Homoeostatic regulation of cytokines to retard liver fibrosis 3

HSC-TARGETED DELIVERY OF HGF TRANSGENE ADMINISTERED VIA BILE DUCT INFUSION ENHANCES ITS LOCALIZATION AT FIBROTIC FOCI & AMELIORATES DMN-INDUCED LIVER FIBROSIS 3.1 AIMS & OBJECTIVES The

CHAPTER 3

3 HSC-TARGETED DELIVERY OF HGF TRANSGENE ADMINISTERED VIA BILE DUCT INFUSION ENHANCES ITS LOCALIZATION AT FIBROTIC FOCI & AMELIORATES DMN-INDUCED LIVER FIBROSIS

3.1 AIMS & OBJECTIVES

The key event of fibrosis progression is the process of activation of quiescent HSCs and portal fibroblasts into activated myofibroblasts, which occurs due to changes in soluble factors, ECM proteins and mechanical stiffness (27,147) Activated myofibroblasts secrete and deposit copious amounts of ECM proteins especially collagens which accumulate to form the fibrous tissue (33) Fibrotic foci are sites of active fibrosis within the affected organ (148) with an increased number of activated myofibroblasts that create a concentrated centre of pro-fibrotic cytokines such as TGF-β1 and accumulation of ECM proteins such as Collagen I

HSCs are one of the major sources of myofibroblasts and abundant at fibrotic foci Recent therapeutics developed for liver fibrosis have targeted HSCs, providing enhanced delivery to the fibrotic foci, and they have largely focused on decreasing/controlling the high levels of TGF-β1 signaling or collagen production (106,149) but they do not show effective hepatic regeneration Anti-fibrotic therapies lacking cell specificity fail to target the fibrotic foci, and lead to adverse effects in healthy non-specific cells surrounding the foci, resulting in changes in their viability, differentiated state and metabolic function of the liver (150) Recent interest in the development of therapeutics targeted to fibrotic foci aims to reduce the doses of the therapeutics, the need for long-term therapeutic expression or engraftment efficiency More importantly, targeted therapies can reduce the adverse side effects that might arise from effects of the non-specific uptake of the therapeutics by healthy cells

(151,152) This led us to investigate targeted HGF-based therapies, both to control the expansion of the fibrotic foci and to ameliorate the hepatocellular damage

HGF (66) has demonstrated anti-fibrotic effects in recent liver fibrosis studies via suppression of TGF-β1 and collagen III expression levels (73,78) Anti-fibrotic therapies with hepatocyte growth factor (79) have not been specifically targeted to the fibrotic foci, and we propose that doing so might increase their therapeutic potential HSCs are one of the major sources of HGF in the liver, under normal and regenerative conditions (153), but sustained activation of HSCs during fibrosis significantly decreases HGF production and inhibits hepatocyte regeneration (154) Although activated HSCs have lower HGF expression, HSCs have higher metabolic activity and are actively proliferating in the fibrogenic state Therefore we investigated whether targeting the HGF gene to HSCs will yield selective enrichment of the therapeutic in the fibrotic foci We hypothesized that HSC-targeted delivery of HGF would decrease collagen accumulation and the number of activated HSCs at fibrotic foci and surrounding regions, compared with untargeted HGF therapy

We have used the retinol-storing function of HSCs as a mechanism for targeted delivery to HSC, as HSCs are the only cells in the liver with retinol-storing capability and with high expression levels of retinol binding proteins (106,152) We cloned the rat HGF gene into a pDsRed2 plasmid DNA vector, and encapsulated the gene construct in Vitamin A-coupled liposomes for specific targeting of HSCs The anti-fibrotic potential of the Vitamin A-Liposome-HGF particles was tested in HSC-T6 (rat hepatic stellate cell line) monocultures, in co-cultures of HSC-T6 and hepatocytes

in vitro, and in DMN-induced fibrotic rat livers Since microvasculature disturbances

in the fibrotic liver and excessive basement membrane deposition lead to constricted hepatic sinusoids and poor hepatic microcirculation and diffusion (155,156), we

delivered all transgene complexes through retrograde intrabiliary infusion, in order to increase the biodistribution to the fibrotic liver

