Genetic Manipulation of Liver Sinusoidal Endothelial Cells

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Genetic Manipulation of Liver Sinusoidal Endothelial Cells

Yoshiyuki Takei

¹

, Kenichi Ikejima

¹

, Nobuyuki Enomoto

¹

, Atsushi Maruyama

²

, and Nobuhiro Sato

¹

Summary. The liver sinusoidal endothelial cells (SECs) possess unique hyaluronan receptors that recognize and internalize hyaluronic acid (HA).

This characteristic was used in the development of a system for targeting foreign DNA to SECs. A gene carrier system was prepared by coupling HA (number-average molecular weight: 1.5 ¥ 10

⁴

) to poly-l-lysine (PLL, number- average molecular weight: 4.6 ¥ 10

⁴

) in a 1 : 1 weight ratio by reductive ami- nation reaction. The resulting copolymer (PLL-g-HA) was mixed with various amounts of DNA in 154 mM NaCl at 4°C. Inter-polyelectrolyte complex formation between PLL-g-HA and DNA exhibited minimal self-aggregation, explaining the highly soluble nature of the complex. The agarose gel retarda- tion assay revealed that the titration point representing the minimum pro- portion of PLL-g-HA required to retard the DNA completely occurred at a 1 : 1 copolymer to DNA charge ratio. Intravenous injection of the [

³²

P]pSV b- Gal plasmid complexed to PLL-g-HA in Wistar rats demonstrated speciﬁc hepatic targeting with >93% of the injected counts taken up by the liver in 1 h. Further, using a ﬂuorescein isothiocyanate-labeled DNA, it was shown that the PLL-g-HA/DNA complex was distributed exclusively in the SECs. Seventy- two hours after injection of 90 m g of pSV b-Gal in a PLL-g-HA-complexed form, a large number of SECs expressing b-galactosidase were detected. Thus, the PLL-g-HA/DNA system permits targeted delivery of exogenous genes selectively to the liver SECs, providing a novel strategy for manipulation of SEC functions.

Key words. Sinusoidal endothelial cell, PLL-g-HA, Triplex DNA, Decoy DNA duplex

129

1

Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

2

Institute of Materials Chemistry and Engineering, Kyushu University, 6-10-1 Hakozaki,

Higashi, Fukuoka 812-8581, Japan

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Introduction

Liver sinusoidal endothelial cells (SECs) play a pivotal role in modulating immunity and inﬂammation [1,2]. Altered immunoresponses of SECs are closely related to pathogenesis of viral hepatitis, fulminant hepatic failure and transplant rejection [3]. Further, interest in xenotransplantation has grown because of the increased demand for donor organs and the possibility of avoiding post-transplant recurrence of viral hepatitis [4]. Xenograft livers usually undergo rapid rejection. It has been proposed that “activation of the SEC” underlies the vascular aspects of discordant xenotransplant rejection.

The phenomena are characterized by rapid gene expression in SECs of pro- coagulant proteins and proinﬂammatory mediators [5].

Given the importance of altered gene expression in SECs, it is possible to envision a new therapeutic strategy for treatment of lethal liver diseases and achievement of graft-speciﬁc immunotolerance through modulation of SEC functions by genetic engineering. However, currently this approach is impos- sible because of lack of modality of selective delivery of nucleotide agents to SECs.

SECs possess unique receptors that recognize and internalize hyaluronic acid (HA) [6,7]. We utilized this characteristic of SECs in the development of a system for targeting foreign DNA to SECs.

Synthesis of PLL-g-HA Comb-Type Copolymers

High molecular weight HA (5.9 ¥ 10

⁵

) was partially degraded by hyalur- onidase to low-molecular weight fragments. Hyaluronidase and HA was added together in 120 ml of water. The solution was stirred at 50°C for a desired time (1–20 h). Then, the solution was boiled for 5 min to terminate the reaction and allowed to cool to room temperature. After the ﬁltration of the cooled mixture through a 0.45 mm ﬁlter (New Steradisc 25, Kurabo, Osaka, Japan), the resulting HA fragments were obtained by freeze-drying.

A gene carrier system (hyaluronate-grafted poly-(l-lysine) copolymer:

PLL-g-HA) was prepared by coupling HA to poly-l-lysine (PLL) in a 1 : 1 weight [8]. The HA fragments (number-average molecular weight: 1.5 ¥ 10

⁴

), obtained as above, were conjugated to PLL (number-average molecular weight: 4.6 ¥ 10

⁴

) by reductive amination using NaBH

3

CN as a reductant.

NaBH

3

CN was added to the mixture of HA (100–300 mg) and PLL (60–120

mg) in 15 ml of sodium borate buffer (0.1 M, pH 8.5) containing 0.4–1 M NaCl

at a ﬁnal concentration of 0.3 M. The reaction mixture was incubated at 40°C

for 3 days while it was stirred constantly, followed by dialysis against a 0.5 M

NaCl aqueous solution using a Spectra/Por 7 membrane (molecular weight

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cutoff of 25 000) to remove unreacted HA. Then, the solution was desalted by dialysis against distilled water, and the resulting copolymer was obtained by freeze-drying.

