PENGARUH KNOCK OUT GEN BMPR2 TERHADAP FUNGSI MAKROFAG RAW264.7 PADA PENSINYALAN SELULER DAN INTEGRITAS PEMBULUH DARAH

BINTANG TATIUS, dr. Widya Wasityastuti, M.Sc, M.Med.Ed, Ph.D; dr. Dwi Aris Agung Nugrahaningsih, M.Sc, Ph.D

2022 | Tesis | MAGISTER ILMU BIOMEDIK

Latar Belakang: Gen BMPR2 berperan dalam menjaga homeostasis fungsi sistem imun. Makrofag merupakan komponen sistem imun yang regulasinya diatur oleh BMPR2. Mutasi BMPR2 pada makrofag tidak hanya mengganggu fungsi, tetapi juga mengganggu berbagai pensinyalan selulernya yang dapat diamati melalui ekspresi CD68, KCNA2, dan CLIC1. Interaksi makrofag disfungsional dengan pembuluh darah dapat mengganggu integritas vaskuler. Tujuan: mengkaji pengaruh knock out (KO) gen BMPR2 terhadap ekspresi mRNA CD68, KCNA2, dan CLIC1 pada makrofag secara in-vitro dan mengkaji perubahan integritas pembuluh darah serta ekspresi mRNA CD68, KCNA2, CLIC1, dan GLUT4 secara ex-vivo sebagai respon terhadap gangguan pensinyalan seluler akibat paparan supernatan makrofag BMPR2 KO Metode: Cell line makrofag RAW 264.7 dilakukan knock out gen BMPR2 menggunakan metode CRISPR/Cas9 (Kelompok BMPR2 KO, n=3), kemudian ekspresi mRNA CD68, KCNA2, dan CLIC1 dibandingkan dengan makrofag BMPR2 wildtype (WT) (n=3). Selanjutnya, enam aorta yang diekstraksi dari mencit jantan usia 3 bulan dibagi menjadi 2 kelompok yang dikultur dalam conditioned medium berisikan supernatan makrofag BMPR2 KO (n=3) dan BMPR2 WT (n=3) dengan konsentrasi 75% supernatan dan 25% DMEM. Setelah 48 jam, gambaran histologis aorta dengan pewarnaan HE dan pengukuran ekspresi mRNA CD68, KCNA2, CLIC1, dan GLUT4 diamati. Pengukuran ekspresi mRNA makrofag in-vitro dan aorta ex-vivo dilakukan dengan RT-qPCR. Hasil: Ekspresi mRNA CD68 dan KCNA2 lebih rendah serta CLIC1 lebih tinggi signifikan (p<0.05) pada makrofag BMPR2 KO dibandingkan BMPR2 WT. Paparan supernatan makrofag BMPR2 KO pada aorta menyebabkan gangguan integritas vaskuler secara histopatologis dan ekspresi mRNA CD68, KCNA2, CLIC1, dan GLUT4 yang lebih tinggi (p<0.05) dibandingkan kelompok kontrol. Kesimpulan: Knock out BMPR2 menyebabkan disfungsi makrofag melalui perubahan ekspresi CD68, KCNA2, dan CLIC1. Akibatnya, pensinyalan seluler pada pembuluh darah mengalami perubahan dan integritas vaskuler terganggu.

Introduction: BMPR2 plays important role in maintaining homeostasis of immune system function. Macrophages are components of the immune system whose regulation is established by BMPR2. BMPR2 mutation in macrophages not only impairs function, but also impair their various cellular signaling which can be observed through the expression of CD68, KCNA2, and CLIC1. The interaction of dysfunctional macrophages with blood vessels can impair vascular integrity. Aim: To study the effect of BMPR2 gene knockout on CD68, KCNA2, and CLIC1 mRNA expression in macrophages in-vitro and investigate changes in blood vessel integrity and ex-vivo expression of CD68, KCNA2, CLIC1, and GLUT4 mRNA in response to cellular signaling disturbances due to exposure to macrophage BMPR2 KO supernatant Methods: RAW 264.7 macrophage cell line was knocked out of the BMPR2 gene using the CRISPR/Cas9 method (BMPR2 KO group, n=3), then mRNA expression of CD68, KCNA2, and CLIC1 was compared with wildtype (WT) BMPR2 macrophages (n=3). Furthermore, six aortas extracted from 3-months-old male mice were divided into 2 groups which were cultured in conditioned medium containing macrophages BMPR2 KO (n=3) and BMPR2 WT (n=3) supernatants with concentration of 75% supernatant and 25% DMEM each. After 48 h, aorta histology slide with HE staining and measurement of CD68, KCNA2, CLIC1, and GLUT4 mRNA expression were observed. mRNA expression of macrophage in-vitro and aorta ex-vivo was performed using RT-qPCR. Results: CD68 and KCNA2 mRNA expression was lower while CLIC1 was higher significantly (p<0.05) in BMPR2 KO macrophages compared to BMPR2 WT. Exposure to BMPR2 KO macrophages supernatant showed impaired vascular integrity and higher mRNA expression of CD68, KCNA2, CLIC1, and GLUT4 in the aorta (p<0.05) compared to control group. Conclusion: BMPR2 knockout causes macrophage dysfunction through altered expression of CD68, KCNA2, and CLIC1. As the result, cellular signaling and vascular integrity of blood vessels are compromised.

Kata Kunci : BMPR2, makrofag, pro-inflamasi, pro-proliferatif, aorta, CD68, KCNA2, CLIC1, GLUT4