Serum Vascular Endothelial Growth Factor

Levels in Various Liver Diseases

FUYUHIKO AKIYOSHI, MD, MICHIO SATA, MD, HIROSHI SUZUKI, MD,

YASUYO UCHIMURA, MD, KEIICHI MITSUYAMA, MD, KATSUHIKO MATSUO, PhD and

KYUICHI TANIKAWA, MD

The clinical signi® cance of circulating vascular endothe lial growth factor (VEGF) in patientswith various liver diseases was investigated. Twenty-one patients with acute hepatitis (AH),40 with chronic hepatitis (CH), 34 with cirrhosis (LC), 16 with fulminant hepatitis (FH), 10with primary biliary cirrhosis (PBC), 12 with autoimmune hepatitis (AIH), and 120 healthyindividuals were included. Serum VEGF leve ls were measured by a chemilumine scenceenzyme-linke d immunosorbe nt assay. The mean value s of serum VEGF levels in the patientswith AH, CH, LC, FH, AIH, PBC, and control were 172.7, 58.0, 44.1, 37.3, 49.7, 74.9, and 65.0pg/ml, respective ly. The patients with AH had a leve l of serum VEGF signi® cantly higherthan that of the control group (P , 0.001) . The serum VEGF levels in survivors of FH weresigni® cantly increased, but not in the nonsurvivors in the recovery phase compared with theleve ls on admission (P , 0.05) . In the LC patients, serum VEGF levels were signi® cantlylower than those of the control group (P , 0.05) . These ® ndings sugge st that serum VEGFleve l may be associate d with hepatocyte regeneration grade .

KEY WORDS: vascular endothelial growth factor; acute hepatitis; fulminant hepatitis; various liver disease s;hepatocyte growth factor.

A speci® c mitogen for endothe lial cells, vascular en-

dothe lial growth factor (VEGF), has recently been

identi® ed (1, 2). By alternating splicing of mRNA,

four different molecular species with 121, 165, 189,

and 206 amino acids were determined, although the

physiological signi® cance of the multiple isoforms has

not been fully establishe d (3, 4). VEGF has been

reported to bind to at least two speci® c receptors

found on endothe lial cells, ¯ t-1 and KDR (5± 7).

VEGF appears to play a role in hypoxia-re lated

angioge nesis occurring during wound healing (8),

prolife rative re tinopathy (9), revascularization of

ischemic areas (10, 11), and tumor progression (12±

15) . In addition to hypoxia-induce d expre ssion of

VEGF, various investigators have also documented in

vivo increased expression of its receptors in patholog-

ical conditions characterized by hypoxia (16, 17) .

MATERIALS AND METHODS

We evaluated 133 patients with various kinds of liverdisease treated at Kurume University Hospital. Twenty-onepatients with acute hepatitis (AH, 12 males and 9 females),40 with chronic viral hepatitis (CH, 25 males and 15 fe -males), 34 with cirrhosis (LC, 15 males and 19 females), 16with fulminant hepatitis (FH, 12 males and 4 females), 10with primary biliary cirrhosis patients (PBC, 2 males and 8females), and 12 with autoimmune hepatitis patients (AIH,1 male and 11 females) were included. We also examined120 healthy individuals as a control group (Table 1). Allpatients were diagnosed by blood biochemical tests andviral markers. AIH was diagnosed by antinuclear antibodyand liver histology. PBC was diagnosed by elevation ofalkaline phosphatase, positive mitochondrial antibody, andcompatible liver histology.

Manuscript receive d April 28, 1997; accepte d September 3, 1997.From the Second Department of Medicine, Kurume Unive rsity

School of Medicine, Kurume , Japan; and Bioscience ResearchDepartment, Toagosei Chemical Industry Tsukuba Research Lab-oratory, Ibaraki, Japan.

Address for reprint requests: Dr. Michio Sata, The SecondDepartment of Medicine , Kurume Unive rsity School of Medicine,67, Asahi-machi, Kurume , Fukuoka 830, Japan.

Digestive Diseases and Sciences, Vol. 43, No. 1 (January 1998), pp. 41± 45

41Digestive Diseases and Sciences, Vol. 43, No. 1 (January 1998)

0163-2116/98/0100-0041$15.00/0 Ñ 1998 Plenum Publishing Corporation

Serum leve ls of alanine aspartate aminotransferase(AST) and aminotransferase (ALT) were measured usingstandard methods. All patients were te sted for hepatitisB surface antigen (HBsAg) by enzyme immunoassay(Q uik Test, Mizuho Medy, Tosu, Japan) and anti-hepatitis C virus (anti-HCV) by second-generation pas-sive hemaglutination assay (Dainabot, Tokyo, Japan) .IgM class hepatitis A and B core antibodies were testedin samples of AH and FH.

We studied the histological index of necroin¯ ammatoryactivity and ® brosis conforming to HAI score in the CH andLC patients.

