Journal of Histochemistry and Cytochemistry, Vol. 45, 559-568, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Immunolocalization of Lipoprotein(a) in Wounded Tissues

Yoko Yanoa, Kuniyasu Shimokawaa, Yume Okadaa, and Akio Nomaa
a Department of Laboratory Medicine, Gifu University School of Medicine, Gifu, Japan

Correspondence to: Yoko Yano, Dept. of Laboratory Medicine, Gifu Univ. School of Medicine, Tukasa-machi 40, Gifu-500, Japan.


  Summary
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Fifty samples from inflamed tissues were examined by immunohistochemical techniques, using antibodies against apo(a), apo B, plasminogen, fibrinogen, proliferating cell nuclear antigen (PCNA), and various components of extracellular matrix. The immunohistochemical features of granulation tissues were characterized by different stages of wound healing. In the first stage, immunoreactivities for anti-apo(a) and anti-apo B were weak and focal, whereas those for anti-plasminogen and anti-fibrinogen were strong and were widespread on the tissue surface. In the second stage, granulation tissues were covered with loose fibrous connective tissue, designated as a "fibrous cap." In this stage, markedly positive staining for lipoprotein(a) [Lp(a)] was observed closer to the surface of the fibrous cap than plasminogen, suggesting that Lp(a) may prevent external fibrinolysis. Lp(a) was also found in endothelial cells and the extracellular space of small vessels underlying the fibrous cap. In the last stage of healing, apo(a) and apo B were not detectable in completely organized tissues. These findings suggest that Lp(a) plays a role in the wound healing. (J Histochem Cytochem 45:559-568, 1997)

Key Words: wound, lipoprotein(a), immunohistochemistry, granulation tissue


  Introduction
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Materials and Methods
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Lipoprotein(a) [Lp(a)] has recently attracted great interest as a potential risk factor for the development of atherosclerotic diseases. Lp(a), originally described by Berg 1963 , is composed of a low-density lipoprotein-like particle to which the glycoprotein apolipoprotein(a) [apo(a)] is attached through a disulfide bridge to apo B-100. Apo(a) is structurally homologous to plasminogen (McLean et al. 1987 ). Plasma Lp(a) concentration, genetically determined as an autosomal co-dominant trait, exhibits wide interindividual variation and is minimally affected by age, sex, diet, drugs, or other environmental conditions. Several reports indicate that high concentrations of Lp(a) are associated with development and progression of coronary artery disease (Hoefler et al. 1988 ; Murai et al. 1986 ; Dahlen et al. 1986 ) and deposition of Lp(a) in atheromatous lesions in the arterial wall (Jurgens et al. 1993 ; Niendorf et al. 1990 ; Rath et al. 1989 ).

We have previously reported transient increases of plasma Lp(a) after acute myocardial infarction and surgical operations (Noma et al. 1994 ; Maeda et al. 1989 ), suggesting that Lp(a) might have some physiological roles in addition to regulating fibrinolysis. This finding, however, is still controversial (Wallberg-Jonsson et al. 1995 ; Crook et al. 1994 ; Slunga et al. 1992 ), and the significance of this transient increase remains unclear.

The purpose of the present study was to clarify the presence of Lp(a) in tissues during healing by immunohistochemical analysis of various stages of tissue repair.


  Materials and Methods
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Materials and Methods
Results
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Tissue Samples
Histological and immunohistochemical studies were performed on sections of 50 formalin-fixed, paraffin-embedded injured tissues. These specimens were obtained during various surgical procedures. They covered all stages of wound healing, from acute or acute on chronic inflammation to scar formation. The anatomic sites of selected samples were as follows: skin (seven cases), external ear (two cases), nasal cavity (11 cases), larynx (nine cases), tongue (six cases), soft palate (five cases), stomach (five cases), colon (four cases), and carotid artery (one case). Some sections of serial tissue specimens (3 µm thick) were stained with hematoxylin and eosin to identify the stage of healing. Others were used for immunohistochemical staining for apo(a) [or apo(a)-laminin double staining], apo B, plasminogen, fibrinogen, proliferating cell nuclear antigen (PCNA), fibronectin, tenascin, large proteoglycan, and dermatan sulfate proteoglycan.

Antibodies
Polyclonal anti-human apo(a) IgG antibody (rabbit) was prepared as previously described (Abe et al. 1988 ). This antibody was purified by affinity chromatography on fluoro-1-methylpyridinium toluene-4-sulfonate (FMP)-activated cellulofine (Seikagaku; Tokyo, Japan) coupled with pooled low Lp(a) and high plasminogen sera. The specificity of purified anti-apo(a) antibody was confirmed by immunofixation and enzyme-linked immunosorbent assay (ELISA). This antibody did not react to apo B, plasminogen, human catalase (Sigma Chemical; St Louis, MO) (Dieplinger et al. 1995 ), or other coagulation factors tested in human plasma.

