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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Cell transplantation-induced hepatic ischemia and recruitment of vasoconstrictors (e.g., endothelin-1; Edn1) leads to clearance of transplanted cells and poses problems for liver repopulation. Therefore, we determined whether darusentan (DAR), which potently blocks Edn1 receptor type A, could benefit cell engraftment. We transplanted primary F344 rat hepatocytes with or without DAR in dipeptidyl peptidase IV–deficient rats. Analysis of microcirculatory events included hepatic ischemia, endothelial injury, including with gene expression arrays, and activations of Kupffer cells (KCs), neutrophils, or hepatic stellate cells (HSCs). The retrorsine-partial hepatectomy model was used for liver repopulation studies. Whether DAR was directly cytoprotective was examined in cultured rat hepatocytes or CFSC-8B rat HSCs. We found that DAR induced hepatic sinusoidal vasodilation, caused more transplanted cells to be deposited in liver parenchyma, and decreased hepatic ischemia and endothelial injury. This lessened perturbations in expression of endothelial biology genes, including regulators of vessel tone, inflammation, cell adhesion, or cell damage, versus drug-untreated controls. Moreover, in DAR-treated animals, cell transplantation-induced activation of KCs, albeit not of neutrophils, decreased, and fewer HSCs expressed desmin. In DAR-treated rats, improvements in cell engraftment led to greater extent of liver repopulation, compared to drug-untreated controls. In cell-culture assays, DAR did not stimulate release of cytoprotective factors, such as vascular endothelial growth factor, from HSCs. Moreover, DAR did not protect hepatocytes from tumor necrosis factor alpha– or oxidative stress–induced toxicity. Endothelin receptor A blockade in vitro did not improve engraftment of subsequently transplanted hepatocytes. Conclusion: Systemic administration of DAR decreases hepatic ischemia-related events and thus indirectly improves cell engraftment and liver repopulation. This vascular mechanism may permit the development of combinatorial drug-based regimens to help optimize cell therapy. (Hepatology 2014;59:1107–1117)

Abbreviations
ALT

alanine aminotransferase

DAR

darusentan

DMEM

Dulbecco's modified Eagle's medium

DPPIV−

dipeptidyl peptidase IV deficient

Edn1

endothelin-1

Ednra/rb

endothelin type A/type B

GGT

gamma-glutamyl transpeptidase

HA

hyaluronic acid

HSC

hepatic stellate cell

IP

intraperitoneally

IS

intrasplenically

KC

Kupffer cell

LSEC

liver sinusoidal endothelial cell

MPO

myeloperoxidase

MTT

methyl thiazol tetrazolium

NIH

National Institutes of Health

OS

oxidative stress

PH

partial hepatectomy

RV

right ventricular

TNF-α

tumor necrosis factor alpha

VEGF

vascular endothelial growth factor

Efficient engraftment of transplanted cells in the liver was apparent early on as a barrier for cell therapy in humans.[1, 2] Cell engraftment requires depositing of cells in liver sinusoids, which causes hepatic ischemia, tissue injury, and inflammation as a result of vaso-occlusion, and 80%-90% transplanted cells are lost within 1-2 days.[3] This cell clearance is mediated, in part, by cytokines, chemokines, and receptors activated by neutrophils, Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), or hepatic stellate cells (HSCs)[3-5] and, in part, by instant blood-mediated reaction involving procoagulant activity and complement.[6] The underlying mechanisms are complex because endothelial damage, without thrombotic occlusion, simultaneously allows transplanted cells to enter liver parenchyma,[7, 8] whereas release by HSC of vascular endothelial growth factor (VEGF), matrix metalloproteinases, and so on, protect transplanted cells and facilitate parenchymal remodeling during cell engraftment.[9] However, on balance, cell transplantation-induced microcirculatory alterations are deleterious[3] and must be overcome. For instance, direct-acting vasodilators (i.e., nitroglycerine, phentolamine, or prostacyclin) improve cell engraftment.[3, 9] Use of such drugs to control harmful microcirculatory events will be highly significant for cell therapy.

