内容请查看原文]
Bax/Bcl2 Expression in Starved FECs When Treated With Beraprost
The mean relative gene expression of pro‐apoptotic Bax in FECs under starvation showed an inverse relationship with the beraprost concentration (Figure 3A). Significant downregulation of Bax expression in starved FECs was apparent after beraprost treatment at 1.0 nM or higher (p < 0.05) compared to the non‐treated group. Treatment with beraprost or its isomers, even at high concentrations, did not cause significant changes in the expression of the anti‐apoptotic Bcl2.
[图：Effects of beraprost against apoptosis‐associated events in FECs. (A) Relative expression of Bax and Bcl2 , apoptosis‐re]
[内容请查看原文]
DNA Fragmentation as Indicated by TUNEL Assay in Starved FECs When Treated With Beraprost
Apoptosis‐associated DNA fragmentation was confirmed in starved FECs without beraprost treatment, based on the percentage of TUNEL‐positive cell nuclei (Figure 3B). Treatment of starved FECs with beraprost significantly reduced the number of TUNEL‐positive apoptotic cells compared to starved FECs without beraprost treatment. At concentrations as low as 0.1 nM, the number of TUNEL‐positive cells was significantly reduced (p < 0.01); however, no further reduction of TUNEL‐positive cells was achieved at beraprost concentrations above 1.0 nM.
[内容请查看原文]
Discussion
The purpose of this study was to establish an in vitro model to evaluate the pharmacological effects of beraprost and its stereoisomers on FECs under starvation conditions and to use this model to assess the efficacy and mode of the anti‐apoptotic effect of beraprost in these cells. The optimal starvation conditions for FECs under which apoptosis‐associated events occurs were identified by measuring caspase 3/7 activity in FECs under various nutrient‐deprived cell culture conditions. When investigating the pharmacological anti‐apoptotic effects of beraprost against starvation‐induced cell death of FECs, the racemic mixture of beraprost and its isomer, beraprost‐314d, exhibited better caspase inhibitory efficacy than beraprost isomer 315d. The anti‐apoptotic effect of beraprost in this FEC model was confirmed using TUNEL staining assay. Furthermore, quantitative analysis of the relative Bax expression revealed a dose‐dependent downregulation with beraprost treatment.
Immortalized cell lines often have higher survival and resistance to apoptosis than freshly isolated cells, and successful attempts have been made to induce apoptosis in immortalized endothelium‐derived cell lines such as human EA.hy926 cells (Jia et al. 2012). However, endothelial cells display different biological behaviours depending on their origin (Thorin and Shreeve 1998). Whether FEC lines created by a relatively robust immortalization protocol via ectopic expression of the SV40LT antigen and hTERT can be used to induce apoptosis under these culture conditions has not been explored. ECGS and FBS contain nutrients such as proteins and growth factors necessary for the maintenance and proliferation of FECs in vitro. The present findings demonstrate that deprivation or reduction of ECGS and FBS activates caspase‐3/7 and induces apoptosis‐associated events in FECs. Although starvation using 0.1% FBS alone was selected as the most suitable condition for the pharmacological study of beraprost, all the presented conditions can be applied to various future pharmacological studies on feline endothelial functions.
Beraprost is a racemate of four enantiomers, and the individual isomers have been shown to represent different affinities and pharmacodynamic properties (Kajikawa et al. 1989; Shen et al. 2019). The previous comparative studies between the pharmacodynamics of beraprost‐314d and beraprost demonstrate the following: (1) the estimated half maximal effective concentration (EC50) for relaxation involving endothelial‐derived NO production in rat pulmonary artery was similar between the groups (beraprost 4.6 µM vs. beraprost‐314d 4.7 µM) and (2) EC50 of elevation of cAMP in human pulmonary arterial smooth muscle cells was 4.6‐fold lower in beraprost‐314d compared to beraprost. Therefore, isomer separation of beraprost to yield beraprost‐314d has been proposed to greatly affect its pharmacology (Shen et al. 2019). However, in human medicine, a randomized, double‐blind, placebo‐controlled phase 3 trial of oral administration of beraprost‐314d combined with inhaled treprostinil was carried out to examine the time to clinical worsening from baseline, but the outcome failed to achieve statistical significance (Oudiz et al. 2019). In the present study, the inhibitory effects of beraprost‐314d on starvation‐induced activation of caspase‐3/7 in FECs were almost consistent with those of beraprost, which correspond to previously described endothelial‐derived NO production in rat pulmonary arteries (Shen et al. 2019). The pharmacological target of beraprost in feline CKD has been assumed to be mainly the endothelium rather than the vascular smooth muscle based on a rodent study (Goto et al. 2014), which is supported by evidence that the clinical dose of beraprost does not show changes in hemodynamic parameters in healthy cats (Takenaka et al. 2018). Consequently, while further in vivo validation is required, our present results lead to the hypothesis that enantiomer isolation may not necessarily provide a substantial advantage in improving the clinical effectiveness of beraprost in patients with feline CKD.
