Human Hepatic Stellate Cells

mRNA treatment produces sustained expression of enzymatically active human ADAMTS13 in mice

Scientific Reports | DOI | Paper 2018 | By Susan Liu-Chen, Brendan Connolly, Lei Cheng, Romesh R. Subramanian, Zhaozhong Han

Background

This proof-of-concept study investigates mRNA-based therapy for thrombotic thrombocytopenic purpura (TTP), a disorder caused by ADAMTS13 deficiency. Lipid nanoparticles deliver mRNA encoding wild-type human ADAMTS13 or an autoantibody-resistant M5 variant to mouse liver. A single intravenous 1 mg/kg dose produced detectable mRNA in liver and plasma, with secreted human ADAMTS13 reaching therapeutically relevant protein levels and enzymatic activity for up to five days in both ADAMTS13-sufficient and -deficient mice. The M5 variant showed higher activity, suggesting mRNA therapy may offer prolonged TTP treatment.

Key Findings

mRNA encoding human ADAMTS13 WT or autoantibody-resistant M5 variant was synthesized, capped, endotoxin-free, and formulated in lipid nanoparticles. In vitro, transfected HeLa, HEK293, and human hepatic stellate cells expressed and secreted enzymatically active rhADAMTS13. In mice, a single 1 mg/kg intravenous dose produced hADAMTS13 mRNA in plasma and liver, confirmed by qRT-PCR and in situ hybridization. Secreted hADAMTS13 reached ~250 ng/mL in sufficient mice and ~140 ng/mL in deficient mice at 24 h; activity was maintained for up to 120 h. The M5 variant showed greater enzymatic activity than WT at equivalent protein levels, supporting mRNA-based enzyme replacement for TTP.

Contribution

Creative Bioarray provided the human hepatic stellate cells used for in vitro transfection experiments. These cells were transfected with ADAMTS13-encoding mRNA, and their supernatants were analyzed for enzymatic activity, helping demonstrate that hepatic/stellate cells can express and secrete active rhADAMTS13.

Targeting integrin αvβ3 by a rationally designed protein for chronic liver disease treatment

Communications Biology | DOI | Paper 2021 | By Ravi Chakra Turaga, Ganesh Satyanarayana, Malvika Sharma, Jenny J. Yang, Shiyuan Wang, Chunfeng Liu, Sun Li, Hua Yang, Hans Grossniklaus, Alton Brad Farris, Jordi Gracia-Sancho, Zhi-Ren Liu

Background

Chronic liver disease is driven by activated hepatic stellate cells (HSCs) and capillarized liver sinusoidal endothelial cells (LSECs), which express high levels of integrin αvβ3. Turaga et al. rationally designed ProAgio, a protein that targets a novel site on integrin αvβ3 and induces caspase-8-mediated apoptosis. In human HSCs and LX-2 cells, ProAgio selectively killed activated HSCs. In two mouse fibrosis models, ProAgio reversed collagen deposition, reduced cross-linking, restored sinusoidal fenestrae and the space of Disse, and improved portal blood flow, suggesting a dual-action therapeutic strategy for chronic liver disease.

Key Findings

ProAgio was designed to bind integrin αvβ3 outside the ligand-binding site and trigger apoptosis by recruiting/activating caspase 8 to the integrin β3 cytoplasmic domain. Integrin αvβ3 was highly expressed in activated human HSCs and LX-2 cells but low in quiescent HSCs. ProAgio induced apoptosis of activated HSCs and LX-2 cells in an integrin β3-dependent manner; β3 knockdown blocked apoptosis, and ProAgio co-immunoprecipitated with integrin β3 but not β1, β5, or β6. In TAA/alcohol- and CCl4-induced mouse liver fibrosis, ProAgio reduced liver weight, serum AST/ALT/ALP, hydroxyproline, collagen deposition, α-SMA, TIMP1/2 and MMP2/9, while increasing collagenase activity. It also decreased collagen cross-linking and lysyl oxidase (LOX) levels/activity. ProAgio reduced CD31-positive angiogenic capillarized LSECs via apoptosis, restored SE-1-positive differentiated LSECs, recovered sinusoidal fenestration and the space of Disse, and improved portal vein blood flow, without obvious toxicity in healthy liver.

Contribution

Creative Bioarray provided primary human hepatic stellate cells (Catalog #CSC-C1496) used to assess ProAgio-induced apoptosis and integrin αvβ3 expression in vitro. These cells supported key mechanistic validation. The article does not indicate Creative Bioarray involvement in study design, data analysis, animal experiments, or manuscript preparation.