Recently, endothelin-1 (Edn1), a potent vasoconstrictor that transduces its effects through type A (Ednra) or type B (Ednrb) receptors, was incriminated in cell transplantation-induced changes.[3] Bosentan, a nonspecific blocker of Ednra/Ednrb, improved cell engraftment, emphasizing the role of Edn1. However, in bosentan recipients, transplanted cells did not proliferate or repopulate the liver. Whether this was the result of displacement by bosentan of harmful ligands that might have produced changes in naïve transplanted cells was possible, e.g., plasma Edn1 levels were elevated in Edn1 receptor knockout mice.[10] This possibility was confirmed when hepatic Edn receptors were blocked beforehand by bosentan in vitro, because transplanted cells could then proliferate and repopulate the liver.[3] Although intracellular signaling from Edn1 receptors is ill-defined, this includes compensatory and/or opposing effects.[11] Of Edn1 receptors, selective blockade of Ednra is considered desirable, because Ednrb may be cytoprotective.[12] Therefore, Ednra blockers were developed, for example, darusentan (DAR), which is in the late clinical phase for vascular conditions[13] and shows promise for liver conditions.[14, 15] Here, we considered that Ednra blockade with DAR will improve cell transplantation-induced microcirculatory changes and thereby cell engraftment. We performed cell transplantation assays in dipeptidyl peptidase IV deficient (DPPIV) F344 rats, including the retrorsine/partial hepatectomy (PH) model of liver repopulation.[3-5, 7-9]

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
DAR and Chemicals

Unless specified, all reagents and chemicals were from Sigma-Aldrich Chemical Co. (St. Louis, MO). DAR (Knoll, Ludwigshafen, Germany) was dissolved to 10 mg/mL of normal saline containing 0.24 mL of 1 N of NaOH for pH 11 with final pH to 7.5 with 0.1 N of HCl.

Animals

Six- to eight-week-old DPPIV rats, weighing 120-150 g, were from the Special Animal Core of Marion Bessin Liver Research Center (Bronx, NY). Donor F344 rats were from the National Cancer Institute (Bethesda, MD). Rats were anesthetized with ketamine and xylazine. The animal care and use committee at Albert Einstein College of Medicine (Bronx, NY) approved protocols per guidelines from the National Institutes of Health (NIH; Bethesda, MD).

Hepatocytes were isolated by collagenase liver perfusion, as described previously (3). For vascular effects of DAR, 2 × 106 15-µm latex microspheres (New England Nuclear, Boston, MA) were injected intrasplenically (IS) 2 hours after 2.5-10.0 mg/kg of DAR or vehicle given intraperitoneally (IP). After 2 hours, rats were killed for microsphere distributions in liver. Right ventricular (RV) pressures were measured by PE50 cannulae in the internal jugular vein before and after DAR. For hepatocyte transplantation, DAR or vehicle was given IP 2 hours before IS injection of 2 × 107 freshly isolated hepatocytes. For KC activity, 0.1-mL carbon particles encriched from India ink (Pelikan no. 17; Pelikan Hannover, Germany) were injected IS 6 hours after cells and rats were killed 30 minutes later.[3] To study the effects of repeatedly administered DAR, 2 × 107 hepatocytes were transplanted twice 10 days apart. For liver repopulation, 30 mg/kg of retrorsine were given IP to 6- and 8 week-old DPPIV rats, followed 4 weeks later by two-thirds PH, when 5 × 106 hepatocytes were injected IS.

Messenger RNA Studies

Total RNA was extracted by Trizol Reagent (Invitrogen, Carlsbad, CA). RNA was reverse-transcribed by the RT[2] PCR Array First Strand Kit, and for reverse-transcription polymerase chain reaction, we used RT[2] Real-Time SYBR Green PCR master mix (SABiosciences, Frederick, MD). Rat endothelial cell biology array with 84 gene probes was used (PARN-015; SABiosciences; http://www.sabiosciences.com/rt_pcr_product/HTML/PARN-015A.html), according to the manufacturer's instructions. We analyzed replicate samples from 3 rats per condition. The 2-ΔΔCt method was used for fold differences. Data were first normalized with β-actin in individual samples. Gene expression differences of >1.5-fold with P < 0.05 were considered significant.