However, the specific mechanism by which beraprost induces its anti‐apoptotic effects was not investigated in this study. Beraprost is involved in endothelial cell maintenance against oxidative stress in vitro (Kainoh et al. 1992) and in vivo in CKD and diabetes rodent models (Goto et al. 2014; Matsumoto et al. 2002). Beraprost has also been shown to stimulate prostacyclin signalling and nitric oxide (NO) production through protein kinase A and cyclic adenosine monophosphate (cAMP) signalling in human and bovine endothelial cells (Niwano et al. 2003). Prostacyclin is released from the endothelium, bound to cellular and nuclear receptors of endothelial cells, and regulates cell survival as an autocrine factor (Narumiya et al. 1999 Lim and Dey 2002; Cines et al. 1998; Hoffmann et al. 2001). CKD is commonly diagnosed in aged cats (Sparkes et al. 2016). Aging of endothelial cells leads to enhanced induction of apoptosis due to reduced NO synthesis, which is proposed to cause further endothelial; cell aging and result in a viscous cycle (Hoffmann et al. 2001). Given the above and the present findings, we hypothesize that the attenuation of apoptosis‐associated events by beraprost may represent a potential mechanism for preserving the capillary network in feline CKD kidneys. However, whether this involves the regulation of the Bax and caspase‐3 pathways via NO or prostacyclin signalling in vivo remains elusive and requires further investigation.
There are several limitations of this study. The in vitro starvation model used in this study does not fully recapitulate the complex microenvironment of feline CKD kidneys, which involves uremic toxins, hemodynamic alterations, and intricate cell–cell interactions. While multiple complementary approaches (caspase‐3/7 activity, TUNEL staining, and Bax expression) were used to evaluate cell death, these assays have limitations and may not definitively rule out other forms of cell death or DNA damage caused by severe starvation stress. While our data clearly demonstrate that beraprost attenuates these apoptosis‐associated events in cultured FECs, it remains to be determined whether this suppression directly contributes to the prevention of capillary rarefaction or the slowing of kidney dysfunction in cats with CKD. Our current findings should be considered preliminary. Further investigations, including in vivo studies and experiments utilizing primary feline renal endothelial cells, are necessary to validate the direct clinical relevance of these findings to feline CKD.
Taken together, these findings indicate that beraprost treatment inhibits apoptosis‐associated events in cultured FECs, even at low concentrations. These findings contribute to the understanding of the mode of action of beraprost in terms of its anti‐apoptotic and endothelial‐protective effects, which may be relevant to cats with CKD. In the future, to further explore the clinical relevance of this study, it will be necessary to confirm the current findings in feline patients with CKD using clinicopathological markers associated with endothelial injury and NO dysregulation, as proposed in humans (Jourde‐Chiche et al. 2019), and pathological biopsies.
[内容请查看原文]
Conclusions
Nutrient deprivation in cell culture media induces caspase‐3/7 activity and increases apoptosis‐associated events in FECs. Beraprost dose‐dependently inhibited caspase‐3/7 activation. The minimum effective dose of the isomer beraprost‐314d was consistent with that of beraprost, whereas that of the isomer beraprost‐315d was higher. Beraprost inhibited caspase 3/7 activity even at low concentrations, downregulated Bax expression, and inhibited FEC death.
Author Contributions
T.M. contributed to data curation, formal analysis, investigation, methodology, validation, visualization, and writing – original draft. C.N.B.B.M. contributed to data curation, formal analysis, investigation, visualization, and writing – original draft. T.Y. contributed to conceptualization, data curation, formal analysis, funding acquisition, supervision, validation, visualization, and writing – review and editing.
Funding
The authors have nothing to report.
Conflicts of Interest
T.M. is an employee of Toray Industries, Inc. The remaining authors declare no conflicts of interest.
Ethics Statement
Since this study was conducted exclusively using an established feline cell line and did not involve any procedures utilizing live animals or primary tissue collections, ethical approval was not required.
Supporting information
[内容请查看原文]
[内容请查看原文]