Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish

Abstract

Alcoholic liver disease (ALD) results from alcohol overconsumption and is among the leading causes of liver-related morbidity and mortality worldwide. Elevated expression of vascular endothelial growth factor (VEGF) and its receptors has been observed in ALD, but how it contributes to ALD pathophysiology is unclear. Here, we investigated the impact of VEGF signaling inhibition on an established zebrafish model of acute alcoholic liver injury. Kdrl activity was blocked by chemical inhibitor treatment or by genetic mutation. Exposing 4-day-old zebrafish larvae to 2% ethanol for 24 h induced hepatic steatosis, angiogenesis and fibrogenesis. The liver started self-repair once ethanol was removed. Although inhibiting Kdrl did not block the initial activation of hepatic stellate cells during ethanol treatment, it suppressed their proliferation, extracellular matrix protein deposition and fibrogenic gene expression after ethanol exposure, thus enhancing the liver repair. It also ameliorated hepatic steatosis and attenuated hepatic angiogenesis that accelerated after the ethanol treatment. qPCR showed that hepatic stellate cells are the first liver cell type to increase the expression of VEGF ligand and receptor genes in response to ethanol exposure. Both hepatic stellate cells and endothelial cells, but not hepatic parenchymal cells, expressed kdrl upon ethanol exposure and were likely the direct targets of Kdrl inhibition. Ethanol-induced steatosis and fibrogenesis still occurred in cloche mutants that have hepatic stellate cells but lack hepatic endothelial cells, and Kdrl inhibition suppressed both phenotypes in the mutants. These results suggest that VEGF signaling mediates interactions between activated hepatic stellate cells and hepatocytes that lead to steatosis. Our study demonstrates the involvement of VEGF signaling in regulating sustained liver injuries after acute alcohol exposure. It also provides a proof of principle of using the zebrafish model to identify molecular targets for developing ALD therapies.

Mannose Phosphate Isomerase and Mannose Regulate Hepatic Stellate Cell Activation and Fibrosis in Zebrafish and Humans

“…A‐SC). To investigate the effects of MPI loss on HSC activation, we isolated livers from control and mpi MO embryos at 5 dpf and used qRT‐PCR to measure expression of genes reflecting activation of fibrotic response . Gene expression for the ECM proteins, collagen, type I, alpha 1b ( col1a1b ), actin, alpha 2, smooth muscle ( acta2 ), platelet‐derived growth factor receptor beta ( pdgfrb ), laminin subunit beta 4A ( lamb4a ), and tissue inhibitor of metalloproteinases 2 ( timp2b ), were significantly increased in mpi MO livers (Fig.…”

Multiplex real-time PCR assay combined with rolling circle amplification (MPRP) using universal primers for non-invasive detection of tumor-related mutations

“…[19][20][21] In the current study, we combined the padlock probe technology with multiplex real-time PCR to detect genetic mutations, and demonstrated the feasibility, great specicity and high sensitivity of the assay.…”

Using Zebrafish to Model Liver Diseases-Where Do We Stand?

“…Transgenic lines that express fluorescent proteins in hepatocytes, intrahepatic biliary cells, endothelial cells, and hepatic stellate cells are available, facilitating the observation of these cells both in live fish and in fixed livers (Table 1). These transgenic zebrafish also allow isolation of different liver cell types through fluorescence activated cell sorting for downstream transcriptome analyses [16]. Commercially available fluorescent lipid analogs, which are designed for visualizing lipid uptake and processing in live fish, have proven to be extremely sensitive in detecting hepatobiliary injuries (Table 1).…”

“…Genes and pathways necessary for alcohol metabolism are highly conserved in zebrafish and they readily take up and metabolize alcohol at as early as 4 days post fertilization [62, 61, 63, 64]. Intriguingly, exposing 4-day-old larvae to 2% ethanol for just 24 hours is sufficient to induce steatosis, angiogenesis, recruitment of macrophages, and activation of hepatic stellate cells [61, 15, 16]. Using this model, the Sadler group showed that the sterol response element binding protein (Srebp) pathway [61] and activation of unfolded protein response (UPR) were required to induce hepatic steatosis in acute ALD liver injury [65, 66].…”

“…Activating transcription factor 6 (Atf6) [65], one of the three UPR sensors, was both necessary and sufficient to cause hepatic steatosis independent of Srebp activity. Our group, on the other hand, investigated the roles of vascular endothelial growth factor (VEGF) signaling in acute alcoholic liver injury [16]. We showed that blocking VEGF receptor Kdrl activity by either a pharmacological inhibitor or genetic mutation suppressed hepatic steatosis, angiogenesis and fibrogenesis induced by acute ethanol treatment.…”