Studies With Cells In Vitro

All conditions were in triplicate and studies were repeated at least twice. For cytotoxicity assays, 1 × 105 hepatocytes were cultured per cm2 plastic dishes in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics. For tumor necrosis factor alpha (TNF-α) studies, cells were preincubated with 200 ng/mL of actinomycin D for 2 hours before 10 ng/mL of TNF-α (Serologicals Corp., Atlanta, GA) for 16-18 hours.[3] For oxidative stress (OS), hepatocytes were cultured for 16-18 hours and exposed to 2 mM of H2O2 for 90 minutes. DAR (in 1-100-μM doses) was added immediately after cell plating. Rat CFSC-8B HSCs were cultured in DMEM. Culture conditions included normoxia (21% O2 and 5% CO2) or hypoxia (5% O2, 5% CO2, and 11% N2).[5] Conditioned medium from CFSC-8B cells was obtained in serum-free DMEM after 16-18 hours. Cell viability was analyzed by thiazolyl blue (methyl thiazol tetrazolium; MTT) utilization.[3] For additional transplantation studies, cells were incubated for 1 hour on ice in serum-free DMEM or DMEM containing 10 μM of DAR.

Tissue Analysis

Multiple samples were analyzed from at least 3-4 rats per condition. Five-micrometer sections were prepared from tissues frozen in methylbutane at −80°C. For gamma-glutamyl transpeptidase (GGT), sections were fixed in chloroform-acetone and reacted with N-γ-L-glutamyl-4-methoxy-β-naphthylamide (Polysciences, Inc., Washington, PA).[6] GGT+ areas were measured in 25-50 fields centered on portal radicles. KCs containing carbon were counted in 50 periportal areas per rat under 200× microscopic magnification. Neutrophils with myeloperoxidase (MPO) activity were localized by peroxidase indicator reagent (Sigma-Aldrich), as previously described.[1] MPO+ cells were counted in 50 areas under 200× microscopic magnification. Sections were stained with desmin antibody (Dako Cytomation, Carpinteria, CA), as previously described.[3] Desmin+ HSCs were counted under 400×. For DPPIV histochemistry, sections were fixed in chloroform-acetone (1:1, vol/vol) with toluidine blue counterstain, as previously described.[3-5, 7-9] DPPIV+ transplanted cells were counted in 100 areas centered on portal radicles under 100× microscopic magnification. After transplanting cells twice, engraftment was analyzed 14 days after the final cell transplantation session. For liver repopulation, 3 weeks after cell transplantation, DPPIV+ cells were analyzed in 25 areas per rat by ImageJ software (NIH, Bethesda, MD).

Hyaluronic Acid and VEGF Measurements

Serum was assayed for hyaluronic acid (HA) and culture medium for VEGF by kits (Corgenix, Inc., Westminster, CO; and Rat VEGF Quantikine ELISA, RRV00; R&D Systems, Minneapolis, MN), as previously described.[3] Serum alanine aminotransferase (ALT) and bilirubin were analyzed by an automated clinical system.

Statistical Analysis

All studies included 3-8 rats per condition. Major findings were reproduced at least twice. Data are expressed as means ± standard deviation. Significances were analyzed by t tests, Mann-Whitney's rank-sum tests, or analysis of variance, including Holm-Sidak's pair-wise comparisons by SigmaStat 3.1 statistical software (Systat Inc., Point Richmond, CA). P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Rats tolerated DAR without morbidity. Administration of DAR (in doses of 2.5-10.0 mg/kg) had no effect on RV pressures. We observed no gross liver abnormalities, histological changes, or differences in serum ALT or bilirubin levels after DAR (not shown).

DAR Improved Cell Engraftment by Altering Sinusoidal Distribution of Transplanted Cells

Dose-ranging studies showed that more latex microspheres were in the distal portions of hepatic sinusoids after 10 mg/kg versus either 1 or 5 mg/kg of DAR (not shown). Consequently, we chose 10 mg/kg of DAR for studies. We observed more transplanted cells in liver sinusoids after DAR (P < 0.05; Fig. 1). Large numbers of transplanted cells (∼90%) were cleared after 6 hours or later in controls as well as DAR-treated rats, with similar kinetics of transplanted cell clearance. However, in DAR-treated rats, more cells engrafted, including after 6 hours, 3 days, and 7 days (P < 0.05). Cumulatively, transplanted cell numbers were 1.5- to 2.5-fold higher in DAR-treated rats (P < 0.05). Greater entry of transplanted cells in sinusoids was reflected by transplanted cells in fewer portal vein radicles after 6 hours (mean of 31% in DAR-treated versus 69% in control rats after 6 hours; P < 0.05). Because transplanted cell numbers in liver were similar after 3 and 7 days, this indicated that cells surviving after initial clearance had engrafted.

