Tsuboya Naoki, Sawada Hirofumi, Mitani Yoshihide, Oshita Hironori, Ohya Kazunobu, Takeoka Mami, Kabwe Jane Chanda, Miyasaka Yoshiki, Ito Hiromasa, Yodoya Noriko, Ohashi Hiroyuki, Maruyama Junko, Okamoto Ryuji, Mashimo Tomoji, Dohi Kaoru, Nishimura Yuhei, Maruyama Kazuo, Hirayama Masahiro
The Department of Pediatrics, Mie University Graduate School of Medicine, Mie, Japan.
The Department of Pediatrics, Nagoya City University School of Medicine, Aichi, Japan.
Cardiovasc Res. 2025 Jul 8;121(7):1076-1090. doi: 10.1093/cvr/cvae244.
We investigated whether the disruption of C-C motif chemokine receptor (CCR) 2 may attenuate the development of pulmonary arterial hypertension (PAH) in any rat models with the reversal of the associated pro-inflammatory state and vascular dysfunction, and synergize with a conventional pulmonary vasodilator.
Using Ccr2(-/-) rats generated by CRISPR/Cas9, we investigated pulmonary hypertension (PH) in Ccr2(+/+) or Ccr2(-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx) and chronic hypoxia (CH). Ccr2(-/-) decreased the right ventricular systolic pressure, an index of right ventricular hypertrophy and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines [interleukin-6, tumour necrosis factor-α, C-C motif chemokine receptor (CCL)-2, interleukin-1β, transforming growth factor-β] in rats 3weeks after MCT injection, but not in SuHx or CH models. Consistently, Ccr2(-/-) decreased indices of pulmonary vascular diseases (PVDs) and perivascular macrophage infiltration, as well as reversed impaired bone morphogenetic protein receptor type 2 signalling, increased endothelial apoptosis and impaired nitric oxide signalling and decreased phosphodiesterase-5 (PDE5) expression in lungs in MCT-treated rats. Gene expression of receptors for prostaglandin I2 and endothelin was not changed by Ccr2(-/-) in MCT-treated rats. In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2(-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. The whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2(-/-) PASMCs, which are related to the regulation of cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2(-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2(-/-) rats but not in Ccr2(+/+) rats. Tadalafil further improved survival in MCT-treated Ccr2(-/-) rats.
The present findings demonstrated that CCR2 disruption ameliorated PAH in MCT-treated rats, which was associated with the reversal of dysregulated inflammatory pathways and vascular dysfunction and synergized with tadalafil. These findings suggest that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.
我们研究了C-C基序趋化因子受体（CCR）2的破坏是否能减轻任何大鼠模型中肺动脉高压（PAH）的发展，逆转相关的促炎状态和血管功能障碍，并与传统的肺血管扩张剂协同作用。
使用通过CRISPR/Cas9技术构建的Ccr2(-/-)大鼠，我们研究了用野百合碱（MCT）、SU5416/低氧（SuHx）和慢性低氧（CH）处理的Ccr2(+/+)或Ccr2(-/-)大鼠的肺动脉高压（PH）情况。Ccr2(-/-)降低了右心室收缩压，这是右心室肥厚和死亡率的一个指标，并逆转了MCT注射后3周大鼠体内炎性细胞因子/趋化因子[白细胞介素-6、肿瘤坏死因子-α、C-C基序趋化因子配体（CCL）-2、白细胞介素-1β、转化生长因子-β]表达的增加，但在SuHx或CH模型中未出现这种情况。同样，Ccr2(-/-)降低了肺血管疾病（PVDs）指标和血管周围巨噬细胞浸润，还逆转了MCT处理大鼠肺中骨形态发生蛋白受体2信号受损、内皮细胞凋亡增加、一氧化氮信号受损以及磷酸二酯酶-5（PDE5）表达降低的情况。在MCT处理的大鼠中，Ccr2(-/-)未改变前列腺素I2和内皮素受体的基因表达。在培养的肺动脉平滑肌细胞（PASMCs）中，Ccr2(-/-)抑制了CCL2诱导的过度增殖和去分化，并逆转了CCL2诱导的PDE5表达降低。全基因组RNA测序分析确定了CCL2刺激的Ccr2(-/-)PASMCs中差异表达的基因，这些基因与细胞分化和收缩的调节有关。基于对大鼠和培养的PASMCs的研究，我们研究了一种PDE5抑制剂他达拉非是否与Ccr2(-/-)协同作用。给予他达拉非改善了MCT处理的Ccr2(-/-)大鼠的PH和PVDs，但对Ccr2(+/+)大鼠无效。他达拉非进一步提高了MCT处理的Ccr2(-/-)大鼠的存活率。
目前的研究结果表明，CCR2破坏改善了MCT处理大鼠的PAH，这与失调的炎症途径和血管功能障碍的逆转有关，并与他达拉非协同作用。这些发现表明，CCR2可能是某些具有特定CCR2相关炎症表型且对传统肺血管扩张剂难治的顽固性PAH患者的治疗靶点。
