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Original Studies |
Departments of Clinical and Laboratory Medicine (M.Nishim., M.U., K.O., M.Y.) and Pediatrics (M.Nishid.), Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-0841; and the Department of Cardiology, Kyoto Second Red Cross Hospital (N.I., A.M., T.M.), Kamaza-Marutamachi, Kyoto 602-8026, Japan
Address all correspondence and requests for reprints to: Masato Nishimura, M.D., Department of Clinical and Laboratory Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan. E-mail: nishim{at}labmed.kpu-m.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retinal arteriosclerosis
One hundred and eighteen out-patients who came to the hospital
of Kyoto Prefectural University of Medicine for routine medical
examination were studied: 59 men (mean age ± SD,
61 ± 11 yr) and 59 women (mean age, 58 ± 12 yr; Table 1
). Subjects who had been receiving
treatment for hypertension or diabetes mellitus or had other
significant medical conditions, including cardiovascular, renal, or
hepatic disorders, were excluded from this study. In addition, subjects
who showed signs of left ventricular hypertrophy on a standard
electrocardiogram were excluded from this study. Blood pressure was
measured in a sitting position in the morning (0900–1100 h) with a
standard sphygmomanometer by nurses. The measurement of blood pressure
was repeated at least three times, and the mean of the last two
measurements was recorded as the actual blood pressure. Photographs of
both optic fundi were taken with a nonmydriatic retinal camera
(CR5–45NM, Canon Co. Ltd., Tokyo, Japan), and arteriosclerotic changes
in the retinal arteries were graded according to Scheie’s
classification (14): grade 0 represents no changes in the arterioles;
grade 1 consists of broadening of the arteriolar light reflex and
simple vein concealment; grade 2 consists of grade 1 changes along with
deflection of veins at arteriovenous crossings (Salus’s sign); grade 3
consists of grade 2 changes as well as the presence of "copper
wire" arterioles and marked arteriovenous crossing changes with
banking of the vein where it appears dilated distal to the crossing
(Bonnet’s sign), tapering of the vein on either side of the crossing
(Gunn’s sign), and right-angled deflection; grade 4 consists of grade
3 changes as well as the presence of silver wire arterioles and severe
arteriovenous crossing changes. Retinal arteriosclerotic changes were
graded in a double blind fashion by two experienced internists and were
double checked by an expert ophthalmologist. Blood was collected after
an overnight fast, and serum was obtained by centrifugation. Serum
concentrations of total cholesterol, high density lipoprotein (HDL)
cholesterol, triglycerides, creatinine, uric acid, total protein,
aspartate aminotransferase, alanine aminotransferase, lactate
dehydrogenase, 
-glutamyltranspeptidase, and alkaline phosphatase
were measured with an automatic analyzer (Ektachem 700 analyzer,
Eastman Kodak Co., Rochester, NY). Erythrocyte counts,
hemoglobin concentrations, and hematocrits were measured by an
autoanalyzer (STKS, Coulter Co., Miami, FL).
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One hundred and ninety subjects who had symptoms of chest pain
and underwent coronary cineangiography in Kyoto Second Red Cross
Hospital were enrolled in this study; the subjects included 142 men and
48 women, and the mean age was 66 ± 10 (±SD) yr.
Blood was collected before cardiac catheterization, and serum was
obtained by centrifugation for measurements of serum concentrations of
hHGF, total cholesterol, HDL cholesterol, triglycerides, creatinine,
and uric acid. Serum concentrations of total cholesterol, HDL
cholesterol, triglycerides, creatinine, and uric acid were measured
with an automatic analyzer (Ektachem 700 analyzer). The subjects were
divided into 4 subgroups according to the findings of coronary
cineangiography: subjects with normal coronary arteries (NCA; n =
22), subjects who had stenotic lesions (>75% stenosis) in 1 of the 3
major branches of the coronary arteries [single vessel disease (SVD);
n = 65], subjects who had lesions in 2 of the 3 major branches of
the coronary arteries [double vessel disease (DVD); n = 63], and
subjects who had lesions in all 3 major branches of the coronary
arteries [triple vessel disease (TVD); n = 40; Table 2].