image

Figure 1. Analysis of cell engraftment in DPPIV rats after Ednra blockade with DAR. (A) Experimental design indicating animals were given IP vehicle or DAR 2 hours before cell transplantation. Groups of rats (6-8 per interval) were killed after 6 hours or 3 and 7 days to collect tissue samples for cell engraftment, gene expression, tissue changes, and serum HA levels. (B) Representative data indicated more transplanted hepatocytes (red color: DPPIV histochemistry) in liver after transplantation. (C) Cumulative morphometric data indicated.that most transplanted cells were cleared from the liver by 3 days in both control and DAR-treated rats. (D) Morphometric analysis indicated that transplanted cells were in fewer portal radicles after 6 hours in DAR-treated rats. *P < 0.05. Original magnification: 200×, toluidine blue counterstain.

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Effects of DAR on Cell Transplantation-Induced Changes

Expression of endothelial biology genes altered the most 6 hours after cell transplantation. In controls or DAR-treated rats, expression was unaltered in 18 of 84 (21%) genes, including vascular genes (e.g., angiotensin receptor 1b, Edn2, Ednra, and so on). By contrast, expression was different by 1.5- to 225-fold in 50 of 84 (60%) genes (P < 0.05; Table 1). These genes included regulators of vessel tone, angiogenic factors, inflammatory cytokines, chemokines, and receptors, cell death or apoptosis, cell adhesion, and so on. In DAR-treated rats, expression of 17 genes up-regulated by cell transplantation decreased, including regulators of vessel tone or angiogenesis (Adam17, Fgf1, Pgf, and transforming growth factor beta 1), apoptosis (Casp1), inflammation (Ccl2, Cx3cl1, Il1b, and Tnfsf10), cell adhesion (Pecam, Sell), or thrombosis (Plg, Thbd). As an exception, Il-6 was up-regulated in DAR-treated rats. Expression in DAR-treated rats of several inflammatory cytokines (Cxcl1, Cxcl2, Il11, Il7, and Tnf) was similar to controls.

Table 1. Differences in Endothelial Cell Biology Gene Expression After Cell Transplantation in Controls and DAR-Treated Rats
Gene DescriptionGene SymbolFold Up or Down in Cells Alone Versus Untreated ControlsFold Up or Down in Cells + DAR Versus Untreated ControlsFold Difference Cells + DAR Versus Cells AloneaComment
  1. a

    Differences considered significant when ≥1.5-fold up or down when cells alone versus cells plus DAR were compared.