Gel permeation chromatography (GPC) was carried out using a Jasco 880- PU pumping system at the ﬂow rate of 1.0 ml/min at 25°C with Ultrahydrogel 250 columns (Japan Waters, Tokyo, Japan). The GPC proﬁle of the reaction mixture and the resulting copolymer showed that, after the reaction, the peak area of PLL increased while that of HA decreased. The molecular weight deter- mined by the light scattering detector increased after the reaction. The incu- bation of HA or PLL alone with NaBH

3

CN under the same conditions caused no change in GPC proﬁle, suggesting that neither degradation nor intermol- ecular side reaction occurred. When HA was incubated with PLL in the pres- ence of 10-fold molar excess of lactose or sucrose with respect to the reducing end of HA, not sucrose, the nonreducing sugar, but lactose, the reducing one, inhibited HA consumption. Therefore it can be concluded that the HA was conjugated with PLL at its reducing end through the reductive amination reaction. The GPC proﬁle also shows that the comb-type copolymer was isolated from unreacted HA by dialysis.

The

¹

H NMR spectra of the comb-type copolymer showed the characteris- tic signals of both PLL and HA moieties: PLL, d1.4–1.8 (b, g, d-CH

2

), 3.0 (e-CH

2

), 4.3 (a-CH); HA, d2.0 (Nac-CH

3

), 3.3–3.9 (H-2,3,4,5,6), 4.4–4.6 (H-1).

From the signal ratio of methyl protons (2.0 ppm) of the N-acetyl groups of the HA-grafts to e-methylene protons (3.0 ppm) of the PLL backbone, the content (wt% and grafting%) of HA in the copolymer was determined. The coupling efﬁciency was more than about 70%. It was determined that one HA chain was grafted onto an average of one of every 150 lysine units.

Consequently, we have prepared the PLL-g-HA comb-type copolymer with a defined density and a defined length of HA side chains. The synthesis of the comb-type copolymer has been achieved by (i) the control of the HA chain length by enzymatic hydrolysis, (ii) the suppression of formation of a polyion complex between HA and PLL in high ionic strength media during coupling reaction, and (iii) the relatively high coupling efficiency of the reductive amination between the reducing end of HA and e-amino groups of PLL.

Estimation of the PLL-g-HA/DNA Interaction

DNA was mixed with PLL-g-HA ([amino group]

copolymer

/[phosphate group]

DNA

= 1), and the ionic strength of the medium was gradually decreased from 1 to

0 M NaCl by step-down dialysis. The transparent solution obtained after dial-

ysis was then lyophilized, dissolved in D

2

H, and analyzed by

¹

H NMR. The

¹

H

NMR spectra of the copolymer with or without DNA were analyzed and it was

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shown that the signals of e-methylene protons of the PLL moiety in the PLL- g-HA broadened in the presence of DNA. This is likely caused by polyanion formation between the PLL moiety and DNA. Although the PLL signal broad- ened, the HA signal remained unchanged, or rather became sharper. The sharp signal of HA in the presence of DNA under low-ionic strength condi- tions seem to be similar to those of PLL-g-HA alone at high ionic strength, where the PLL-g-HA did not form polyion complex; i.e., HA chains were free.

These results suggest that the PLL backbone selectively formed the polyanion complex with DNA even in the presence of the HA side chains.

Complex formation between PLL-g-HA and DNA was further assessed with a gel retardation assay. Increasing the proportion of the PLL-g-HA copolymer in the DNA samples affected plasmid DNA migration in an agarose gel. The titration point representing the minimum proportion of PLL-g-HA required to retard the DNA completely occurred at a 1 : 1 copolymer (based on PLL) to DNA charge ratio.

Based on the results, it is postulated that the PLL-g-HA/DNA complex forms the multiphase structure in which the hydrophobic PLL-g-HA complex is surrounded by the hydrated shell of free HA. The presence of free HA chains is essential for directing the complex to the target cells.

Targetability of the PLL-g-HA/DNA Complex to SECs

To determine whether the PLL-g-HA/DNA complex was recognized by SEC HA receptors in vivo, Wistar rats were injected intravenously via the tail vein with PLL-g-HA complexed to

³²

P-labeled pSV b-Gal (an expression plasmid encoding lacZ). One hour postinjection, more than 90% of the injected radioactivity remained in the liver. Administration of the PLL-g-HA com- plexed to a ﬂuorescein isothiocyanate-labeled DNA revealed that the carrier- DNA complex was distributed exclusively in SECs [9]. Polymerase chain reaction (PCR) ampliﬁcation detected the presence of the transgene in the cytosolic fraction 3 h after transfection only from the liver of the rats injected with PLL-g-HA/pSV b-Gal, but not with pSV b-Gal alone. A large number of cells expressing b-galactosidase were detected in SECs when transfected with the PLL-g-HA/pSV b-Gal complex.