3.2 MATERIALS & METHODS

3.2.1 In Vitro Cultures

For monoculture studies, HSC-T6 cells were seeded at 2x105 cells per 35mm collagen-coated dish and cultured for 3 days in DMEM (Sigma) with 10% FBS to allow for activation On the fourth day, the HSC-T6 monocultures were transfected with different liposome/DNA complexes Co-cultures were established with hepatocytes and HSC-T6 cells at the ratio of 1:10 (i.e., for every 1 hepatocyte, 10 hepatic stellate cells are seeded) Hepatic stellate cells were seeded 3 days prior to the co-culture in DMEM with 10% FBS at the density of 2x105 cells per 35mm collagen-coated dish to induce the activation process Three days later, primary rat hepatocytes

in an appropriate number according to the above ratio, were seeded in Williams E medium with serum (Primary rat hepatocytes were isolated from male Wistar rats by a 2-step collagenase perfusion method as described previously (133); the isolation procedure was approved by the IACUC of National University of Singapore) The monocultures and co-cultures were transfected with Vitamin A-coupled liposomes (VAL)/ Vitamin A-coupled liposomes with pDsRed2 alone (VALD)/ Vitamin A-coupled liposomes with pDsRed2-HGF (VALH) particles with 3µg plasmid DNA Media was changed to DMEM with serum 4 hrs after transfection Cell culture supernatants were collected 24 hrs after treatment and the cells were lysed for RNA isolation

3.2.2 Measurement Of Hepatocyte Proliferation

The isolated hepatocytes were seeded at 2x105 cells per 35mm collagen-coated dish (IWAKI) in Williams E (Sigma) with 10% fetal bovine serum (FBS, Sigma) After 4 hrs, media was changed to Williams E without serum After overnight serum starvation, the cells were treated with filtered conditioned media from transfected HSC-T6 monocultures Cells from the collagen-coated dishes were collected by treatment with 0.1% SDS and samples were lysed and centrifuged at 11,000g for 10 min The supernatant was diluted 10x and the DNA quantity was assayed by incubation with equal amount of picogreen dsDNA dye for 5 min The fluorescence was measured at 520nm and a standard curve was used to calculate the number of cells from the observed DNA quantity

3.2.3 Transgene Validation By Transfection In HEK-293T Cells

HEK-293T cells were seeded at 5x105 cells per 35mm culture dish in DMEM with serum One day later transfection was carried out with 1µg plasmid DNA using Lipofectamine LTX reagent according to the manufacturer’s protocol Media was changed 4 hrs after transfection and the samples were collected 24 hrs after transfection Transfection efficiency as measured by the DsRed fluorescence was found to be nearly 8% in transfected cell cultures

3.2.4 Transgene Construction And Encapsulation In Vitamin A-Coupled Liposomes

Rat mRNA was isolated from freshly isolated hepatocytes and the mRNA was converted to cDNA using a HGF gene specific primer (HGF reverse primer in Table 4) The HGF gene was isolated using highly specific primers (Table 4) in a PCR reaction using Phusion DNA polymerase at primer melting temperature 62°C The PCR product was run on a 0.8% agarose gel The band observed near the 2000bp

mark was extracted; purified, expanded using pJET1.2 cloning vector (Fermentas) and TOP10 cells (Invitrogen) The purified plasmid sequence was verified using HGF specific sequencing primers designed in-house using Oligo7 software (See Table 4;sequencing primers)

Table 4 List of primer sequences used for HGF gene isolation and PCR

plasmid DNA was encapsulated with the liposomes at the N:P ratio of 1:11.5 The plasmid DNA:Liposome particle size was determined by particle size analyzer, as shown in Fig 21 Apart from the particle size of the complexes, the surface charge also contributes equally to their effective uptake by the liver Neutrally charged particles have the lowest uptake (157) whereas positive or negatively charged particles have a better uptake efficiency, Therefore, we analyzed the surface charge of

negatively charged particles is known to have better ability to be taken up the liver, (158) we believed that these particles could be easily taken up by the liver if directed appropriately