Angiotensin I converting enzyme (peptidyl-dipeptidase A) 1Ace3.22.50.8No effect
A disintegrin and metalloproteinase domain 17 (tumor necrosis factor, alpha, converting enzyme)Adam171.4−2.00.4Down-regulation
Angiotensinogen (serpin peptidase inhibitor, clade A, member 8)Agt2.01.50.7No effect
Angiopoietin 1Angpt1−1.4−2.10.7No effect
Annexin A5Anxa51.71.50.9No effect
Bcl2-associated X proteinBax1.81.40.8No effect
B-cell leukemia/lymphoma 2Bcl22.01.80.9No effect
Bcl2-like 1Bcl2l11.8−1.20.6No effect
Baculoviral IAP repeat-containing 1bBirc1b−1.5−2.80.5Down-regulation
Burkitt lymphoma receptor 1Blr14.8−2.20.7No effect
Caspase 1Casp11.3−13.60.09Down-regulation
Caspase 3, apoptosis-related cysteine proteaseCasp31.71.20.7No effect
Caspase 6Casp61.61.20.7No effect
Chemokine (C-C motif) ligand 2Ccl29.44.30.5Down-regulation
Cadherin 5 (predicted)Cdh5_predicted1.1−1.20.3Down-regulation
CASP8- and FADD-like apoptosis regulatorCflar2.01.30.6No effect
Procollagen, type XVIII, alpha 1Col18a11.61.10.6No effect
CASP2 and RIPK1 domain-containing adaptor with death domain (predicted)Cradd−1.1−2.20.5Down-regulation
Colony-stimulating factor 2 (granulocyte-macrophage)Csf213.515.61.2No effect
Chemokine (C-X3-C motif) ligand 1Cx3cl13.81.10.3Down-regulation
Chemokine (C-X-C motif) ligand 1Cxcl1181.0130.40.7No effect
Chemokine (C-X-C motif) ligand 2Cxcl2224.9267.51.2No effect
Endothelial cell growth factor 1 (platelet-derived)Ecgf1−4.0−5.60.7No effect
Endothelin 1Edn15.46.31.2No effect
Fibroblast growth factor 1Fgf1−1.3−5.50.2Down-regulation
FMS-like tyrosine kinase 1Flt1−1.2−2.00.6No effect
Fibronectin 1Fn12.0−1.30.6No effect
Intercellular adhesion molecule 1Icam14.83.20.7No effect
Interleukin-11Il111.11.41.2No effect
Interleukin-1 betaIl1b3.51.60.5Down-regulation
Interleukin-6Il620.946.02.2Up-regulation
Interleukin-7Il73.62.90.8No effect
Integrin alpha 5Itga5−1.1−1.90.6No effect
Integrin beta 1 (fibronectin receptor beta)Itgb11.9−1.30.6No effect
Integrin beta 3Itgb31.71.20.7No effect
Kinase insert domain protein receptorKdr−2.6−3.90.7No effect
V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologKit−1.3−1.90.7No effect
Matrix metallopeptidase 9Mmp923.214.60.6No effect
Nitric oxide synthase 2, inducibleNos255.639.50.7No effect
Natriuretic peptide receptor 1Npr1−1.4−1.60.9No effect
Platelet-derived growth factor receptor, alpha polypeptidePdgfra1.9−1.30.6No effect
Platelet/endothelial cell adhesion moleculePecam1.2−1.50.4Down-regulation
Chemokine (C-X-C motif) ligand 4Cxcl42.22.31.1No effect
Placental growth factorPgf13.67.10.5Down-regulation
Plasminogen activator, tissuePlat2.11.90.9No effect
Plasminogen activator, urokinasePlau2.22.41.1No effect
PlasminogenPlg1.1−2.10.3Down-regulation
Prostaglandin I2 (prostacyclin) synthasePtgis5.83.60.6No effect
Inositol 1,3,4,5,6-pentakisphosphate 2-kinaseIppk2.01.60.8No effect
Ras homolog gene family, member BRhob8.95.50.6No effect
Selectin, lymphocyteSell12.83.50.3Down-regulation
Selectin, plateletSelp40.531.70.8No effect
Serine (or cysteine) peptidase inhibitor, clade E, member 1Serpine1181.0144.00.8No effect
Endothelial-specific receptor tyrosine kinaseTek−1.8−2.30.8No effect
Transforming growth factor, beta 1Tgfb11.8−1.90.5Down-regulation
ThrombomodulinThbd5.22.60.5Down-regulation
Thrombospondin 1Thbs119.118.91.0No effect
Tissue inhibitor of metallopeptidase 1Timp119.519.81.0No effect
Tumor necrosis factor (TNF superfamily, member 2)Tnf9.08.30.9No effect
Fas (TNF receptor superfamily, member 6)Fas2.11.60.8No effect
Tumor necrosis factor (ligand) superfamily, member 10Tnfsf10−1.3−3.00.4Down-regulation
Fas ligand (TNF superfamily, member 6)Faslg−1.2−1.50.8No effect
TNFAIP3 interacting protein 2Tnip23.12.10.7No effect
OccludinOcln1.0−1.70.4Down-regulation
Vascular cell adhesion molecule 1Vcam12.21.30.6No effect
Von Willebrand factorVwf−1.2−1.70.7No effect

Tissue analysis confirmed hepatic ischemia after cell transplantation. In controls, hepatic GGT was expressed, carbon uptake increased in KC, and MPO+ neutrophils appeared after 6 hours; also, desmin+ HSCs appeared after 3 days (Fig. 2). Serum HA levels in rats 6 hours after cell transplantation were higher (70 ± 8 versus 35 ± 6 ng/mL in healthy rats; P < 0.05), confirming endothelial injury. By contrast, in DAR-treated rats, hepatic ischemia and related events decreased because GGT expression declined by 50% (P < 0.05), numbers of KCs with carbon declined by 23% (P < 0.05), and serum HA levels decreased to 55 ± 5 ng/mL (P < 0.05); also, 40% fewer desmin+ HSCs were observed (P < 0.05). However, the number of MPO+ neutrophils was unchanged in DAR-treated rats.

