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,
,2
* Division of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachussetts, USA;
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA;
Harvard-MIT Division of Health Sciences and Technology, Boston, Massachusetts, USA
2Correspondence: Harvard Medical School, Division of Matrix Biology, Beth Israel Deaconess Medical Center, Department of Medicine, Dana 514, 330 Brookline Ave., Boston, MA 02215, USA. E-Mail: rkalluri{at}bidmc.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: transforming growth factor ß • hepatocytes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, BMP-7, is now being recognized as multifunctional cytokine, which mediates growth and differentiation of many different cell types (2
3
4
5)
. Among its various functions during development, BMP7 mediates sprouting of the liver bud from the central foregut endoderm, which eventually gives rise to hepatocytes (6
, 7)
. In the adults BMP-7 is predominantly produced by the kidney and the bone (2)
. BMP-7 circulates in the blood at a concentration range of 100–300 pg/m (8)
. Therefore, we hypothesized that BMP-7 might function as an endogenous regulator of liver health.
The unique capacity of the liver to regenerate following acute mass ablation has been recognized for more than 3000 yr now, but the complex underlying regenerative mechanisms are not yet fully understood (9)
. In normal adults, hepatocytes have a quiescent, highly differentiated phenotype and rarely divide in human or animal livers (10)
. However, their innate capacity to replicate is not lost and is readily activated after liver resection or on acute liver injury due to chemicals or viruses (11)
. If the loss of functional liver parenchyma does not reach a critical level, the liver can completely regenerate its functional mass (12)
. However, if the number of viable hepatocytes falls under a critical level (acute liver failure), the regenerative capacity cannot compensate for the acute loss of liver mass (13)
. In this regard, acute liver failure, defined by the occurrence of hepatic encephalopathy (due to loss of synthetic and metabolic functions of the liver), results in significant mortality (40–80%), and donor organs for curative orthotopic liver transplantation are still very limited (14)
. Therefore, there is a renewed interest in therapeutic alternatives for patients with ALF to either allow for accelerated liver regeneration.
In the present study, we demonstrate that therapeutic administration of rhBMP-7 significantly enhances liver regeneration and function after partial hepatectomy in mice. Additionally, we demonstrate that neutralization of endogenous BMP-7 levels result in impaired regeneration of the liver. Collectively, our data suggest that BMP-7 is a physiological regulator of hepatocyte health.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Partial hepatectomy
Male CD1 mice (
30g) were deprived of food for 12 h before partial hepatectomy (PH) surgery. Animals were hepatectomized by the method of Higgins and Anderson with minor modifications (21)
. Briefly, anesthesia was induced by peritoneal injection of ketamine (50 mg/kg of body wt). The large median lobe (including right central lobe and left central lobe) and left lateral lobe, which constitute 70% of the total liver, were separately ligated and resected. For sham surgery, the abdominal wall was opened and the xyphoid process was removed before the abdominal wall was closed. The mass of the resected liver tissue was measured after surgery, and that of the remnant liver was determined after sacrificing the animals at 2 days (n=14, including sham=4, BMP-7 administered=5 and control=5), 4 days (n=15, including sham=4, BMP-7 administered=6 and control=5), 7 days (n=11, including sham=3, BMP-7 administered=4 and control=4), or 10 days (n=21, including sham=5, BMP-7 administered=9 and control=7) after surgery. For rhBMP-7 treatment, soluble human recombinant BMP-7 (300 µg/kg body wt) was given intraperitoneally (i.p.) within 6 h after surgery and then every other day. The equivalent volume of PBS was administered instead of rhBMP-7 in control groups. All animals were injected i.p. with BrdU (50 µg/g body wt) 2 h before sacrifice. All protocols were approved by the Institutional Animal Care and Use Committee.
Infusion of mice with anti-BMP-7 Ab or ALK3/Fc chimera
One day before the surgery, anti-BMP-7 Ab (273 ng/30 g bodyweight) or ALK3/Fc chimera (200 ng/ 30 g body weight) was injected intravenously (i.v.) through retro-orbital venous plexus to neutralize endogenous BMP-7 activity. After 70% hepatectomy, anti-BMP-7 Ab or ALK3/Fc chimera was given i.v. once every day until the day of sacrifice. The mice were sacrificed 2, 4, and 10 days after the surgery.