Figure 21: Liposome/DNA complex sizes Particle size measurement of particles

dissolved in deionized water as measured in particle size analyzer n=3

3.2.5 Protein Measurements By Western Blot

For Western blots, liver tissue sections were homogenized; protein levels quantified and equal amounts of protein samples were separated on a 10% SDS PAGE in 1x Tris Glycine, and transferred onto a 0.22µm nitrocellulose membrane overnight in 1x Tris-buffered saline (TBS) with 10% methanol The membrane was then treated with

blocking buffer (2% skimmed milk in 1x TBS with 0.01% Tween 20 (1x TBST)) for

1 hour Later the membrane was washed 3 times in 1x TBST; treated with mouse SMA antibody (Sigma; 1:200), mouse β-actin antibody (Sigma; 1:40000), or goat HGF antibody (Santacruz; 1:100) in 1x TBST for 2hrs; washed 3 times in 1x TBST and treated with goat anti-mouse IgG-HRP (Santacruz; 1:10000 in blocking buffer) or mouse anti-goat antibody (Santacruz; 1:10000 in blocking buffer) for 1 hour The membrane was developed using SuperSignal West Pico Chemiluminescent solution (Thermo Scientific)

α-3.2.6 DMN-Induced Liver Disease

Male Wistar rats (250g) were administered 1% N-nitroso dimethylamine (10mg/kg; Wako) intraperitoneally for 3 consecutive days each week for 4 weeks, to establish liver fibrosis Fresh liver tissue samples were collected and frozen immediately in liquid nitrogen for further RNA and protein analysis Simultaneously, liver tissue sections were fixed in 10% formaldehyde and processed for histopathology Blood was collected from the heart by cardiac puncture and the separated blood serum was stored at -20°C till further processing

3.2.7 Retrograde Intrabiliary Infusion

Fibrotic rats with DMN administration for 3 consecutive weeks and not administered with DMN for the next seven days were anaesthetized with Ketamine/Xylazine before the procedure The common bile duct was canulated and the area sutured with 5-o-silk sutures The protocol for retrograde intrabiliary infusion was adapted from our previous work (159) Liposome with pDsRed2-HGF (Lip-HGF) or VALD or VALH (200µg plasmid DNA) were administered at a constant flow rate of 0.2ml/min with a syringe pump using a 32G needle (Hamilton) fitted through Ext-12 (DiLab) as shown

in Figs 29 A & 29B After retrograde infusion, the bile duct was ligated for 10mins to

prevent immediate backflow The abdominal wall of the operated rats was sutured with 3-o-prolene sutures and the rats were administered baytril and buprenorphine for

3 days after surgery All animal procedures were carried out under the premises of the IACUC protocol approved by the Biological Resource Centre, Biopolis

3.2.8 Gene Expression Analysis - RT-PCR

mRNA was isolated from the cells/homogenized tissue using RNeasy mini kit (Qiagen), 1µg of mRNA from each sample set was converted to cDNA (Invitrogen, Superscript Reverse Transcriptase III) and real-time PCR reaction (Roche, Sybr Green Master mix) was carried out for HGF, α-SMA, Collagen I (Col I), Col IV, TGF-β1, TSP-1, PAI-1, TIMP-1, β-actin and GAPDH (primer sequences listed in Table 3 in chapter 2 and Table 4) The fold change in gene expression values were determined by the Del-Del CT relative quantitation method (138); the target CT

values were normalized to the endogenous reference β-actin (for in vitro samples) and GAPDH (for in vivo samples),

3.2.9 Active TGF-β1 Measurement

For cell culture supernatants, equal volumes of sample were assayed for active β1 ELISA (Promega TGF-β1 Emax Immunoassay (137)); according to the

TGF-manufacturer’s protocol For in vivo blood samples, protein levels were quantified

using the Bradford assay (Biorad) and equal amounts were assayed for active TGF-β1 levels

3.2.10 Liver Protein Levels

Liver proteins such as alanine transaminase, aspartate aminotransferase, lactate dehydrogenase, and albumin in the blood serum were quantified with Vitros DT kits using Johnson & Johnson DT 60 analyzer

3.2.11 Collagen Imaging – Non Linear Microscopy And Image Acquisition

Deparaffinized, unstained liver tissue sections of 50µm thickness were imaged with second harmonic generation microscopy (SHG) using a modified Carl Zeiss LSM 510 system as described previously (160) Data was acquired using both Two photon excited fluorescence and SHG imaging modes A total of four images (3x3 tile scans, 3072x3072 pixels and 3.2µs well time) were collected for each tissue specimen, and two specimens were extracted from each animal The SHG images were analyzed using the direct segmentation method (Gaussian mixture model) The method is based

on the assumption that the distribution of intensities is a mixture of several Gaussian distributions, each corresponding to a separate tissue class In an SHG image, the intensity of pixels is modeled as the mixture of two Gaussian distributions, one representing collagen area with strong SHG signals and the other representing the background