Se rum VEGF leve ls were measured in duplicate by acolorime tric e nzyme -l inke d imm unosorbe nt assay(ELISA) with slight modi® cations of a chemilumines-cence enzyme immunoassay method previously describedby Hanatan i et al (18) . An anti-human VEGF polyclonalantibody was prepared from rabbit serum immunizedwith re combinant human V E GF1 2 1 ± g lutath ione -S-transferase fusion prote in. Nine ty-six-we ll microtiterplates (Combiplate Breakable EB, Labsystems, Helsinki,Finland) were coated with 5 m g/ml of the puri ® ed anti-VEGF antibody in 0.1 M NaCl and 0.25 M carbonatebuffe r (pH 9.5) , 0.1 M NaCl, and 0.1% NaH3. For theassay, 100 m l of samples and serially diluted VEGF(standards) were added to the wells and incubated for 1hr at 22°C. Afte r washing the we lls six times, 100 m l ofperoxisidase -conjugate d Fab 9 of the anti-V EGF antibodywas added to each well and incubated for 1 hr at 22°C.We lls were washed eight time s, and then the enzymereaction was carried out at 22°C for 30 min with o-phenylenediamine (Sigma Chemical Company, St. Louis,Missouri) as a substrate . The enzyme reaction was ter-minated by adding 100 m l of 2 M H2SO 4 to each well, theabsorbance at 490 nm of each well was measured with amicroplate reader (Molecular Device s, California), andthe VEGF content of the samples was e stimate d from thestandard curve de termined from the serially dilutedVEGF121 . The analytical sensitivity of this assay wasassessed by measuring serially diluted recombinant hu-man VEGF1 21 ranging from 1000 pg/ml to 0 pg/ml. Theintra- and interassay coef® cients of variation were bothless than 10% throughout the range . Because the thawingcaused a maximal loss of VEGF immunoreactivity in theserum of 50% , repeated frozen samples were not used inthis study.

Serum hepatocyte growth factor (HGF) leve ls were mea-sured by an ELISA kit (Otsuka Assay Laboratory, To-kushima, Japan) (19) .

The statistical analysis was done using the Mann-WhitneyU test, Wilcoxon’s single-rank test, and chi-squared test;P , 0.05 was considered to indicate a signi® cant difference.

RESULTS

Serum VEGF Levels in Various Liver Diseases.

The mean value s of serum VEGF leve ls in the pa-

tients with AH, CH, LC, FH, AIH, and PBC were

172.7, 58.0, 44.1, 37.3, 49.7, and 74.9 pg/ml, respec-

tively (Figure 1). The mean value of serum VEGF in

the control group was 65.0 pg/ml. The patients with

AH had signi® cantly higher levels of VEGF than did

the control group (P , 0.001) or other groups (P ,0.05± 0.001) . The patients with FH and LC had sig-

ni® cantly lower leve ls of VEGF than did the control

group.

Change in Serum VEGF Levels of AH During Clin-

ical Course. We compared the serum VEGF levels in

the same AH patients when they were in the acute

phase and when they were in the convale scent phase .

Elevated serum VEGF leve ls in the acute-phase re-

turned almost to normal range in the convale scent

phase . The difference in serum VEGF leve ls between

the acute and convale scent phase s was signi® cant

(Figure 2, P , 0.001) .

TABLE 1. CLINICAL AND LABORATORY FINDINGS OF PATIENTS WITH LIVER DISEASES

AH CH LC FH PBC AIH Control

Patients (N) 21 40 34 16 10 12 120

Age (yr) 39.8 6 3.3 45.4 6 1.9 58.6 6 1.6 47.1 6 4.4 52.6 6 3.8 46.0 6 3.4 47.9 6 5.5Sex (M/F) 12/9 25/15 15/19 12/4 2/8 1/11 68/52

Etiology (A/B/C/others) 12/4/5/0 0/14/26/0 0/5/27/2 1/7/0/8AST (IU/liter) 1087.4 6 367.5 90.5 6 18.2 74.3 6 5.8 2115.4 6 734.4 48.0 6 6.9 202.1 6 65.4 23.5 6 3.6

ALT (IU/liter) 1514.0 6 374.9 128.1 6 33.0 57.9 6 5.8 1474.4 6 444.4 39.7 6 9.5 256.2 6 115.4 21.5 6 4.8Serum VEGF (pg/ml) 172.7 6 25.8 58.0 6 9.6 44.1 6 6.0 37.3 6 7.0 49.7 6 9.4 74.9 6 16.2 65 6 4.3

range 74± 462.6 0± 252.9 2.5± 179.3 0± 90.3 0.8± 151.4 4.2± 104.6 0± 224.1

Fig 1. Serum VEGF levels of various liver disease s.

