The Anti-angiogenic And Anti-fibrotic Effect Of Carvedilol And Intrinsic Mechanisms Study

BackgroundLiver fibrosis is a major cause of morbidity and mortality worldwide due to chronic viral hepatitis. Hepatic stellate cell activation represents a critical event in fibrosis because these cells become the primary source of extracellular matrix in liver upon injury. As pathways of fibrogenesis are increasingly clarified, the key challenge will be translating new advances into the development of antifibrotic therapies for patients with chronic liver disease. Inflammatory cytokines play a key role in fibrosis. PDGF-BB has been regarded the most potent mitogen for hepatic stellate cells (HSCs), inhibition of the PDGF receptor is the potential target in anti-fibrosis therapies.Angiogenesis, the growth of new blood vessels from pre-existing ones, is an essential process during embryonic and postnatal development. Data from the literature during the last decade have unequivocally linked angiogenesis with liver fibrogenesis and chronic liver disease progression, suggesting that angiogenesis may favor fibrogenesis. Liver injury leads to distinct morphological abnormalities such as loss of sinusoidal fenestration, vasoconstriction, and angiogenesis as well as molecular changes, which may exacerbate liver fibrosis in return. Experimental anti-angiogenic therapies have resulted in significant inhibition of fibrogenic progression.Exosomes are extracellular membrane vesicles which are produced in multivesicular bodies or at the plasma membrane. Recent studies have explored potential roles for exosomes in the pathogenesis of liver inflammation, fibrosis, and portal hypertension. An increase in this extracellular vesicle subtype has been postulated in patients with cirrhosis. However, the mechanisms by which exosomes achieve their effects on target cells are not known, especially in the context of endothelial cell regulation of HSC migration.Carvedilol is a third-generation, nonselective-blocker that also possesses al-adrenergic blocking, antioxidant, and calcium antagonist properties. Its analogue propranolol was reported to inhibit human umbilical vascular endothelial cell (HUVEC) proliferation and angiogenesis effect. Carvedilol has also been reported could attenuate CC14 induced liver fibrosis in mice. However, there is lack of evidence that carvedilol could inhibit angiogenesis or inhibit HSCs activation.In the present study, we investigated whether carvedilol could inhibit HSCs activation directly, then observed the effect of carvedilol on angiogenesis using HUVEC model. Finally, we investigated the effect of endothelial cells derived exosomes on HSCs and the intrinsic mechanisms.Part 1. Carvedilol may inhibit Hepatic Stellate Cells Proliferation and Activation via PDGFR/AKT pathwayObjectivesThe activation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of hepatic fibrosis. Platelet-derived growth factor-BB (PDGF-BB) is the strongest stimulator of HSC proliferation and intracellular signaling with concurrent predominant expression of PDGF receptor beta (PDGFR-β) subunits in activated HSCs. For these reasons, targeting PDGF, PDGFR and molecules related to the PDGF intracellular signaling-transduction pathway is attractive for therapeutic intervention in hepatic fibrosis. At present, beta-blockers such as carvedilol remain the medical treatment of choice for protection against variceal bleeding and other complications. Several studies revealed that carvedilol could attenuate myocardial degeneration and fibrosis. It has also been reported that carvedilol exerted antifibrotic effects in chronic CC14-induced liver damage. However, the underlying mechanisms have yet to be fully defined. Thus, our aim was to determine whether carvedilol could inhibit PDGF-BB induced HSC activation and define the inner mechanisms.Methods1. To investigate the effect of carvedilol on HSCs viability variation by CCK-8.According to group designing, HSCs were planted in 96-well plate.24h later, CCK-8 method was employed to measure the changes of cell proliferation.2. To investigate the effect of carvedilol on HSCs migration and invasion.Wound healing assay was employed to detect the effect of carvedilol on HSCs migration ability. Transwell assay was used to detect the effect of carvedilol on HSCs invasion level.3. To examine the effect of carvedilol on HSCs fibrogenesis effect.Use real-time PCR to detect mRNA expression of FN and collagen I in HSCs after treated with PDGF-BB and carvedilol. Use western blot to detect protein expression of FN and a-SMA in HSCs after treated with PDGF-BB and carvedilol.4. To examine the phosphorylation level of signaling pathway related molecules by western blotWestern blot was employed to examine the phosphorylation level of PDGFR-β, PI3K, Akt and Erkl/2 proteins.Results1. Carvedilol inhibited HSCs proliferation in a dose-dependent manner (IC50 = 28.42 μM).2. Carvedilol reduced PDGF-BB induced HSCs migration and invasion in a dose-dependent manner.3. PDGF-BB increased FN and collagen I mRNA expression, FN and a-SMA protein expression, in HSCs. Carvedilol significantly attenuate these fibrosis related molecules expression increase. 4. PDGF-BB increased PDGFR-β and Akt tyrosine phosphorylation in HSCs.Carvedilol significantly inhibited the phosphorylation.ConclusionCarvedilol could inhibit HSCs proliferation, migration and invasion, it could also inhibit PDGF-BB induced fibrosis effect of HSCs. This inhibitory effect may be mediated by PDGF-BB-induced PDGFRβ/Akt signaling pathway.Part2. Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathwayObjectiveTo investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways.Methods1. To investigate the effect of carvedilol on HUVECs viability variation by CCK-8.According to group designing, HUVECs were planted in 96-well plate.24h later, CCK-8 method was employed to mesure the changes of cell proliferation.2. To assess the effect of carvedilol on cell cycle progression by flow cytometryAccording to group designing, HUVECs were planted in 6-well plate. After carvedilol treatment, cells were collected and fixed. Then, the cells were stained with propidium iodide (PI) and RNase A. Then cell cycle was determined by flow cytometry.3. To investigate the effect of carvedilol on HUVECs migration and invasion.Wound healing assay was employed to detect the effect of carvedilol on HSCs migration ability. Transwell assay was used to detect the effect of carvedilol on HSCs invasion level.4. To investigate the effect of carvedilol on HUVECs tube formation effect.96-well plate was coated with Matrigel. Carvedilol and the VEGF-treated cell suspensions were added to the wells and were incubated at 37℃. Tube formation was imaged after 8h.5. To examine the phosphorylation level of signaling pathway related molecules by western blotWestern blot analysis detected the phosphorylation levels of three cell signaling pathway proteins, VEGFR-2, Src, and extracellular signal-regulated kinase (ERK). The specific Src inhibitor PP2 was used to assess the role of Src in the VEGF-induced angiogenic pathway.6. To investigate the effect of carvedilol on SKI expression of HUVECsUse real-time PCR and Western Blot to detect mRNA and protein expression ofSKI in HUVECs after treated with carvedilol respectively.Results1. Carvedilol inhibited HUVEC proliferation in a dose-dependent manner (IC50= 38.5 μM).2. The distribution of cells in the S phase decreased after treated by carvedilol for 24h.3. VEGF-induced HUVEC migration and invasion was inhibited by carvedilol treatment.4. VEGF-induced tube formation was also reduced significantly by carvedilol treatment.5. VEGF-induced VEGFR-2, ERK1/2 and Src phosphorylation was inhibited by carvedilol treatment. VEGF-induced ERK phosphorylation could also be decreased by Src kinase inhibitor (PP2).6. VEGF induced SKI expression was also inhibited by carvedilol treatment in both mRNA and protein levels.ConclusionCarvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced VEGFR2-Src-ERK1/2 signaling pathways. Carvedilol may exert anti-fibrosis effect by the anti-angiogenic effect on endothelial cells.Part3. Exosome adherence and internalization by hepatic stellate cells triggers sphingosine 1-phosphate dependent migrationObjectiveTo determine whether endothelial cell (EC) derived exosomes could regulate the phenotype of hepatic stellate cells (HSC).1. Isolation of Mouse liver endothelial cells (LEC)Liver tissue was perfused, harvested, dissected, minced, digested, incubated with immunomagnetic Dynabeads. Cells were separated with a magnet and plated on collagen I-coated dishes.2. Site-directed Mutagenesis and Generation of Stable Cell LinesLentivirus was generated using 293T cells and used to transduce TSEC and establish stable cell lines that express SKI wild type, SKI dominant negativeD81A and SKI constitutive active G113A mutants.3. Exosome purification and fluorescent labelingExosomes were purified following gradient centrifugation method. Isolated exosomes were characterized by Western blot (WB), nanoparticle tracking analysis, and electron microscopy immunogold labeling.4. Immunofluorescence, confocal microscopy, and image quantificationHSCs were first fixed, incubated with primary antibodies and appropriate Alexa Fluor-conjugated second antibodies, counterstained with DAPI. Then viewed with Fluorescence confocal microscopy5. Nanoparticle tracking analysisThe presence, size distribution and concentration of vesicles were assessed by Nanoparticle Tracking Analysis (NTA).6. Western Blot and Real-time PCRWestern Blot and Real-time PCR were performed following standard protocols.7. Transwell migration assaysExosomes and varies reagents were used to assess HSC migration measured by Transwell Assay.8. Transmission Electron Microscopy and immune-gold labelingThe specimens were resuspended and fixed, deposited onto EM grids, grids were washed, blocked, incubated with antibody, washed, incubated with specific gold-labeled secondary antibody, washed, post-fixed, stained and then viewed for TEM.9. Scanning Electron MicroscopySamples were fixed, post-fixed, washed, dehydrated, mounted and gold-palladium sputter coated, and then viewed by SEM.10. Biotinylation Assay for Plasma Membrane Integrin ActivityAfter HSCs treated with exosome, cell surface proteins were labeled with biotin. Then cells were lysed and cell lysates were subjected to protein G sepharose beads pull down followed by WB for HUTS4 and CD29.11. Animals and proceduresC57BL/6 mice were given carbon tetrachloride (CC14) and BDL treatment. Mice also received S1PR2 inhibitor according to requirements.12. Sirius Red StainingLiver paraffin sections were stained with picrosirius red and counterstained with fast green. Then take pictures and quantify with ImageJResults1. Initial microarray studies showed fibroblast growth factor-2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SKI). Exosomes derived from an SKI overexpressing EC line increased HSC migration by 3.2-fold. Migration was not conferred by the dominant negative SKI exosome.2. Incubation of HSC with exosomes was also associated with an 8.3-fold increased phosphorylation of AKT and 2.5-fold increased migration.3. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot and transmission electron microscopy. Blockade of FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration.4. Inhibitions of endocytosis with transfection of dynamin siRNA, dominant negative dynamin GTPase construct Dyn2K44A, or by the pharmacological inhibitor Dynasore, significantly attenuated exosome-induced AKT phosphorylation.5. SKI levels were increased in serum exosomes derived from mice with experimental liver fibrosis and SKI mRNA levels were upregulated in human liver cirrhosis patient samples by 2.5-fold.6. S1PR2 inhibition protected mice from CC14-induced liver fibrosis.ConclusionEC-derived SKI-containing exosomes regulate HSC signaling and migration through FN-integrin dependent exosome adherence and dynamin dependent exosome internalization. These findings advance our understanding of EC/HSC crosstalk and identify exosomes as a potential target to attenuate pathobiology signals.