Clinicopathologic evaluation of liver regeneration
To assess liver regeneration, we established a regeneration curve by sacrificing mice 2, 4, 7, and 10 days after partial hepatectomy. Separately, sham-operated mice were sacrificed at each time point as a control. Histochemical staining, including hematoxylin and eosin (H&E) and BrdU staining were performed to investigate regenerative changes in the liver. The activities of serum alanine transaminase (ALT), aspartic transaminase (AST), and total serum bilirubin were assessed to evaluate for liver function. Procedural details are described below.
Determination of liver function
Blood samples were obtained from orbital venous plexus of mice under anesthesia just before sacrifice. The activities of serum ALT and AST, and total serum bilirubin were measured by Sigma Diagnostics kits according to the manufacturer’s protocols.
Determination of hepatic regeneration by liver mass index
In preliminary experiments, the resected liver and the remaining livers were weighted in mice after the partial hepatectomy. The percentage of resected liver weight relative to the original whole liver was 68.3% ± 6.1% (means±SD, n=9) as described previously (22)
. Thus, we calculated the original whole liver weight relative to the resected liver weight (resected liver wt x100/68.3). When mice were sacrificed at the indicated times after the partial hepatectomy, the remnant liver was excised and weighed, and the liver regeneration rate (%) was expressed as follows: remnant liver wt/[original whole liver wt] 5100.
SDS-PAGE electrophoresis and Western blot analysis
SDS-PAGE electrophoresis and immunoblotting were performed as described previously (23
, 24)
. Frozen liver samples obtained from the remnant liver (right lobe) were pooled and homogenized in cold homogenizing buffer containing 150 mM NaCl, 1% Triton X-100, 10 mM Tris HCl (pH 7.6), 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 0.5% Nonidet P-40, 25 mM NaF, and 0.2 mM PMSF. The homogenate from each group sample was sonicated, and the protein concentration was determined using BCA protein assay kit. Twenty micrograms of protein solution per lane was used for SDS-PAGE electrophoresis on 10% gels. The separated proteins were transferred to a nitrocellulose membrane and blocked with 5% milk solution for 30 min on a shaker at room temperature. After blocking, the blot paper was incubated with first antibodies in TBS. Subsequently, the blot was washed thoroughly with washing buffer and incubated with a secondary Ab conjugated to horseradish peroxidase for 1 h at room temperature on a shaker. Positive reaction products were identified by enhanced chemiluminescence (ECL) (Amersham Pharmacia, Piscataway, NJ, USA), according to the manufacturers’ protocol.
Determination of hepatic ALK3 and phosphorylated Smad1 by immunohistochemistry
The liver tissues from the remnant right lobes were fixed with 4% paraformaldehyde in PBS, embedded in paraffin and sectioned at 4 µm thick. Paraffin wax was removed from liver section with xylene and rehydrated with decreasing graded ethanol washes. After blocking of nonspecific sites with 2% BSA in PBS, the sections were incubated subsequently with primary antibodies and secondary antibodies. PBS was used instead of the first Ab in the negative control.
Determination of proliferation by BrdU incorporation
Hepatic proliferation was detected by incorporation of BrdU in proliferating cells in paraffin sections, as described previously with minor modifications (25)
. The mice were subjected to an i.p. injection of BrdU (50 µg/mg body wt), 2 h before sacrifice. The liver tissue from remnant right lobes were fixed with 4% paraformaldehyde in PBS, embedded in paraffin, and sectioned at 4-µm thickness. Paraffin wax was removed from liver with xylene and rehydrated with decreasing graded ethanol washes, before sections were digested with 0.4% trypsin in PBS and denatured with 2 M HCl containing 0.1%Triton X-100. After blocking of nonspecific sites with 2% BSA in PBS, the sections were incubated first with antibromodeoxyuridine Ab and then with rhodamine-conjugated second Ab. Slides were covered with Vectashield mounting medium with 4',6'-diamidino-2-phenylidole (DAPI) (4',6-diamidine-2'-phenylindole dihydrochloride). A total of 12 visual fields (total of 10,000 nuclei) were randomly selected and the number of BrdU-positive nuclei, which were regarded as proliferating cells, was counted at a x400 magnification.