AKIYOSHI ET AL

42 Digestive Diseases and Sciences, Vol. 43, No. 1 (January 1998)

Change in Serum VEGF Levels and Serum HGF

Levels in FH Patients During Clin ical Course. The

FH patients were divide d into two groups: seven

nonsurvivors and six survivors. The serum VEGF

leve ls in the survivors were signi® cantly increased

compared with their levels on admission (P , 0.05) .

In the nonsurvivors, the serum VEGF levels de-

creased signi® cantly during the illne ss (P , 0.05,

Figure 3A).

The serum HGF leve ls in the FH survivors were

signi® cantly decreased compared with the ir leve ls on

admission (P , 0.05, Figure 3B).

Serum VEGF Levels in CH and LC Patien ts in

Relation to Histological Activity Index. The serum

VEGF levels did not correlate with histological activ-

ity conforming to HAI score in the CH and LC

patients. No appare nt correlation was found between

serum VEGF leve ls and the score of necroin¯ amma-

tory change (total score of HAI 1± 3 compartment).

There was no corre lation between serum VEGF lev-

els and ® brosis score (HAI 4 compartment).

DISCUSSION

The VEGF gene is expressed in a wide varie ty of

normal adult guine a pig and human tissue s (20) . As

assessed by Northern analysis, the highe st leve ls of

VEGF mRNA were found in normal lung, kidne y,

heart, and adrenal gland. Lower but still readily de-

tectable VEGF transcript leve ls were found in live r,

spleen, and gastric mucosa and to a lesser extent in

the breast (20) . VEGF is a glycoprote in that selec-

tively induce s endothe lial prolife ration, angioge nesis,

and capillary hyperpermeability (12, 13) . In agree-

ment with the report of Monacci et al in the rat (21) ,

in situ hybridization studies of normal human live r

revealed that VEGF mRNA was located in hepato-

cytes and a small number of scattered Kupffer cells

(22) . The function of hepatocyte -derived VEGF is

not clear. The purpose of the present study was to

explore the clinical signi® cance of circulating VEGF

in patients with various live r diseases. We attempted

to detect circulating VEGF by using a highly sensitive

ELISA in healthy individuals and various live r disease

patients.

The patients with AH had signi® cantly highe r leve ls

of VEGF than those of the control group. The serum

VEGF leve l did not correlate with serum aminotrans-

ferases. The elevation of serum VEGF might not be

the outcome of release from damage d hepatocyte s.

The characte ristics of an acute -phase response after

Fig 2. Comparison of serum VEGF levels between acute and

convale scent phases of acute hepatitis.

Fig 3. Comparison of serum VEGF levels (A) and serum HGF

leve ls (B) in FH patients during the clinical course ; the FH patientswere divided into two groups: seve n nonsurvivors and six survivors.

SERUM VEGF IN VARIOUS LIVER DISEASES

43Digestive Diseases and Sciences, Vol. 43, No. 1 (January 1998)

local injury include the release of cytokine s such as

IL-1, IL-6, IL-11, and TNF- a , which in turn induce a

systemic reaction manifested by, eg, fever, e levated

secretion of glucocorticoid s, and change s in the con-

centration of a speci® c set of plasma prote ins, termed

acute -phase proteins, which are mainly produced in

the liver (23, 24) . These acute -phase proteins are

eithe r up-regulate d or down-regulate d during the

acute -phase response (17) . VEGF induce s capillary

hyperpermeability, which contribute s to acute in¯ am-

matory change . Thus, VEGF may be associate d with

an acute -phase prote in.

No elevation of the serum VEGF leve l was ob-

served in the nonsurvivors of FH, but a gradually

increasing VEGF level was observed in the FH sur-

vivors. Activation of the host immune system in re-

sponse to viral infection results in the production of

many cytokine s that act as mediators of disease activ-

ity. The serum levels of TNF-a , IL-6, and IL-1Ra are

signi® cantly corre lated with parameters of hepatocyte

injury and acute hepatic prote in synthe sis in patients

with FH and AH (25, 26). Therefore , the serial mea-

surement of serum VEGF may be useful for following

the clinical course of active live r diseases.

In the LC patie nts, the serum VEGF leve ls were

signi® cantly lowe r than those of the control group.

There was no corre lation between VEGF leve l and

ne crosis of hepatocyte s in the compartment of HAI

score . Despite the necroin¯ ammatory change , the

se rum VEGF leve ls in the pre sent LC patie nts

remaine d at a low leve l. The VEGF leve l decreased

according to histological progre ssion in Pugh-Child

classi® cation (data not shown) . These ® ndings sug-

gest that a low serum VEGF leve l in an LC patie nt

may re¯ ect the degre e of live r dysfunction and may

be associated with the grade of hepatocyte regen-

eration.

The regulatory mechanisms of serum VEGF in

live r diseases appear to be complex. Furthe r investi-

gation is required to elucidate the mechanisms of

VEGF elevation in live r diseases.

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