Several lines of evidence shown in this study indicate that the complex was

transferred successfully to the SECs via HA receptor-mediated endocytosis

[9]. (i) Intravenous administration of the PLL-g-HA/[

³²

P]pSV b-Gal resulted

in accumulation of >90% of the radioactivity in the liver. The PLL-g-HA/DNA

complex was localized exclusively in the sinusoidal lining cells (i.e. SECs). (ii)

The transgene was restored from the cytosolic fraction of the liver and lacZ

gene expression was demonstrated in the SECs following transfection with

the PLL-g-HA/pSV b-Gal complex.

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Stabilization of Triplex DNA

The ability to target specific sequence of DNA through oligonucleotides-based triplex formation provides a powerful tool for genetic manipulation. Triplex- forming oligonucleotides (TFOs) can be targeted for its sequence-specific binding to the major groove of homopurine–homopyrimidine stretch in target genes. Triplex-forming oligonucleotides can inhibit or enhance DNA transcription, generate site-specific mutations, cleave DNA, and induce homologous recombination. However, instability of triplex DNA under phys- iological conditions, due to the electrostatic repulsion between a TFO and the duplex as well as pH-dependence and K

⁺

sensitivity, limits in vivo application of the triplex strategy. We previously reported that comb-type copolymers composed of PLL and hydrophilic polysaccharides signiﬁcantly increased thermal stability of triplex structure.

In this study, we used PLL-g-HA, a comb-type copolymer having HA as a side chain, and evaluated its ability to stabilize triplexes. Triplex formation was analyzed by electrophoretic mobility shift assay (EMSA) using a 30-mer target duplex from rat a1 (I) collagen gene promoter (T-1/T-2 duplex, T-1: 5¢-CCTTTCCCTTCCTTTCCCTCCTCCCCCCTC-3¢;

T-2: 3 ¢-GGAAAGGGAAGGAAAGGGAGGAGGGGGGAG-5¢); Pu-20 (5¢-

GGAAAGGGAAGGAAAGGGAGGAGGG-3¢) and Py-20 (5¢-

CCCTTCCTTTCCCTCCTCCC-3¢) as TFOs. PLL-g-HA diminished remarkably K

⁺

inhibition of purine motif triplex formation with Pu-20 as well as pH- dependence of pyrimidine motif triplex formation with Py-20 [9]. Moreover, thermal denaturation (UV-Tm) and circular dichroism experiments revealed that PLL-g-HA signiﬁcantly increased the thermal stability of triplex struc- ture. Thus, the PLL-g-HA/DNA carrier system stabilizes triplex DNA.

Inhibition of TNF a-Induced ICAM-1 Expression in SECs Using a Double-Strand Decoy DNA Oligomer for NF- kB

Altered gene expression of SECs is associated with impaired immune response. We determined if the decoy technique could effectively suppress tumor necrosis factor a (TNFa)-induced intercellular adhesion molecule 1 (ICAM-1) expression in SECs. A nuclear factor (NF)-kB decoy (NF-kB31:

5 ¢-TGGGGACTTTCCAGTTTCTGGAAAGTCCCCA-3¢), which contains a con-

sensus sequence for NF-kB, was synthesized [10]. NF-kB31 was complexed to

PLL-g-HA. Controls were oligodeoxynucleotides (ODNs) with reversed and

scrambled sequences. The PLL-g-HA/NF-kB31 complex was added to the

culture media of LSE cells, a human SEC-derived cell line, and cultured for

3 h. Then, cells were stimulated for 1–4 h with TNFa (5 ng/ml). Tumor necro-

sis factor a caused a dramatic translocation of NF-kB protein into the nuclei

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of LSE cells followed by marked upregulation of ICAM-1 on the cell surface.

PLL-g-HA/NF-kB31, but not control ODNs, inhibited the intranuclear local- ization of NF-kB induced by TNFa, with almost complete inhibition at 2.5 mg/ml. NF-kB31 attenuated the increase in ICAM-1 mRNA as well as protein levels in LSE cells. The decoy technique in combination with PLL-g-HA may provide a novel strategy for manipulation of SEC functions.

Conclusion

The PLL-g-HA/DNA carrier system permits targeted delivery of exogenous genes selectively to the liver SECs. The gene carrier system in combination with the decoy or triplex DNA approaches may lead to a new and more effec- tive strategy for manipulation of SEC functions.

Acknowledgments. This work was supported by the Grants-in-Aid for the Development of Scientiﬁc Research (B 07557045 and C2 15590690) from the Ministry of Education and Science, Japan, and by grants from the Uehara Memorial Foundation and Kanae Medical Foundation.

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