Polyclonal anti-apoB IgG (rabbit), anti-fibrinogen IgG (rabbit), anti-fibronectin, and anti-plasminogen IgG (rabbit) were purchased from Dako Japan (Kyoto, Japan). We confirmed by immunofixation that the anti-plasminogen IgG did not react to apo(a). Monoclonal anti-PCNA IgM was obtained from Coulter Immunology (Hialeah, FL). Polyclonal anti-laminin (rabbit) and anti-tenascin (rabbit) were obtained from Chemicon International (Temecula, CA) and monoclonal anti-large proteoglycan IgG (2-B-1) and anti-dermatan sulfate proteoglycan IgG (6-B-6) from Seikagaku.

Immunohistochemistry
The experimental procedures were carried out at room temperature in a controlled humidity chamber. Immunohisto-chemical analyses of apo(a), plasminogen, fibrinogen, PCNA, laminin, fibronectin, tenascin, large and dermatan sulfate proteoglycans were performed according to the avidin-biotin complex (ABC) method (Vectastain ABC kit: Vector Laboratories, Burlingame, CA). PBS 0.15 M, pH 7.4, was used for washing of specimens and dilutions of antibodies. Deparaffinized sections were blocked for endogenous peroxidase activity in 0.3% H2O2 in methanol for 30 min. Tissue sections were washed for 20 min and preincubated with normal goat serum for 20 min to reduce nonspecific staining. Then they were treated with the specific primary antibodies for 1 hr. After washing three times for 10 min, they were incubated for 30 min with the biotin-conjugated secondary antibody. After rinsing in the same way, avidin-biotin complex was applied for 60 min and diaminobenzidine (DAB) (20 mg DAB in 100 ml PBS + 1 ml of 0.3% H2O2) was added for visualization. Finally, sections were counterstained with hematoxylin, dehydrated in alcohol, and mounted on glass slides. To exclude crossreaction with plasminogen, sections were treated with sheep anti-plasminogen IgG antibody (The Binding Site; San Diego, CA) for 60 min before incubation with apo(a) antibody, and apo(a) antibody that had been preincubated with plasminogen was used as primary antibody. Conversely, plasminogen antibody that had been preincubated with Lp(a) was also used as primary antibody to exclude crossreaction with apo(a). Development of PCNA staining was carried out in DAB solution containing 100 µl of 1 M imidazole to increase color substrate reactivity. For apo(a)-laminin double staining, apo(a) first was stained in the usual manner, followed by staining for laminin after treatment with pepsin (400 mg pepsin in 100 ml of 0.01 N HCl) at 37C for 2 hr. Laminin staining was developed with 1% CoCl2 in DAB solution (Co-DAB), which was mixed for 30 min, filtered, and then added to 1 ml 0.3% H2O2 . After development, methyl green was used for counterstaining. Staining for large and dermatan sulfate proteoglycans required pretreatment with chondroitinase ABC (Seikagaku) after deparaffinization to reveal the core protein recognized by the antibodies. The pretreatment consisted of two steps: 1 hr incubation in Tris-acetate (0.1 M, pH 8.0) at 37C and 1 hr incubation with chondroitinase ABC (5 U/ml in 0.1 M Tris-acetate, pH 8.0) at 37C. In addition, protein digestions with 0.1% trypsin (100 mg trypsin in 100 ml of 0.1% CaCl2, pH 7.8) at 37C were performed for fibronectin staining for 15 min and for tenascin staining for 2 hr.When staining results were compared in formalin-fixed, paraffin-embedded sections with those in frozen sections, only apo B staining was significantly reduced in the former. Therefore, immunohistochemical analysis of apo B was performed according to the standard labeled streptavidin-biotin (LSAB) method (Dako LSAB kit; Dako Japan) to increase reactivity. The washing and dilution buffer was Tris-HCl (0.05 M, pH 7.6). After deparaffinization and blocking of endogenous peroxidase, sections were preincubated with normal bovine serum for 5 min, followed by incubation with polyclonal anti-apo B antibody for 1 hr. After washing three times for 15 min, sections were incubated again with biotinylated antibody for 10 min, followed by incubation with streptavidin-labeled peroxidase for 10 min. Finally, sections were rinsed, developed with DAB, counterstained with hematoxylin, and mounted as for the ABC method. Immunostaining for negative controls was performed by replacing the primary antibodies with normal rabbit or mouse serum at the same dilution as the primary antibodies.

Evaluation of staining results was performed as described by Hazelbag et al. 1995 . The staining results of specimens were evaluated by three independent investigators. When scoring results diverged, averages were used. The response of each antibody was evaluated semiquantitatively in a 0 to 4 score: 0, no staining; 1, poor; 2, average; 3, good; 4, strong. The scoring results were analyzed with ANOVA, followed by post hoc multiple comparison analysis with Fisher's least significant difference test.


