Platelet-derived growth factor A mRNA in platelets is associated with the degree of hepatic fibrosis in chronic hepatitis C

Hepatic fibrosis, a consequence of various chronic stimuli, including infection with hepatitis C virus (HCV), is characterized by the progressive accumulation of extracellular matrix (ECM) components in the hepatic parenchyma, which leads to a progressive replacement of the functional tissue with scar tissue¹.

Hepatic stellate cells (HSCs) are considered the main cells producing ECM proteins² and their activation and proliferation are mediated by two cytokines, transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor (PDGF). Both are secreted by HSCs, platelets, and other cells^3–4.

Transforming growth factor beta 1 is the major profibrogenic cytokine in the liver because of its contribution to fibrosis development via HSC activation and the regulation of gene expression associated with signaling cascades related to fibrosis^5–6.

Platelet-derived growth factor is the most potent mitogen for HSCs⁷. There are four different PDGF isoforms⁸ and the PDGFB isoform is considered the most important mitogen for HSCs^7–9. However, studies have demonstrated that PDGFA can also be considered an important profibrogenic agent, acting via TGFB1 induction¹⁰.

Considering that platelets are major sources of TGFB1 and PDGF, which are growth factors involved in the development of liver fibrosis associated with chronic hepatitis C, and that studies have already demonstrated that platelets that accumulate in the liver during chronic hepatitis may be involved in hepatic fibrosis¹¹, the aim of this study was to evaluate the expressions of PDGFA, PDGFB, and TGFB1 in hepatic tissue and platelets from HCV-infected patients presenting with varying degrees of hepatic fibrosis.

Aliquots of ethylenediaminetetraacetic acid (EDTA)-anticoagulated peripheral venous blood and fragments of hepatic tissue were collected from 43 HCV-infected patients who presented at the Department of Internal Medicine, Gastroenterology Division, Botucatu Medical School, São Paulo State University [Universidade Estadual Paulista (UNESP)], Botucatu, SP, Brazil. The inclusion criteria were naïve patients infected with HCV genotype 1 who were aged >18 years, had undergone a liver biopsy before the start of antiviral treatment, and had signed informed consent. The exclusion criteria were hepatitis B virus- or human immunodeficiency virus-positive serology, antiviral treatment before liver biopsy, the presence of other hepatic diseases, and pregnant women. The 43 patients included in this study were representative of patients seen in the service globally who have the characteristics of the inclusion criteria.

The patients were divided into two groups according to the degree of fibrosis determined in liver biopsies. The fragments were analyzed by a pathologist who used the METAVIR score system to evaluate fibrosis progression¹². The patients were assigned to group 1 (G1) (patients with lower stages of fibrosis – F1 or F2) or group 2 (G2) (patients with higher degrees of fibrosis – F3 or F4). Information about sex, age, HCV genotype, fibrosis degree, and alcohol abuse (defined as >40g per day for women and >80g per day for men) was obtained from the patient’s medical registers.

Whole-blood samples were aliquoted and plasma was isolated after centrifugation at 1,312 × g for 3 min. Platelet pellets were obtained according to Padovani et al.¹³.

Hepatic tissue samples were collected using RNAlater ^® (Thermo Fisher Scientific, Inc., CA, USA) and stored at -70^oC until ribonucleic acid (RNA) extraction. RNA from hepatic tissue was isolated using the spin (SV) Total RNA Isolation System kit (Promega Corporation, Madison, WI, USA), according to the manufacturer’s specifications.

Ribonucleic acid from platelets was obtained according to Padovani et al.¹³. RNA extracted from hepatic tissue and platelets was used as a source to perform reverse-strand complementary deoxyribonucleic acid (cDNA) synthesis using the High-Capacity cDNA Archive kit (Thermo Fisher Scientific, Inc., CA, USA), according to the manufacturer’s specifications.

TaqMan Universal polymerase chain reaction (PCR) Master Mix (Thermo Fisher Scientific, Inc., CA, USA) was used to evaluate the relative mRNA expression levels of PDGFA, PDGFB, and TGFB1 in the hepatic tissue and platelets. Primers and probes for the target genes (PDGFA: Hs00964426_m1; PDGFB: Hs00966522_m1; TGFB1: Hs00998133_m1) and the reference gene (ACTB: Hs01060665_g1) were obtained commercially (TaqMan^® Gene Expression Assays; Thermo Fisher Scientific, Inc., CA, USA). An endogenous reference gene (β-actin) was used to normalize the expression levels of target genes. All measurements were performed in duplicate.

Relative quantification of RNA in hepatic tissue and platelets from each sample was performed using a pool of RNA as the control; this was denominated the F1 pool, which was extracted from hepatic tissue obtained from treatment-naïve patients with HCV genotype 1 and mild fibrosis (F1) (the available group closest to fibrosis absence).

Relative RNA quantification was performed by calculating the S/C ratio, in which S was the expression level of the target gene and C was the expression of the control gene in hepatic tissue or platelets. If this ratio was >1, the target gene was considered to be overexpressed (OE); otherwise it was under-expressed (UE)

The data were initially analyzed by descriptive statistical techniques and an exploratory analysis was performed to observe the frequency distributions of the variables. Pearson’s chi-squared test or Fisher’s exact test were used to verify possible associations between the fibrosis degree (G1 and G2); alcohol abuse; and the expression levels of PDGFA, PDGFB, and TGFB1 in the hepatic tissue and platelets. The level of significance for all statistical tests was set at 0.05. Data were analyzed using R software version 3.2.0¹⁴.

A descriptive analysis was performed to evaluate the relative expression levels of the target genes in platelets, using as a control the expression of the same gene in hepatic tissue from the same patient.