BMP-7 KO/KO embryos
BMP-7 KO/KO embryos (18)
were obtained by timed matings of BMP-7 KO/wt mice (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Genotyping was performed according to the provider’s recommendation.
Real-time quantitative PCR
Real-time PCR (RT-polymerase chain reaction) primer sequences, as well as FAM-labeled probe 5' CCGGGAAAACGCTGGGCGT 5' were designed to RNA encoding BMP-7 using the program Primer Express 1.5. PreDeveloped TaqMan Assay Reagents, 18S rRNA (Applied Biosystems, Foster City, CA, USA) was used as an internal standard. RT-polymerase chain reaction (RT-PCR) was carried out in a 7000 Sequence Detector System (Applied Biosystems), and measurements were standardized to the 18S rRNA reaction using standard calculation methods, as described previously (26)
.
Determination of hepatocyte proliferation
Proliferation assay was performed using [3H]-labeled thymidine, as described previously, with minor modifications (24)
. Primary mouse hepatocytes suspended with F12 medium containing 10% FBS and 2 µg/ml insulin. 3.5 x 105 cells per well were used in 24-well plates. Plates were incubated for 24 h in a cell incubator. On the following day, the medium was replaced with 1 ml F12 medium/0.1% FBS, containing TGF-ß1 and/or rhBMP-7, and the cells were incubated for an additional 12 h. 0.1 µC of [3H]-labeled thymidine was added into each well, and the cells were incubated for an additional 24 h. The next day, cells were lysed by incubation for 30 min at 60°C in 0.3 M NaOH. The resulting solution was analyzed using a scintillation counter.
Statistical analysis
Results are expressed as means ± SD Multiple comparisons were performed by one-way ANOVA in statistical Packages for the Social Sciences (SPSS) 10.0 for Windows. P values lower than 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The residual lobes enlarge to compensate for the removed mass, but the excised lobes do not grow back. To evaluate the potential of rhBMP-7 to enhance liver regeneration, we administered CD1 mice after partial hepatectomy (resection of the large median lobe, the right central lobe, the left central lobe, and the left lateral lobe) with 300 µg/kg rhBMP-7 every other day [which was determined as the optimum therapeutic dose in previous studies (15)
] and compared the liver regeneration rate to that of vehicle-administered (control) mice and sham-operated mice. In control mice, after partial hepatectomy, DNA synthesis started 12–16 h after the surgery. About 10 days after partial hepatectomy, the regenerated liver reached the original weight of the preoperative liver mass (±10%). In rhBMP-7-administered mice, the regeneration rate was significantly enhanced as compared to control mice. In rhBMP-7 administered mice the liver wt reached 57% of its initial wt after 2 days compared to 42% in the vehicle-administered (PBS) control mice, 78% after 4 days (48% in control mice) and 89% after 7 days (control 62%) (Fig. 1)
. After 10 days, the liver weight reached 90% of its initial weight in the rhBMP-7-administered group, while liver weight in control mice reached 80% of its initial weight (Fig. 1)
. After 20 days, livers in BMP-7 administered as well as PBS-administered groups were completely regenerated.
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To evaluate global liver function, we measured ALT, AST, and total bilirubin in the blood of CD1 mice 2, 4, 7, and 10 days after partial hepatectomy. In the PBS-administered control mice, ALT was significantly elevated two days after partial hepatectomy (280 U/l), decreased to 230 U/l (17.8% decrease), and returned to baseline levels (50 U/l) after 10 days (Fig. 1)
. In the rhBMP-7-administered mice, ALT levels were 240 U/l 2 days post partial hepatectomy and decreased to 100 U/l (58.3% decrease) at day 4 and reached normal levels after 7 days (Fig. 1)
. Similarly, AST and total bilirubin were significantly lower in the rhBMP-7-administered mice compared to the vehicle-administered group, 4 days after partial hepatectomy (Fig. 1)
.