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Patients who had been diagnosed with Henoch-Schönlein purpura, an allergic vasculitis of systemic arterioles, in the pediatric department of Kyoto Prefectural University of Medicine were enrolled in this study. Subjects were in the acute phase of Henoch-Schönlein purpura [n = 14, 7 males and 7 females; mean age, 10 ± 3 (±SD) yr; serum C-reactive protein, 3.94 ± 0.27 mg/dL] or the remission phase (n = 19, 11 males and 8 females; mean age, 9 ± 3 yr; serum C-reactive protein, 0.22 ± 0.06 mg/dL). Paired sera from the acute and remission phases were obtained from 9 patients presenting in the acute phase of Henoch-Schönlein purpura (5 males and 4 females; mean age, 8 ± 3 yr), and serum from the subacute phase was obtained from 4 of these 9 patients. All patients in the acute phase of Henoch-Schönlein purpura had typical cutaneous and joint manifestations of Henoch-Schönlein purpura, which were due to systemic arteriolitis. In the subacute phase, the cutaneous manifestations and joint symptoms began to resolve, but did not disappear. The remission phase was defined as the absence of all symptoms of Henoch-Schönlein purpura. None of the patients with Henoch-Schönlein purpura in this study presented with apparent renal or hepatic complications, as determined by urinalysis, blood examination, and ultrasonography. As controls, we examined serum hHGF concentrations in 17 children without any medical disease (9 males and 8 females; mean age, 10 ± 3 yr).
Measurement of serum hHGF
Serum concentrations of hHGF were measured with a specific enzyme-linked immunosorbent assay kit (Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan); the intra- and interassay variations were 2.9% and 2.6%, respectively.
Statistical analysis
Data are expressed as the mean ± SEM. The significance of differences between groups was evaluated by ANOVA followed by Duncan’s multiple range test. Differences in mean serum hHGF concentrations among the acute, subacute, and remission phases were evaluated by Student’s paired t test. Simple regression analyses were used to assess the relationship between hHGF and other parameters. The criterion for statistical significance was P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retinal arteriosclerotic changes were seen in 24 of 59 male
participants and in 26 of 59 female participants (Table 1
). Differences
in age, systolic or diastolic blood pressure, body mass index, and
serum concentrations of total cholesterol, triglycerides, or creatinine
were not seen between subjects with different grades of retinal
arteriosclerosis. The grade 3 group showed lower serum concentrations
of HDL cholesterol and higher concentrations of serum uric acid than
did subjects with changes of grades 0–2. Higher mean concentrations of
serum hHGF were seen with more advanced grades of retinal
arteriosclerosis [grade 0, 0.053 ± 0.005 ng/mL (n = 68);
grade 1, 0.144 ± 0.022 ng/mL (n = 21; P
< 0.01 vs. grade 0); grade 2, 0.338 ± 0.036 ng/mL
(n = 20; P < 0.01 vs. grade 0 or 1);
grade 3, 0.526 ± 0.051 ng/mL (n = 9; P <
0.01 vs. grade 0, 1, or 2); Fig. 1]. Serum concentrations of hHGF were
positively correlated with serum concentrations of uric acid
(r = 0.269; P = 0.003; n = 118) and
were weakly correlated with age (r = 0.298;
P = 0.024; n = 118), but were inversely correlated
with serum concentrations of HDL cholesterol (r = -0.279;
P = 0.002; n = 118). All participants whose serum
hHGF concentrations were more than 0.2 ng/mL (n = 35) showed the
presence of arteriosclerotic lesions in the retinal arteries (grade 1,
n = 7; grade 2, n = 19; grade 3, n = 9), although they
were not hypertensive [systolic blood pressure, <160 mm Hg (range,
92–154 mm Hg); diastolic blood pressure, 
90 mm Hg (range, 60–90 mm
Hg)].
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Coronary atherosclerosis and hHGF
Higher mean concentrations of serum hHGF were seen with more
advanced grades of coronary atherosclerosis [NCA, 0.188 ± 0.022
ng/mL (n = 22); SVD, 0.243 ± 0.011 ng/mL (n = 65;
P < 0.05 vs. NCA); DVD, 0.330 ± 0.013
ng/mL (n = 63; P < 0.01 vs. NCA or
SVD); TVD, 0.379 ± 0.023 ng/mL (n = 40; P <
0.05 vs. DVD; P < 0.01 vs. NCA
or SVD); Fig. 2]. Serum concentrations
of hHGF were positively correlated with serum concentrations of uric
acid (r = 0.324; P = 0.0001; n =
190), but were inversely correlated with serum concentrations of HDL
cholesterol (r = -0.452; P = 0.0001; n
= 190). Serum HDL cholesterol concentrations were lower in subjects
with coronary atherosclerosis than in those without, but no significant
differences were found in age or serum concentrations of other
biochemical factors among the NCA, SVD, DVD, or TVD subgroups (Table 2). The reproducibility of serum hHGF
values measured 2 weeks apart in the same person with coronary
atherosclerosis was 95.2 ± 6.8% (n = 22).
