SIRT1 ameliorates premature senescence-induced defenestration in hepatic sinusoidal endothelial cell

Premature senescence, linked to progerin, involves in endothelial dysfunction and liver diseases. Activating sirtuin 1 (SIRT1) ameliorates liver fibrosis. However, the potential mechanisms of premature senescence in defenestration in hepatic sinusoidal endothelial cells (HSECs) and how SIRT1 affects fenestrae remains elusive. Our study showed that in vivo, premature senescence occurred, with decrease of SIRT1, during CCl4-induced defenestration in HSECs and liver fibrogenesis; whereas overexpressing SIRT1 with adenovirus vector lessened progerin-associated premature senescence to relieve CCl4-induced defenestration and liver fibrosis. In vitro, fenestrae in HSECs disappeared, with progerin-associated premature senescence; these effects aggravated by H2O2-induced oxidative damage. Nevertheless, knockdown of NOX2 or overexpression of SIRT1 with adenovirus vector reduced progerin-associated premature senescence to maintain fenestrae through deacetylating p53. Furthermore, more Ac p53 K381 and progerin co-localized with accumulation of actin filament (F-actin) in the nuclear envelope of H2O2-treated HSECs; in contrast, these effects were rescued by overexpressing SIRT1. In conclusion, NOX2-dependent oxidative damage aggravates defenestration in HSECs via progerin-associated premature senescence; SIRT1-mediated deacetylation of p53 maintains fenestrae and attenuates liver fibrogenesis through inhibiting premature senescence.

Fuzhenghuayu Decoction ameliorates hepatic fibrosis by attenuating experimental sinusoidal capillarization and liver angiogenesis

Fuzhenghuayu (FZHY) is a compound extracted from natural plants. Its anti-fibrotic effect has been confirmed in experimental and clinical studies. However, precise effects and underlying mechanisms of FZHY in liver angiogenesis largely remain understood. In this study, we investigated the effects of FZHY on sinusoidal capillarization and angiogenesis with mice challenged for Carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN), in vitro human hepatic sinusoidal endothelial cells (HHSEC) and Human Umbilical Vein Endothelial Cell (HUVEC) 3D fibrin gel model. Besides its anti-fibrotic effect, FZHY ameliorated CCl4 and DMN-induced sinusoidal capillarization, angiogenesis and expression of angiogenesis-associated factors, i.e. CD31, VEGF, VEGF receptor II, phosphor-ERK and HIF-1α. Consistent with the findings based on animal models, inhibitory effects of FZHY on capillarization and angiogenesis were further confirmed in HHSEC and the HUVEC 3D fibrin gel model, respectively. These data suggest that FZHY ameliorates not only liver fibrosis but also vessel remodeling in experimental models. Therefore, FZHY might be a potentially useful drug to treat liver cirrhosis in clinical practice.

Development of a Human in Vitro Cell Model for Haemophilia a

Aim Haemophilia A is an X-chromosome linked hereditary bleeding disorder caused by loss-of-function mutations in the clotting factor VIII (FVIII) gene resulting in either a deficit or a total lack of the corresponding activity. Although the quality of the standard replacement therapy has improved significantly over the last decades, patients still require FVIII administration at regular intervals and frequently develop an immune response against the exogenous protein that interferes with FVIII function. FVIII gene therapy could provide a less intrusive and perhaps safer alternative to current FVIII replacement therapy. Here, we aimed to develop a human based cell model to facilitate future effective development and evaluation of therapeutic gene therapy approaches for haemophilia A. In order to closely model key challenges for such a development, we chose to introduce patient-specific FVIII mutations into immortalized human hepatic sinusoidal endothelial cells, which are the cells that naturally secret FVIII. Methods First, we immortalized primary human hepatic sinusoidal endothelial cells (HHSEC), the natural cell of FVIII synthesis in humans, by lentiviral transduction of a doxycycline-inducible SV40-Large T oncogene. After establishment of the HHSEC cell line, we chose from the FVIII gene variant database (European Association for Haemophilia and allied disorders) five different FVIII mutations resulting in a severe form of haemophilia A. All of the selected variants were small deletions resulting in frameshift mutations, leading to a loss-of-function FVIII gene product. The FVIII mutations were introduced by lentiviral transduction of sgRNAs causing DNA double strand breaks at the respective positions together with the RNA-guided CRISPR/Cas9 (saCas9) endonuclease. Characterization and verification of immortalized HHSEC with and without FVIII mutation were performed by sequencing, immunofluorescence and aPTT-based FVIII activity assays. Results Our first experiments resulted in the successful immortalization of primary HHSEC cells. Since the cells were conditionally immortalized, they returned to the primary cell state upon removal of doxycycline. By western blotting we could demonstrate SV40-Large T oncogene expression under doxycycline conditioning, and a decrease in expression after doxycycline withdrawal. Additionally, using cumulative proliferation assays (cell counting assay), we could estimate a doubling time of almost 2 days for the immortalized HHSEC under doxycycline treatment and strong proliferative disadvantage in absence of doxycycline. Subsequently, we examined the FVIII activityof immortalized HHSEC using immunofluorescence microscopy and an aPTT-based FVIII clotting assay. Immunohistochemical staining for FVIII in the immortalized HHSEC revealed a strong signal resembling vesicular structures as expected. Using the established aPTT-based FVIII clotting assay, which is also used in the clinic to estimate FVIII residual activities, we could demonstrate functional FVIII activity of 75% up to 99% of normal human plasma. FVIII activity was measured in medium supernatant after 24h of incubation, at least at three independent days. The FVIII activity showed always similar values and stayed constant over the time. We successfully introduced two out of five haemophilia A patient-specific mutations into the FVIII gene of the HHSEC. Sequence analysis revealed for one mutant cell line a predominant deletion of seven base pairs and for the other cell line a mixed indel generation both resulting in frameshift mutations. FVIII clotting assays showed a reduction of FVIII activity to 40%. Likewise, the immunohistochemical staining showed reduced intensity. In ongoing experiments, we currently generate a full FVIII knock-out cell line by subcloning, in order to get cell lines strongly resembling FVIII activity levels that are seen in severe haemophilia A patients (<1%). Conclusion By immortalization and transduction of HHSECs we generated a human haemophilia A cell line model based on mutagenesis of the endogenous genetic FVIII locus in the cell line, which mainly accounts for FVIII production in humans. This cell model will be used to study FVIII synthesis, signaling and processing and, in addition, aids in the development of new strategies for haemophilia A gene therapy or new FVIII preparations. Disclosures Miesbach: Bayer, Chugai, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire, UniQure: Speakers Bureau; Bayer, BioMarin, CSL Behring, Chugai, Freeline, Novo Nordisk, Octapharma, Pfizer, Roche, Takeda/Shire, UniQure: Consultancy; Bayer, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire: Research Funding.