3.2.12 Immunohistochemistry

Liver tissue sections of 5µm thickness were stained with Masson’s Trichrome stain to assess collagen deposition The next serial section was stained with Haematoxylin & Eosin (H&E) to determine necrosis levels and Knodell score

3.2.13 Immunofluorescence

Serial sections from the liver tissue were deparaffinized with Xylene and ethanol gradient washes for 3mins each Then the sections were permeabilized, treated with blocking buffer followed by treatments with primary antibodies for α-SMA (Sigma); TGF-β1 (Santacruz); Collagen I (Millipore), HGFα (Santacruz); DsRed2 (Santacruz); platelet endothelial cell adhesion molecule-1, PECAM-1 (Abcam); von Willebrand Factor, vWF (Santacruz); hyaluronic acid receptor, CD44 (Abcam) Later, the tissue sections were incubated with appropriate secondary antibodies (Santacruz) for 2hrs

After washing, the stained tissue sections were imaged with an Olympus FluoView FV1000 confocal laser-scanning microscope Marker intensity and colocalizated points were quantified from the images using ImageJ software

3.2.14 Scanning Electron Microscopy For SECs

Liver tissue sections (precision cut to 1mm x 1mm x 1mm size) were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight and stained with 1% osmium tetroxide for 1hr The stained tissue sections were then dehydrated step-wise with ethanol gradient (25%, 50%, 75%, 90% and 100%) for 10 min each, and vacuum dried overnight (161) After gold sputter coating, liver sinusoids were imaged using FE-SEM (JEOL JSM-6701F) at 10 kV

3.2.15 Statistical analyses

Statistical analyses were performed using Graphpad Prism 5 software The results are

expressed as mean ± SEM For all in vitro experiments n = 4; for all animal studies, n

≥ 5 Comparisons between different groups were performed with unpaired Student’s t-test Differences were considered significant when the p-value < 0.05

3.3 RESULTS

3.3.1 pDsred2-HGF Gene Construction & In Vitro Validation

The HGF gene was isolated from rat cDNA and incorporated into the pDsRed2C1 vector to enable visualization of the delivered gene (Fig 22A) In order to validate the expression and functionality of the transgene, HEK-293T cells were transfected with pDsRed2 vector alone or with the pDsRed2-HGF combined construct, and both transfections produced high levels of DsRed2 fluorescence (Fig 22B) The cells transfected with the pDsRed2-HGF combined construct expressed significantly higher

levels of the HGF gene (Fig 22C) compared to the cells transfected with pDsRed2 vector alone

Figure 22: Validation of the pDsRed2-HGF construct in HEK-293T cells Vector

diagram of pDsRed2-HGF transgene construct (A) HEK-293T cells transfected with pDsRed2 or pDsRed2-HGF plasmid DNA (B) show DsRed2 autofluorescence Scale bar: 100 µm Significant increase in HGF gene expression with the transfection of pDsRed2-HGF construct in HEK-293T cells; normalized to β-actin (C) Co-localization of DsRed2 and HGF proteins in transfected cells (D) Scale bar: 50 µm, DsRed2: Red, HGF: Green, DAPI: Blue Functional test for secreted HGF protein showed significant increase in primary rat hepatocyte proliferation (Picogreen assay)

with conditioned media from pDsRed2-HGF transfected cells (E) * p < 0.05, ** p <

0.01

The cells expressing high levels of HGF protein also showed high levels of DsRed2 (Fig 22D) To evaluate the function of the transgene, primary rat hepatocytes were treated with conditioned media from transfected cells and assessed for proliferation The media from pDsRed2-HGF-transfected cells was significantly more hepatotrophic than cells transfected empty vector (Fig 22E)