The Key Glycolytic Enzyme PFKFB3 is Up-Regulated During Hepatic Stellate Cell Activation and Its Blockade Attenuates Glycolysis

Activated hepatic stellate cells (HSCs) exhibit increased proliferation and matrix production. To meet these energy demands, they likely enhance aerobic glycolysis, driven by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3), which produces fructose-2,6-bisphosphate (F2,6BP). The study began by characterizing the expression pattern of the critical glycolytic enzyme PFKFB3 during the activation of hepatic stellate cells (HSCs), which are primarily responsible for liver fibrosis.

Using freshly isolated mouse primary HSCs, they observed that PFKFB3 expression increased rapidly within 24 hours of culture-induced activation, confirmed by co-immunofluorescence with alpha-smooth muscle actin (α-SMA) (Fig. 1A and B). These results were corroborated in the human LX2 HSC line (Fig. 1A), a well-characterized model of human activated HSCs. LX2 cells were cultured for up to 7 days on plastic surfaces of cell culture dishes to mimic the molecular changes in HSCs during liver injury. Immunoblot analysis at different times demonstrated that PFKFB3 protein upregulation was an early and sustained event during HSCs activation, coinciding with increased expression of myofibroblastic markers, including α-SMA, platelet-derived growth factor (PDGF), PDGF receptor-ß, and collagen type I (Fig. 1C).

To investigate the biological effects of enhanced glycolysis, they examined the impact of the PFKFB3 small molecule inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) on HSCs (Fig. 1D). LX2 cells were cultured for 3 days in a medium containing either a low dose of 3PO or vehicle (DMSO). Inhibition of PFKFB3 activity led to a significant 51% reduction in F2,6BP levels (Fig. 1D), a surrogate marker for glycolytic rate, without inducing cell death, consistent with previous studies. These findings suggest that HSCs activation involves a PFKFB3-regulated glycolytic reprogramming.

Fig. 3. PFKFB3 protein expression is up-regulated during HSCs transdifferentiation, and PFKFB3 blockade attenuates glycolysis in HSCs. (Mejias, M., Gallego, J., et al., 2020)

HSCs Can Express KIAA1363, and Pharmacological Inhibition of KIAA1363 Impairs RE Degradation upon Serum Starvation in LX-2 Cells and Primary Human HSCs

Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, they identified KIAAl363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase.

To investigate KIAA1363 expression in liver, they performed qPCR and Western blotting on total liver, primary murine hepatocytes, and HSCs. Purity of primary murine hepatocytes and HSCs was confirmed by high mRNA expression of the biomarkers (Fig. 2A). KIAA1363 showed highest mRNA expression in HSCs compared to primary hepatocytes and total liver. Western blot revealed KIAA1363 protein bands at ~50 kDa in HSCs, but not in total liver or primary hepatocytes (Fig. 2B). HSCs cultured on plastic surfaces can be activated and show high α-SMA expression. They examined KIAA1363 mRNA levels in quiescent HSCs and found an approximately 4-fold increase in KIAA1363 mRNA levels after 2 and 14 days of culture (Fig. 2C, left). Expression of α-SMA was initially low in resting HSCs and increased with time in culture (Fig. 2C, middle), contrasting with Lrat mRNA, which decreased (Fig. 2C, right).

Since they observed that KIAA1363 contributes to RE hydrolysis in murine HSCs, they next asked whether this was also conserved in human HSCs including LX-2 and primary human HSCs. Western blot analysis showed KIAA1363 expression in LX-2 cells but not in HepG2 cells (Fig. 3A). Using the KIAA1363-specific inhibitor JW480 and general serine hydrolase inhibitor Orlistat, they found that JW480 significantly reduced RE hydrolase activity by 80% in LX-2 cells (Fig. 3B). In primary human HSC lysates, JW480 led to a 92% reduction in RE hydrolase activity, while Orlistat had no effect (Fig. 3C). Pulse-chase experiments showed that JW480 completely inhibited RE degradation in LX-2 cells and primary human HSCs, whereas Orlistat had no significant impact (Fig. 3D). Similarly, in primary human HSCs, no detectable RP was found under basal conditions. Loading HSCs with ROH and oleic acid increased RP, which decreased by 83% after serum starvation. Orlistat had no effect on RP degradation, while JW480 resulted in 4-fold higher RP levels compared to DMSO control (Fig. 3E). These findings indicate that KIAA1363 plays a major role in RE hydrolase activity and metabolism in human HSCs.

Fig. 4. KIAA1363 exhibits low expression in whole liver but is highly expressed in primary murine HSCs (Wagner, C., Hois, V., et al., 2022).

Fig. 5. Pharmacological inhibition of hKIAA1363 impairs RE degradation in LX-2 cells and primary human HSCs upon serum starvation (Wagner, C., Hois, V., et al., 2022).