Treatment with rhBMP-7 enhances hepatocyte proliferation in regenerating liver
Liver regeneration after partial hepatectomy is predominantly contributed by proliferation of the mature resident cells, such as hepatocytes (10
, 16)
. The peak hepatocyte proliferation appears between 24 and 48 h after partial hepatectomy (10)
. We assessed their proliferation during liver regeneration in control and rhBMP-7-administered mice after partial hepatectomy. In control mice, the proliferation index (% of nuclei with bromodeoxyuridine (BrdU) incorporation) peaked 48 h after partial hepatectomy (23%) and decreased (12% after 4 days) until it reached baseline levels (3%) after 10 days (Fig. 2)
. In rhBMP-7-administered mice the proliferation index was significantly increased compared to control mice after 2 days (150% higher than control) and 4 days (200% increase compared to control) until it reached normal levels after 10 days (Fig. 2)
.
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Liver regeneration is associated with increased hepatic ALK3 expression.
We hypothesized that administration of rhBMP-7 enhances liver regeneration, as it uses an existing physiological pathway. To test this hypothesis we determined expression of endogenous BMP-7 and of its functional receptors in control and regenerating livers. By Western blot analysis, using specific antibodies to BMP-7, we failed to detect expression of BMP-7 in lysates from sham-operated mice or from mice following partial hepatectomy (Fig. 3
A), confirming previous publications that demonstrated absence of endogenous BMP-7 expression in adult liver tissue (17)
. To confirm absence of BMP-7 expression in the liver, we performed real-time polymerase chain reaction (PCR) on RNA isolated from livers and kidneys from sham-operated mice, from vehicle-administered mice with partial hepatectomy, and from BMP-7-administered mice with partial hepatectomy (Fig. 3B
). BMP-7 expression was 10-fold higher in kidneys than in livers, while partial hepatectomy or treatment with rhBMP-7 had no significant effect on renal BMP-7 expression levels. To determine whether the BMP-7 expression detected in adult levels are of biological relevance, we used liver from E18.5 fetal BMP-7KO/KO mice as control (Fig. 3C
). BMP-7 mRNA expression levels in livers from adult C57BL/6 mice equaled the detected signal in fetal BMP-7KO/KO livers, suggesting that the minimal detected levels were irrelevant to background (Fig. 3C
) (18)
. BMP-7 mediates its action via binding to BMP type II receptors, which form a complex with activin-like kinase (ALK) receptors, ALK3 and ALK6 type I serine/threonine kinase receptors (19)
. We evaluated the expression of ALK3 and ALK6 in sham-operated and regenerating liver by immunoblot and immunohistochemistry. ALK3 expression was significantly increased in regenerating livers as compared to sham control (Fig. 3D
). Administration of rhBMP-7 had an insignificant effect on the expression of ALK3 (Fig. 3D
). ALK6 expression was not detected in the normal adult liver or in the regenerating livers (Fig. 3D
). To confirm the up-regulation of ALK3 expression in hepatocytes, we performed ALK3/ ASGP-R (hepatocyte-specific marker) double-staining experiments. Colocalization of ALK3 and ASPG-R revealed that ALK3 was mainly expressed on hepatocytes (Fig. 3E
). To confirm functional relevance of increases ALK3 expression in regenerating livers, we performed immunofluorescence labeling using specific antibodies to phophorylated Smad1 (nuclear translocation of phosphorylated Smad1 indicates active BMP signaling). We detected nuclear pSmad1 in 19% of cells sham-operated livers compared to 72% in regenerating livers (4 days after partial hepatectomy) (Fig. 3E
).
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Neutralization of physiological levels of endogenous circulating BMP-7 retards liver regeneration.