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Patients in the acute phase of Henoch-Schönlein purpura
showed higher (P < 0.01) mean serum hHGF
concentrations (0.347 ± 0.038 ng/mL; n = 14) than those in
the remission phase (0.097 ± 0.017 ng/mL; n = 19) or control
group (0.049 ± 0.011 ng/mL; n = 17; Fig. 3A). Measurement of hHGF concentrations
in paired sera from the acute and remission phases showed that serum
hHGF concentrations were decreased in the remission phase in subjects
whose serum hHGF concentration was higher than 0.30 ng/mL in the acute
phase (Fig. 3B). In addition, mean serum hHGF concentrations in the
subacute phase (0.345 ± 0.024 ng/mL; n = 4) showed a
tendency (P < 0.1) to be lower than those in the acute
phase (0.439 ± 0.027 ng/mL; n = 4), and mean serum hHGF
concentrations in the remission phase (0.153 ± 0.049 ng/mL;
n = 4) were lower (P < 0.01) than those in either
the acute or subacute phase. Of 19 subjects in the remission phase, 5
of 7 patients (71.4%) with serum hHGF concentrations over 0.10 ng/mL
and all patients with serum hHGF concentrations over 0.20 ng/mL had
relapse of Henoch-Schönlein purpura within 3 months after
measurement of serum hHGF, whereas relapse was seen in only 2 of 12
patients (16.7%) whose serum hHGF concentrations were less than 0.10
ng/mL in the remission phase.
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Discussion |
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Two hypotheses may explain the origin of increased serum concentrations of hHGF in subjects with vascular lesions. One is that increased serum hHGF is derived from enhanced hHGF production in vascular tissues with lesions of either arteriosclerosis, atherosclerosis, or arteriolitis. Immunoreactive hHGF is reportedly found in spindle-shaped and mononuclear cells in sites surrounding newly formed arterioles or capillary vessels, but not in the walls of blood vessels in the skin of patients with psoriasis, which is a common inflammatory skin disease characterized by prominent epidermal hyperplasia and neovascularization in the dermal papillae (16). In our recent study, high concentrations of hHGF were found in the vitreous of patients with retinal neovascularization associated with proliferative diabetic retinopathy (12). Therefore, production of hHGF is likely to be enhanced in sites with neovascularization. In contrast, local HGF production is reported to be inhibited in balloon-injured rat arteries compared with that in intact vessels, accompanied by a reduction of HGF messenger ribonucleic acid (mRNA) (17). Therefore, hHGF synthesized in injured vascular tissues may not significantly contribute to the increased serum hHGF concentrations in subjects with vascular lesions of arteriosclerosis, atherosclerosis, or arteriolitis.
The other hypothesis involves an endocrine mechanism through which hHGF is supplied from extravascular tissues or organs such as the liver, kidney, or spleen, via blood circulation. Increased HGF mRNA expression after liver injury occurs in extrahepatic organs as well as in the liver; 6–12 h after 70% partial hepatectomy in rats, HGF mRNA in the kidney and spleen increases 3- to 5-fold (18). In addition, HGF gene expression in the spleen increases after the onset of renal injury caused by unilateral nephrectomy (19). Cytokines or growth factors, such as platelet-derived growth factor (20), basic fibroblast growth factor (20, 21), or, in particular, interleukin-1 (20, 21, 22, 23, 24), are candidate promoters of HGF synthesis in noninjured organs. HGF produced in noninjured organs may be involved in regeneration of the liver or kidney through an endocrine mechanism (25). The precise mechanism underlying the increase in serum hHGF concentrations in subjects with vascular lesions is not clear from this study; however, the production of cytokines such as interleukin-1 is likely to be enhanced in endothelial cells, VSMC, or blood cells such as monocyte/macrophages at the sites of vascular lesions, and increases in these cytokines in circulating blood may induce up-regulation of hHGF production in extravascular tissues. Further studies are needed to clarify the origin and mechanism of increased serum hHGF concentrations.