image

Figure 2. Cell transplantation-induced changes in liver and effects of DAR. Experimental design was as described in Fig. 1. (A) Panels showing representative data from vehicle-treated controls and DAR-treated rats 6 hours (GGT, KC, and MPO+ cells) or 3 days (desmin+ cells) after cell transplantation. GGT staining indicated hepatic ischemia after cell transplantation. Activated KCs with carbon (arrows) are shown adjacent to DPPIV+ hepatocytes (red color); inset shows magnified view of KCs with carbon, MPO+ neutrophils (arrows), and desmin+-activated HSCs (arrows). (B) Cumulative morphometric data indicated that DAR decreased hepatic GGT expression (ischemia), as well as numbers of activated KCs and HSCs, but not activation of neutrophils. *P < 0.05. Original magnification: KCs and GGT, 200×; MPO and desmin, 400×. Toluidine blue (for KCs) or hematoxylin counterstain.

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These tissue findings corresponded to gene expression differences in DAR-treated rats. Less GGT expression and KC or HSC activity were reflected in lower expression of genes regulating vessel tone, angiogenesis, cell adhesion, thrombosis, apoptosis, or macrophage/monocyte-associated cytokines (Ccl2 and Cx3cl1), whereas expression of other cytokines (e.g., Tnf, Il6, Cxcl1, and Cxcl2) likely reflected continued neutrophil activation.

Repeated Hepatocyte Transplantation and DAR

Rats tolerated multiple doses of DAR without morbidity. In drug-untreated controls, 2 weeks after two doses of 2 × 107 cells, transplanted cell numbers increased by 2-fold versus single dose of 2 × 107 cells (P < 0.05; Fig. 3). In DAR-treated rats, transplanted cell numbers increased by 4- and 2-fold versus one or two doses of cells in controls, respectively (P < 0.05). This indicated that DAR was repeatedly beneficial for cell engraftment.

image

Figure 3. Effects of DAR on repeated hepatocyte transplantation. (A) Experimental design indicating cell transplantation in vehicle- or DAR-treated rats at 10-day intervals followed 2 weeks later by analysis of cell engraftment. (B) Representative data showing more transplanted cells (red color: DPPIV histochemistry) in DAR-treated rat liver. The difference in number of transplanted hepatocytes was >2-fold above controls, as shown in chart. *P < 0.05. Original magnification: 200×, toluidine blue counterstain.

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DAR and Transplanted Cell Proliferation

We used retrorsine/PH-preconditioned rats for liver repopulation with cells transplanted immediately after isolation or after incubation with DAR or vehicle in vitro (Fig. 4A). Engraftment of cells transplanted immediately was similar in DAR-treated retrorsine/PH-preconditioned rats and DAR-treated nonpreconditioned rats (P = not significant). In retrorsine/PH-preconditioned rats without DAR, 7% ± 2% of liver was repopulated after 3 weeks. In DAR-treated retrorsine/PH-preconditioned rats, 4.4-fold more liver was repopulated (31% ± 13%; P < 0.05; Fig. 4B). Therefore, DAR did not interfere with hepatocyte proliferation. By contrast, transplantation of cells incubated in vitro produced different results. First, engraftment of DAR-incubated cells was only 33% ± 6% of vehicle-treated cells; second, proliferation of vehicle-incubated cells was inferior to cells transplanted immediately after isolation; and third, proliferation of DAR-incubated cells was worse than vehicle-incubated cells (P < 0.05) in each case (Fig. 4C).

image

Figure 4. Proliferation of transplanted hepatocytes. (A) Experimental design indicating preconditioning with retrorsine/PH followed by vehicle or DAR and hepatocyte transplantation. Liver repopulation was analyzed after 3 weeks. (B) Representative liver sections with greater area of transplanted cells (red color) in DAR-treated rats. Morphometric analysis confirmed liver repopulation was 3- to 4-fold greater after DAR. (C) Results after transplantation of cells treated with vehicle or DAR in vitro. In this situation, cell engraftment and proliferation were inferior under both conditions, compared with cell transplantation without incubation in vitro. *P < 0.05. Original magnification: 200×, toluidine blue counterstain.