On the basis of our findings that BMP-7 enhances liver regeneration and that repair of the liver is associated with increased ALK3 expression, we hypothesized that endogenous circulating BMP-7 is a physiological regulator of hepatocyte proliferation. As BMP-7 null mice are not viable and the heterozygous mice continue to express normal levels of BMP-7, we neutralized endogenous circulating BMP-7 by administration of soluble Alk3/FC chimera (to act as a BMP-7 trap) to determine the role of physiological levels of BMP-7 on liver regeneration. ALK3/Fc Chimera was given to mice at a molar concentration to neutralize a possible 400 pg/ml of circulating BMP-7. In ALK3/Fc chimera -infused mice, liver regeneration was significantly impaired, when compared to the control group. The liver weight reached 52% of its initial weight after 2 days compared to 62% of PBS-administered control mice, 58% after 4 days (control 76%), and 85% after 10 days (control 113%) (Fig. 4
). Because Alk3/Fc chimera has the capacity to bind other BMPs with equal affinity, we performed a second set of experiments, in which we used a neutralizing antibody (Ab) (equimolar administration to neutralize 400 pg/ml of BMP-7) that is specific for BMP-7. This Ab does not bind to other BMPs, which bind ALK3 receptor (data not shown). Neutralization of endogenous circulating BMP-7 with anti-BMP-7 Ab equally inhibited spontaneous liver regeneration after partial hepatectomy. In mice, which received BMP-7 antibodies, the liver weight reached 56% of its initial weight after 2 days compared to 56% of PBS-administered control, 66% after 4 days (control 71%) and 83% after 10 days (control 100%) (Fig. 4)
, demonstrating a contribution of endogenous BMP-7 in the regeneration of liver. Lastly, we performed rescue experiments, in which injection of BMP-7-neutralizing antibodies was followed by injection of equimolar amount of rhBMP-7. In these experiments, rhBMP-7 reversed the effect of the BMP-7-neutralizing Ab, further confirming a specific role of endogenous BMP-7 on hepatocyte proliferation and liver regeneration (Fig. 4)
. In these experiments, altered liver regeneration rate was reflected by decreased BrdU incorporation (in mice that received neutralizing BMP-7 antibodies) and normalized BrdU incorporation in the rescue experiments, further implicating the contribution of BMP-7 on proliferation of hepatocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Several studies with acute loss of liver mass have established that the regenerative response of the liver is proportional to the extent of the lost liver mass until the liver reaches its correct weight, which is precisely regulated in proportion to the body size (10)
. Despite this regenerative capacity of the liver, acute liver failure is still associated with a high mortality (13)
. Further knowledge about positive and negative regulators of liver regeneration is of high interest, because supportive therapy to enhance liver regeneration could potentially help to decrease mortality and the need for orthotopic liver transplantation (13)
. Here, we report a role for endogenous BMP-7 as a physiological regulator of hepatocyte proliferation and health. In the mouse partial hepatectomy model, liver mass is spontaneously regenerated within 10 days after surgery. During the first 48 h post partial hepatectomy, liver regeneration is predominantly dependent on proliferation of hepatocytes, which lead to formation of hepatocyte clumps around capillaries. Four to ten days after partial hepatectomy, the clumps then transform into lobules typical of mature liver. Administration of rhBMP-7 significantly enhances liver regeneration within the first 4 days after partial hepatectomy, suggesting that it primarily functions by enhancing the proliferation of hepatocytes.
BMP-7 is an endogenous circulating protein, raising the possibility that the therapeutic effect of exogenous rhBMP-7 observed in our studies is due to a preexisting physiological role of endogenous BMP-7. We demonstrate that liver regeneration is associated with increased ALK3 expression and increased nuclear translocation of phosphorylated Smad1, suggesting that endogenous BMP-7 is involved in liver regeneration. However, we did not detect hepatic BMP-7 expression, either in livers from sham-operated mice or in mice with partial hepatectomy, confirming previous studies that demonstrated that in adults, BMP-7 expression is predominantly confined to the kidney. It has been proposed that BMP-7 is a renal hormone, which is constantly released by the kidney into the circulation (20)
. In this regard, Hruska and coworkers demonstrated that loss of renal mass in 5/6 nephrectomized mice led to vascular calcification and osteodystrophy, both of which could be prevented by administration of rhBMP-7, even at low doses of 10 µg/kg (5
, 20)
. Our own studies, in which we injected mice with ALK3-Fc chimera or neutralizing BMP-7 antibodies at equimolar doses to neutralize 400 pg/ml of circulating BMP-7 in mice, results in an inhibition of liver regeneration. On the basis of these findings, we suggest that circulating BMP-7 serves as a regulator of hepatocyte health and is an endogenous regulator of hepatocyte function. Because this effect is mediated via circulating BMP-7 produced likely in the kidney and bone, we propose that BMP-7 serves as a liver hormone.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 3, 2006. Accepted for publication August 22, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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