A significant correlation between serum hHGF concentrations and blood pressure was not seen in the present study; this finding differs from a previous study by Nakamura et al. that did show such a correlation (15). One possible reason for the discrepancy between Nakamura’s study and ours is the difference in hHGF assay methods between the two studies; we measured the active form of serum hHGF, whereas Nakamura et al. measured total serum hHGF, which includes active and inactive forms of hHGF. Further studies are necessary to clarify the precise relationship between blood pressure and serum HGF concentrations.
There is a significant overlap between serum hHGF concentrations observed in subjects with or without vascular lesions, although mean hHGF concentrations are statistically different between these two groups. One possible reason is that various humoral factors that affect hHGF production may be involved in this overlap of serum hHGF concentrations; transforming growth factor-ß and angiotensin II inhibit hHGF production (17, 26, 27, 28, 29), whereas interleukin-1 (20, 21, 22, 23, 24), interleukin-6 (17), basic fibroblast growth factor (20, 21), platelet-derived growth factor (20), and hHGF activator, which changes inactive hHGF to active hHGF (30), promote hHGF synthesis. Differences in these factors at the sites of production of hHGF may result in overlap of serum hHGF concentrations between subjects with or without vascular lesions. Overlap of serum hHGF concentrations between subjects with or without vascular lesions was more marked in the subjects with coronary atherosclerosis than in the patients with arteriosclerosis or arteriolitis. The presence of retinal arteriosclerosis is presumed to represent the degree of arteriosclerosis in systemic organs and tissues (31), and arteriolitis due to Henoch-Schönlein purpura is systemically distributed over a wide range of tissues during the active stage, whereas vascular lesions may be limited to the coronary arteries in subjects with coronary atherosclerosis. Thus, the increase in serum hHGF concentration is probably smaller in subjects with coronary atherosclerosis than in those with systemic arteriolar lesions.
The correlation of serum hHGF concentration with changes in disease state is an important issue. The plasma half-life of HGF is reported to be 4 min in rats (32). This rather short half-life and high reproducibility of serum hHGF values measured at different times in subjects with either retinal arteriosclerosis or coronary atherosclerosis indicate that secretion of hHGF into the circulation is constitutively enhanced in subjects with these vascular lesions, because both retinal arteriosclerosis and coronary atherosclerosis reflect rather stable vascular lesions. On the other hand, serum hHGF concentrations appeared to change with disease activity in the subjects with Henoch-Schönlein purpura, because serum hHGF concentrations in the subacute phase tended to be lower than those in the acute phase, and serum hHGF concentrations were significantly decreased in the remission phase. It is not clear from this study whether an increase in the serum hHGF concentration precedes the onset of Henoch-Schönlein purpura, but relapse occurred with high frequency in subjects with serum hHGF concentrations higher than 0.10 ng/mL in the remission phase. Arteriolitis due to Henoch-Schönlein purpura may still be active even in the remission phase when serum hHGF concentrations are over 0.10 ng/mL regardless of the disappearance of symptoms or the serum inflammatory response.
In conclusion, serum hHGF concentrations over 0.2 ng/mL in adult subjects without apparent systemic diseases such as diabetes mellitus are likely to indicate the presence or development of systemic arteriosclerosis regardless of blood pressure. Changes in serum hHGF concentrations in children with Henoch-Schönlein purpura may reflect the degree or activity of systemic arteriolitis, and serum hHGF concentrations over 0.10 ng/mL in the remission phase may indicate the risk of relapse of this disease within several months. Increased serum hHGF may not be sufficient to completely repair injured endothelial cells in the vascular lesions associated with these disorders, because hHGF concentrations higher than 1.0 ng/mL are needed to induce human endothelial proliferation (10). However, our results in the present study show that serum concentrations of hHGF may be a useful biochemical indicator for the presence or development of systemic arteriolar lesions, although it is uncertain whether serum hHGF concentrations accurately reflect the severity of localized atherosclerotic lesions such as coronary arterial disease.
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Acknowledgments |
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Footnotes |
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Received January 4, 1999.
Revised March 11, 1999.
Accepted March 19, 1999.
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References |
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-stimulated
human embryonic lung fibroblasts. Biochim Biophys Acta. 1244:357–362.[Medline]
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