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Effects of DAR on Cells In Vitro

Cell viability was unchanged after culture of hepatocytes with vehicle or 1-100 μM of DAR. Hepatocyte viability declined by approximately 50% after culture with TNF-α or H2O2 (P < 0.05; Fig. 5). In hepatocytes cultured with 1-100 µM of DAR plus TNF-α or H2O2, viability neither improved nor worsened.

image

Figure 5. Effects of DAR on cytotoxicity in cultured cells. Cell viability with MTT assays is shown as percent of controls. (A) TNF-α-induced cytotoxicity in rat hepatocytes. DAR neither improved nor worsened cell viability. (B) H2O2-induced cytotoxicity in rat hepatocytes. Again, DAR did not alter cell viability. (C) Effect of DAR on hypoxia-induced VEGF release in medium of CFSC-8B cells over 3 days. (D) Effect of conditioned medium harvested under hypoxia from CFSC-8B cells with or without DAR on TNF-α cytotoxicity in rat hepatocytes.

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To determine whether DAR could have affected release of paracrine factors from HSCs, which benefited cell engraftment in the case of bosentan,[3] we studied rat CFSC-8 HSCs. Culture of these cells under hypoxia increased VEGF secretion into medium (22 ± 4 vs. 44 ± 10 pg/mL; P < 0.05). The conditioned medium protected hepatocytes from TNF-α toxicity. Culture of CFSC-8 cells with DAR under hypoxia led to secretion of only 12 ± 7 pg/mL of VEGF (P < 0.05); moreover, this medium did not additionally protect hepatocytes from TNF-α. Therefore, HSCs did not likely contribute to cell engraftment from release of hepatoprotective paracrine factors in DAR-treated rats.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

We established that DAR improved transplanted cell engraftment without interfering with transplanted cell proliferation and liver repopulation. These benefits were realized with relatively small doses of DAR. Previously, DAR was found to be safe for cardiovascular applications in much higher doses, for example, 50 mg/kg daily (in rats) and up to approximately 6 mg/kg or more daily in humans over extended periods.[16, 17] Even 2-to 3-fold greater initial cell engraftment has enormous benefit on the kinetics of liver repopulation, as shown here and in previous studies.[3-5, 7-9] The mechanisms by which DAR was helpful involved direct effects on microcirculation, rather than indirect effects on either transplanted cells or native cells (e.g., HSCs). Therefore, systemic administration will be necessary of DAR for benefits.

In DAR-treated rats, cell transplantation-induced vascular alterations improved, including at gene expression and tissue levels, although not all perturbations were resolved (e.g., inflammation continued). After cell transplantation, hepatic ischemia occurs immediately, but other changes evolve over hours, as shown here. After cell transplantation, hepatic blood flow was restored by sinusoidal vasodilatation, as documented with live video microscopy in nitroglycerine-treated rats.[9] Similarly, directly acting drugs, such as prostacyclin, and the alpha-adrenergic blocker, phentolamine, decreased cell transplantation-induced vasoconstriction. Several classes of vascular drugs were ineffective (e.g., β-blockers, calcium-channel blockers, or angiotensin-converting enzyme inhibitors) because these drugs dilated large vessels, but not hepatic sinusoids.[3, 9] In the case of DAR, hepatic sinusoidal vasodilatation occurred, as indicated by entrapment of fewer cells in portal radicles, the presence of more cells in distal sinusoidal locations, and decreases in hepatic GGT expression, which was originally demonstrated to be activated by ischemic occlusion of hepatic sinusoids,[18] and has since been well-established as a convenient reporter of cell transplantation-induced ischemia.[3, 9] Moreover, expression of several genes regulating vessel tone, cell adhesion, endothelial damage, thrombosis, and so on, was decreased in recipients of DAR, consistent with the role of Edn1-mediated vascular events after cell transplantation. It should be noteworthy that DAR blocks Edn1-induced responses in vascular smooth muscle cells,[19] which regulate vessel tone, and also has direct effects on LSECs because endothelial fenestrae are dilated.[20] Similarly, by improving sinusoidal perfusion, DAR decreased hepatic ischemia-reperfusion injury,[14, 15] which will be in agreement with sinusoidal vasodilatation after cell transplantation in DAR-treated rats.

