Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue

3-D structure of extracellular matrix regulates gene expression in cultured hepatic stellate cells to induce process elongation

HSCs showed myofibroblast-like shapes when cultured on polystyrene surface or on type I collagen-coated surface, whereas HSCs cultured on type I collagen gel were induced to elongate cellular processes, suggesting that HSCs recognize 3-D structure of extracellular type I collagen fibrils and change their morphology and function. In this study we examined the differentially regulated gene expression by extracellular matrix (ECM) components by PCR-differential display (PCR-DD) analysis followed by cloning and FASTA homology search, and identified the mRNA species as a transcription factor SP1, breast cancer resistant protein (BCRP), dystonin, and KAP3B. Regulation of dystonin and KAP3B expression was confirmed by RT-PCR analysis. Thus, cell surface-binding to extracellular interstitial collagen may trigger intracellular signaling and alteration in gene expression, and HSCs not only produce various ECM components but also change their morphology and gene expression in response to ECM components adhering to the cells

Intercellular Adhesive Structures Between Stellate Cells – An Analysis in Cultured Human Hepatic Stellate Cells

To investigate whether or not hepatic stellate cells can form intercellular junctions with each other, we cultured human stellate cells (LI90) on different kinds of substrata. Intercellular junctions were detected between these cultured stellate cells by transmission electron microscopy (TEM). The molecular components of the intercellular adhesive structures were identified by immunofluorescence microscopy. Immunofluorescence for cadherin and catenins was detected at the adhesion sites between the cultured stellate cells. Thus, the intercellular junctions were indicated to be adherens junctions at the molecular level. The junctions developed in the cultured stellate cells irrespective of the type of substratum. These data suggest that the junctional formation between the stellate cells occurs in vivo as well as in vitro

Droplet digital PCR assay provides intrahepatic HBV cccDNA quantification tool for clinical application

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance

Intralobular Distribution of Vitamin A-Storing Lipid Droplets in Hepatic Stellate Cells with Special Reference to Polar Bear and Arctic Fox

We examined the liver of adult polar bears, arctic foxes, and rats by gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, hematoxylin-eosin staining, staining with Masson's trichrome, Ishii and Ishii's silver impregnation, and transmission electron microscopical morphometry. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. These results indicate that the hepatic stellate cells of the polar bears and arctic foxes possess heterogeneity of vitamin A-storing capacity in their liver lobules

Neutralization against Omicron sublineages (BA.2/BA.5/BQ.1.1/XBB/XBB.1.5) in bivalent BNT162b2-vaccinated HCWs with or without risk factors, or following BT infection with Omicron

Abstract SARS-CoV-2-BA.4/5-adapted-bivalent-BNT162b2-vaccine (bvBNT), developed in response to the recent emergence of immune-evasive Omicron-variants, has been given to individuals who completed at least 2-doses of the monovalent-BNT162b2-vaccine (mvBNT). In the present cohort study, we evaluated neutralization-titers (NT50s) against Wuhan-strain (SCoV2Wuhan) and Omicron-sublineages including BA.2/BA.5/BQ.1.1/XBB/XBB.1.5, and vaccine-elicited S1-binding-IgG in sera from participants-vaccinated with 5th-bvBNT following 4th-mvBNT. The 5th-bvBNT-dose elicited good protective-activity against SCoV2Wuhan with geometric-mean (gMean)-NT50 of 1966–2091, higher than the peak-values post-4th-mvBNT with no statistical significance, and favorable neutralization-activity against not only BA.5 but also BA.2, with ~ 3.2-/~ 2.2-fold greater gMean-NT50 compared to the peak-values post-4th-mvBNT-dose, in participants with or without risk factors. However, neutralization-activity of sera post-5th-bvBNT-dose was low against BQ.1.1/XBB/XBB.1.5. Interestingly, participants receiving bvBNT following breakthrough (BT) infection during Omicron-wave had significantly enhanced neutralization-activity against SCoV2Wuhan/BA.2/BA.5 with ~ 4.6-/~ 6.3-/~ 8.1-fold greater gMean-NT50, respectively, compared to uninfected participants receiving bvBNT. Sera from BT-infected-participants receiving bvBNT had enhanced neutralization-activity against BQ.1.1/XBB/XBB.1.5 by ~ 3.8-fold compared to those from the same participants post-4th-mvBNT-dose, and had enhanced gMean-NT50 ~ 5.4-fold greater compared to those of uninfected-participants’ sera post-bvBNT. These results suggest that repeated stimulation brought about by exposure to BA.5’s-Spike elicit favorable cross-neutralization-activity against various SARS-CoV-2-variants