4121 The beneficial, anti-fibrotic effects of chemokine receptor 2 and 5 antagonists on fat-exposed mouse primary hepatic stellate cells (pHSCs)

• Published in: Journal of clinical and translational science 2020-06, Vol.4 (s1), p.102-103
• Main Authors: Kruger, Annie J., Bergman, Kruger, Gay, Martha, Cao, Hong, Tucker, Robin, Shivapurkar, Narayan, Smith, Jill P.
• Format: Article
• Language: English
• Subjects: Actin; CC chemokine receptors; CCR2 protein; CCR5 protein; Cell viability; Chemokines; Choline; Cirrhosis; Collagen; Collagen (type I); Drug dosages; Gelatinase B; Gene expression; High fat diet; Immunosuppressive agents; Leukocyte migration; Liver cirrhosis; Matrix metalloproteinase; Mechanistic Basic to Clinical; Metalloproteinase; Monocyte chemoattractant protein 1; Nutrient deficiency; Population studies; Smooth muscle; Stellate cells; Tissue inhibitor of metalloproteinase 1; Transforming growth factor-b
• ISSN: 2059-8661; 2059-8661
• DOI: 10.1017/cts.2020.317

OBJECTIVES/GOALS: Non-alcoholic steatohepatitis (NASH) is a leading cause of cirrhosis in the world for which no anti-fibrotic therapies exist. We hypothesized that BMS-22 and maraviroc (MVC), chemokine receptor 2 (CCR2) and 5 (CCR5) antagonists, respectively, would diminish the fibrogenic activity of "fat-exposed" murine pHSCs. METHODS/STUDY POPULATION: pHSCs were isolated from livers of 6 week old male mice following 4 weeks on a NASH-inducing choline-deficient high fat diet (CDAHFD, “fat-exposed”) or standard diet (SD) and passaged in vitro . Early passage (6-12) pHSCs were plate-adhered and TGF-b-treated (10ng/mL) to maximally activate their pro-fibrogenic genes, collagen 1α1 ( Col1A1), tissue inhibitor of metalloproteinase 1 ( TIMP1) , or α-smooth muscle actin (ACTA2 ). CDAHFD and SD pHSCs were then treated for 48 hours with increasing doses of BMS-22 or MVC (range: 0.3-120ng/mL) to determine (1) the degree of attenuation of the pro-fibrogenic response as measured by qPCR of fibrogenic genes ( Col1A1, TIMP1,ACTA2); (2) enhancement of a fibrolytic response as measured by qPCR of matrix metalloproteinases (MMP ) 2, 9 and 13 genes; and (3) pHSC migration using the scratch assay. Cell viability and CCR2 and CCR5 gene expression in response to escalating doses of antagonists were also measured. RESULTS/ANTICIPATED RESULTS: Plate- and TGF-b activated CDAHFD pHSCs had a 2-fold greater, dose-dependent attenuation of their pro-fibrogenic activity in response to BMS-CCR2-22 and MVC, when compared with plate- and TGF-b activated SD pHSCs, as measured by reductions in collagen 1α1 ( Col1A1 ) and α-smooth muscle actin ( ACTA2 ) gene expression. TIMP1 gene expression was unaffected by drug treatment for 48 hours. Cell viability was not affected up to doses of 30ng/mL of each drug. pHSCs also demonstrated a dose-dependent increase in CCR2, CCR5 and MMP-9 gene expression in response to surface receptor antagonism. Migration assays comparing CDAHFD and SD pHSCs in response to escalating doses of MVC and BMS-22 are ongoing and expected to demonstrate a significantly decreased migratory capacity of CDAHFD pHSCs than SD pHSCs in response to therapy, reflecting the increased susceptibility of the “fat-exposed” pHSCs to anti-fibrotic therapy than normal pHSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: Anti-fibrotic drugs that dampen pro-fibrogenic activities of “fat-exposed” pHSCs are urgently needed. CCR2 and CCR5 antagonists, BMS-22 and MVC, respectively, can selectively dampen