However, the effects of DAR in the cell-transplantation setting differed from other drugs, including nitroglycerine and prostacyclin, and, particularly, bosentan,[3] as summarized in Table 2. Whereas DAR and bosentan both dilated hepatic sinusoids with restoration, at least partly, of endothelial biology gene expression, only DAR decreased hepatic ischemia, endothelial injury, and KC activation, as judged by decreased GGT expression, serum HA levels, and carbon uptake in KCs. These vascular effects of DAR were similar to the effects of nitroglycerine and prostacyclin on hepatic GGT expression, endothelial injury (lower serum HA levels), and activation of KC after cell transplantation.[3] Whether DAR will modulate procoagulant activity of cells after transplantation for further benefits is unknown.

Table 2. Differences in Hepatic Effects of DAR and Bosentan in Cell Transplantation Setting
ProcessEffects of DAREffects of BosentanaComment
  1. a

    Data from Raper et al. 1996.1

Hepatic sinusoidal vasculatureVasodilationVasodilationConcordant effect
Hepatic ischemia (GGT expression)DecreasedNo changeDiscordant effect
LSEC injury (increased serum HA levels)DecreasedNo changeDiscordant effect
Endothelial biology gene expressionDecreases in regulators of vessel tone, cell damage, adhesion, thrombosisDecreases in regulators of vessel tone, cell damage, adhesion, thrombosisConcordant effect
Activation of neutrophilsNo changeNo changeConcordant effect
Macrophage (KC) responseDecreasedNo changeDiscordant effect
Activation of HSCsDesmin expression decreased; no stimulation of VEGF releaseDesmin expression increased; VEGF release stimulatedDiscordant effect
Transplanted cell engraftmentIncreasedIncreasedConcordant effect
Benefits for repeated cell transplantationYesUnknownNeeds further study
Transplanted cell proliferationNo interferenceInterferenceDiscordant effect
In vitro drug manipulation of cellsbefore transplantationNo benefitBeneficialDiscordant effect

Of note, neither DAR nor bosentan prevented cell transplantation-induced neutrophil activation. This inability to inhibit neutrophil activation was shared by nitroglycerine and prostacyclin.[3] Continuing expression of several inflammatory cytokines and chemokines, including Il6, Tnf, and so on, which is associated with activated neutrophils, despite DAR (or other vascular drugs), was in agreement with this finding. Previously, Edn1 was incriminated in transcriptional regulation of inflammatory cytokine expression by Ednra, along with Ednrb,[21, 22] because these receptors share intracellular signaling pathways.[11] Displacement of Edn1 from its receptors by DAR or bosentan could certainly have amplified such intracellular inflammatory perturbations.

Differences also emerged in cell transplantation-induced activation of HSCs. DAR decreased desmin expression in HSCs in vivo without inducing VEGF release in vitro, whereas bosentan and nitroglycerine increased desmin expression in HSCs in vivo and bosentan induced VEGF release in vitro.[3] This difference was significant because release of VEGF and other hepatoprotective substances was beneficial for cell engraftment.[5]

Other differences concerned roles of DAR and bosentan in protecting hepatocytes from cytotoxic injury. After Ednra blockade by DAR in vitro, we did not observe cytoprotection, whereas after Ednra/Ednrb blockade by bosentan in vitro, cells resisted cytotoxic insults.[3] Similarly, treatment of CFSC-8B cells with DAR did not elicit paracrine benefits by VEGF release, which was different from bosentan treatment of CFSC-8B cells.[3] While bosentan interfered with transplanted cell proliferation, DAR did not interfere with proliferation of transplanted cells.

These differences in the effects of DAR and bosentan provided insights into the role of Edn1 and Ednra/Ednrb in cell transplantation. For cell therapy strategies, incubating cells with bosentan for its cytoprotective effect, along with administration of DAR to animals for hepatic sinusoidal dilatation, should be helpful. This combined approach should be effective for cell transplantation on multiple occasions. Meanwhile, other mechanisms, for example, to inhibit expression of deleterious inflammatory cytokines, promote endothelial permeability without thrombosis, manipulate extracellular matrix components, and induce release of cytoprotective factors from HSC, should help achieve further gains in cell engraftment and liver repopulation.

The general clinical safety of DAR over the long term has been established.[13, 17] Longer half-life of DAR (approximately 24 hours), compared with short-acting vasodilators (e.g., nitroglycerine, prostacylin, and so on[3, 9]), will be advantageous, because after cell transplantation, hepatic vasoconstriction persisted for up to 24 hours.[9] Single doses of DAR needed for cell transplantation should be without toxicity.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Ms. Chaoying Zhang provided technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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