Titanium Dioxide Nanoparticles Enhance Leakiness and Drug Permeability in Primary Human Hepatic Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) represent the permeable interface that segregates the blood compartment from the hepatic cells, regulating hepatic vascular tone and portal pressure amidst changes in the blood flow. In the presence of pathological conditions, phenotypic changes in LSECs contribute to the progression of chronic liver diseases, including the loss of endothelial permeability. Therefore, modulating LSECs offers a possible way to restore sinusoidal permeability and thereby improve hepatic recovery. Herein, we showed that titanium dioxide nanoparticles (TiO2 NPs) could induce transient leakiness in primary human hepatic sinusoidal endothelial cells (HHSECs). Interestingly, HHSECs exposed to these NPs exhibited reduced protein kinase B (Akt) phosphorylation, an important protein kinase which regulates cell attachment. Using a 3D co-culture system, we demonstrated that TiO2 NPs diminished the attachment of HHSECs onto normal human hepatic cell LO2. To further illustrate the significance of leakiness in liver sinusoids, we showed that NP-induced leakiness promoted Sunitinib transport across the HHSEC layer, resulting in increased drug uptake and efficacy. Hence, TiO2 NPs have the potential to modulate endothelial permeability within the specialized sinusoidal endothelium, especially during events of fibrosis and occlusion. This study highlighted the possible use of inorganic NPs as a novel strategy to promote drug delivery targeting the diseased liver.

Evidence of Amniotic Epithelial Cell Differentiation toward Hepatic Sinusoidal Endothelial Cells

Amniotic epithelial cells (AECs) represent a useful and noncontroversial source for liver-based regenerative medicine, as they can differentiate into hepatocytes upon transplantation into the liver. However, the possibility that AECs can differentiate into other liver cell types, such as hepatic sinusoidal endothelial cells (HSECs), has never been assessed. In order to test this hypothesis, rat- and human-derived AECs (rAECs and hAECs, respectively) were subjected to endothelial cell tube formation assay in vitro. Moreover, to evaluate differentiation in vivo, the retrorsine (RS) model of liver repopulation was used. Pyrrolizidine alkaloids (including RS) are known to target both hepatocytes and endothelial cells, inducing cell enlargement and inhibition of cell cycle progression. rAECs and hAECs were able to form capillary-like structures when cultured under proangiogenic conditions. For in vivo experiments, rAECs were obtained from dipeptidyl peptidase type IV (DPP-IV, CD26) donors and were transplanted into the liver of recipient CD26 negative animals pretreated with RS. rAEC-derived cells were engrafted in between hepatocytes and resembled HSECs as assessed by morphological analysis and the pattern of expression of CD26. Donor-derived CD26+ cells coexpressed HSEC markers RECA-1 and SE-1, while they lacked expression of typical hepatocyte markers (i.e., cytochrome P450, hepatocyte nuclear factor 4α). As such, these results provide the first evidence that AECs can respond to proangiogenic signals in vitro and differentiate into HSECs in vivo. Furthermore, they support the conclusion that AECs possesses great plasticity and represents a promising tool in the field of regenerative medicine both in the liver and in other organs.