Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Escuela de Química y Farmacia, Facultad de Medicina, Universidad Andres Bello, 2520000, Viña del Mar, Chile.
Mol Biol Rep. 2019 Oct;46(5):5197-5207. doi: 10.1007/s11033-019-04977-3. Epub 2019 Jul 15.
Cardiac myofibroblast (CMF) are non-muscle cardiac cells that play a crucial role in wound healing and in pathological remodeling. These cells are mainly derived of cardiac fibroblast (CF) differentiation mediated by TGF-β1. Evidence suggests that bradykinin (BK) regulates cardiac fibroblast function in the heart. Both B1 and B2 kinin receptors (B1R and B2R, respectively) mediate the biological effects of kinins. We recently showed that both receptors are expressed in CMF and its stimulation decreases collagen secretion. Whether TGF-β1 regulates B1R and B2R expression, and how these receptors control antifibrotic activity in CMF remains poorly understood. In this work, we sought to study, the regulation of B1R expression in cultured CMF mediated by TGF-β1, and the molecular mechanisms involved in B1R activation on CMF intracellular collagen type-I levels. Cardiac fibroblast-primary culture was obtained from neonatal rats. Hearts were digested and CFs were attached to dishes and separated from cardiomyoctes. CMF were obtained from CF differentiation with TGF-β1 5 ng/mL. CF and CMF were treated with B1R and B2R agonists and with TGF-β1 at different times and concentrations, in the presence or absence of chemical inhibitors, to evaluate signaling pathways involved in B1R expression, collagen type-I and prostacyclin levels. B1R and collagen type-I levels were evaluated by western blot. Prostacyclin levels were quantified by an ELISA kit. TGF-β1 increased B1R expression via TGFβ type I receptor kinase (ALK5) activation and its subsequent signaling pathways involving Smad2, p38, JNK and ERK1/2 activation. Moreover, in CMF, the activation of B1R and B2R by their respective agonists, reduced collagen synthesis. This effect was mediated by the canonical signaling pathway; phospholipase C (PLC), protein kinase C (PKC), phospholipase A (PLA), COX-2 activation and PGI secretion and its autocrine effect. TGF-β1 through ALK5, Smad2, p38, JNK and ERK1/2 increases B1R expression; whereas in CMF, B1R and B2R activation share common signaling pathways for reducing collagen synthesis.
心肌成纤维细胞（CMF）是一种非肌肉心脏细胞，在伤口愈合和病理性重塑中起着至关重要的作用。这些细胞主要来源于转化生长因子-β1（TGF-β1）介导的心肌成纤维细胞（CF）分化。有证据表明，缓激肽（BK）调节心脏中的心肌成纤维细胞功能。B1 和 B2 激肽受体（B1R 和 B2R）分别介导激肽的生物学效应。我们最近表明，这两种受体都在 CMF 中表达，其刺激可减少胶原蛋白的分泌。TGF-β1 是否调节 B1R 和 B2R 的表达，以及这些受体如何控制 CMF 中的抗纤维化活性，目前还知之甚少。在这项工作中，我们试图研究 TGF-β1 介导的培养 CMF 中 B1R 表达的调节，以及 B1R 激活对 CMF 细胞内 I 型胶原蛋白水平的分子机制。从新生大鼠中获得原代心肌成纤维细胞培养物。将心脏消化并将 CF 附着在培养皿上，与心肌细胞分离。用 5ng/ml TGF-β1 诱导 CF 分化获得 CMF。用 B1R 和 B2R 激动剂以及不同时间和浓度的 TGF-β1 处理 CF 和 CMF，在存在或不存在化学抑制剂的情况下，评估参与 B1R 表达、I 型胶原蛋白和前列环素水平的信号通路。通过 Western blot 评估 B1R 和胶原蛋白 I 型水平。通过 ELISA 试剂盒定量测定前列环素水平。TGF-β1 通过 TGFβ 型 I 受体激酶（ALK5）的激活及其随后涉及 Smad2、p38、JNK 和 ERK1/2 激活的信号通路增加 B1R 表达。此外，在 CMF 中，B1R 和 B2R 的各自激动剂的激活减少了胶原蛋白的合成。这种作用是通过经典信号通路介导的；磷脂酶 C（PLC）、蛋白激酶 C（PKC）、磷脂酶 A（PLA）、COX-2 激活和 PGI 分泌及其自分泌效应。TGF-β1 通过 ALK5、Smad2、p38、JNK 和 ERK1/2 增加 B1R 表达；而在 CMF 中，B1R 和 B2R 的激活共享减少胶原蛋白合成的共同信号通路。