3.3.2 Effects Of Vitamin A-Liposome-HGF On Fibrotic Cultures In Vitro

The pDsRed2 empty vector and pDsRed2-HGF constructs were encapsulated in VitaminA-coupled liposomes by gentle mixing for one hour at 4°C Hepatic stellate cells, HSC-T6, (Fig 23A) were transfected with the prepared VitaminA-Liposome–pDsRed2 (VALD) or VitaminA-Liposome–pDsRed2-HGF (VALH) particles Conditioned media from VALH-treated HSCs also showed significantly higher levels

of hepatotrophic potential toward primary rat hepatocytes (Fig 23B) HSC-T6 cells effectively took up VALH particles, and the transfected cells produced significant levels of functional HGF To assess the impact of VALH particles on HSC expression

of fibrotic factors, we measured the concentration of active TGF-β1 protein by ELISA VALH particles caused a strong decrease in the levels of active TGF-β1 (Fig 23C) VALH-treated HSCs showed significantly higher HGF protein expression levels than VALD-treated HSCs (Fig 23D)

Figure 23: Evaluation of the anti-fibrotic effects of VALH particles in HSC-T6 cells HSC-T6 monoculture (A; Scale bar: 60 µm) transfected with VALH complexes

secreted functional HGF as assessed by the induction of proliferation in primary rat hepatocytes after treatment with the conditioned media from these cells (B) and also expressed lower active TGF-β1 levels (C) Increased HGF expression as measured in

culture supernatants of transfected HSC-T6 monocultures (D) * p < 0.05, ** p <

Figure 24: Evaluation of the anti-fibrotic effects of VALH particles in HSC-T6: Hepatocytes co-culture Co-culture of HSC-T6 cells and hepatocytes (A; Scale bar:

60 µm) transfected with VALH complexes showed a decline in the gene expression of fibrotic markers compared to VALD-transfected co-culture; normalized to β-actin (B)

and a decline in the protein levels of active TGF-β1 (C); * p < 0.05, ** p < 0.01, ***

p < 0.001 compared to VALD

To assess the impact of VALH particles on fibrotic co-cultures of HSC-T6 cells and hepatocytes, we measured the expression of several fibrotic genes by RT-PCR, and found significant decreases in the expression levels of various fibrosis-associated genes, such as myofibroblast marker, α-SMA; ECM protein, Col I; basement membrane marker, Col IV; fibrotic factors, TGF-β1, TSP-1, PAI-1, and TIMP-1 (Fig 24B) VALH-transfected hepatic co-cultures also showed decreased active TGF-β1 levels (Fig 24C) In summary, the VALH particles demonstrated the ability to control

the levels of fibrotic factors in vitro

3.3.3 Establishment Of DMN-Induced Liver Fibrosis

0In order to induce liver fibrosis, male Wistar rats were injected with 1% DMN for 3 consecutive days a week, for 4 weeks DMN-induced fibrotic rats exhibited a high degree of hepatic necrosis, as seen in Masson’s trichrome staining (Fig 25A) In particular, the necrosis percentage levels increased sharply from 7-10% in weeks 1-2,

to nearly 25% by week 4 (Fig 25B) The Knodell score, used to categorize the extent

of liver damage (quantified from H&E stained liver tissue sections), showed a steep increase between weeks 2 and 4 (Fig 25C)

A

D

Figure 25: Structural changes in the rat liver with DMN induction DMN

administration caused an increase in necrosis as observed in the Masson Trichrome images of liver tissue sections (A & B; p < 0.05 compared to week 2 controls) Scale bar 100 µm Knodell score measured from Masson’s trichrome and H&E stained liver tissue sections (C) Increased collagen accumulation in DMN-treated rats as quantified by SHG imaging, collagen (green; SHG), hepatic parenchyma (red; TPEF)

(D & E) Scale bar 50 µm* p < 0.05, *** p < 0.001

SHG microscopy of liver tissue sections (Fig 25D) shows the amount of collagen increased during disease progression, as quantified from the SHG images (Fig 25E) Gene expression profiles of fibrotic factors α-SMA, Col I, Col IV, TGF-β1, TSP-1, PAI-1 and TIMP-1 increased with 4 weeks of DMN treatment, while HGF gene expression decreased, relative to the levels in week 0 control rats (Fig 26)

As fibrosis developed, tissue protein levels showed suppression of HGF and increased expression of α-SMA (Fig 27A) Liver function was also affected, as shown by a decrease in serum albumin, and an increase in alanine transaminase (ALT) (Fig 27B) Thus DMN-induced rat livers exhibited numerous molecular and functional features, characteristic of fibrotic disease

E