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Stages of Healing
The immunohistochemical features of granulation tissues were strikingly distinctive in different stages of wound healing. Tissue specimens in all healing stages were used in the present study. We classified four stages of healing as described (Marchesi 1985 ).

In the first healing stage, fibrin clots with entangled blood cells cover the denuded surface of wounds (Ia), after infiltration of inflammatory cells (Ib). In the second stage, the base of the coagulum is replaced by granulation tissue, which is produced by fibroblasts, endothelial cells, and vascular sprouts from adjacent viable tissues, probably induced by growth factors released during the first stage. In this stage, granulation tissue is often covered with loose fibrous connective tissue with various thickness, designated as a "fibrous cap." Angiogenesis also takes place in this stage. The epithelial sheets are spread to cover the granulation tissue in the third stage. We divided the third stage into two substages according to the thickness of epithelium: partial or thin (IIIa) or thick (IIIb) epithelial sheet covers on granulation tissue. In the last stage, collagen fibers replace the granulation tissue, resulting in reduction of wound size. Finally, the healing process is completed by replacement of granulation tissue with new epithelium or by organization.

To investigate characteristics of each healing stage, the specimens were stained for PCNA and various components of the extracellular matrix by immunohistochemical techniques, in 110 sections from 50 tissue specimens. The pathological diagnoses and healing stages of all specimens are shown in Table 1 and the results of semiquantitative analysis of the immunohistochemical stainings are summarized in Table 2. Expression of PCNA, a marker of cellular proliferation and DNA synthesis, was occasionally observed in the first stage. Staining for PCNA was positive in endothelial cells, epithelial cells, and fibroblasts (Figure 1A) in both the second and third stages but was negative in scar tissues in the fourth stage. On the other hand, the reactivity for dermatan sulfate proteoglycan, which is found in non-neoplastic connective tissues, was absent in the first stage but was strongly present in the second (Figure 1B) and subsequent stages. In contrast, the reactivity for large proteoglycan, which is involved in cell adhesion and is found in immature tissues where cell proliferation and morphologic change occur, was consistently present from the first to third stages, with the maximum in the second stage (Figure 1C). Staining for tenascin and fibronectin was positive throughout all stages.



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Figure 1. Features of immunohistochemical staining for PCNA (A), dermatan sulfate proteoglycan (B), and large proteoglycan (C) in granulation tissue (Stage II) of nasal cavity. Fibroblasts, endothelial cells, and epithelial cells show positive reactions with PCNA antibody, extracellular space widely with the dermatan sulfate proteoglycan antibody, and subepithelium (arrowhead) and the extracellular space around both the vessels (arrows) and the glands (double arrows) with large proteoglycan antibody. Bars = 160 µm.


 
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Table 1. Pathological diagnoses and stages of sample diagnosis/stages


 
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Table 2. Quantitative analysis of staining scores using antibodies against PCNA and extracellular matrices in healing tissuesa

Plasminogen and Fibrinogen
The reactivities for plasminogen and fibrinogen antibodies in the first stage were strong and widely distributed on the tissue surface (Figure 2C and Figure 2D). In the second stage, the staining for plasminogen was diffuse on the fibrous cap except for its surface (Figure 4E), whereas fibrinogen staining was detected on the surface of the fibrous cap. Reactivity gradually declined towards the third stage and was completely absent in the last organized stage (Table 3). In addition, similar results were obtained even when the antibody for plasminogen was preabsorbed by purified Lp(a).



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Figure 2. Serial sections of tongue ulcer tissue with necrotic debris in the first healing stage. Immunostaining of apo(a) (A) is localized to extracellular spaces in area of necrotic debris near the surface; enlarged in E (upper box in A), and an area of inflammatory cell infiltration, small vessels, and the adjacent extracellular space; enlarge in F (lower box in A). Immunostaining of apo B (B) is closely co-localized with apo(a) (A). Plasminogen (C), and fibrinogen (D) are more extensively distributed than apo(a) and apo B. Bars: A-D = 280 µm; E,F = 70 µm.



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Figure 3. Higher magnification views of sections shown in Figure 2A and Figure 2D. The immunoreaction pattern for apo(a) (A) is granular, whereas that for fibrinogen (B) is diffuse (left) and lace-like (right). Bars = 140 µm.

Figure 4. Photomicrographs showing vocal cord granulation tissue with a thick fibrous cap. Immunostaining of apo(a) (A) and apo B (D) is co-localized to the surface stratum of the fibrous cap, and to endothelial cells and the extracellular space underlying the fibrous cap (arrows). Bars = 140 µm. B and C are higher magnifications of the surface and the arrowed portion in A, respectively. Bars = 70 µm. Immunostaining of plasminogen (E) is localized in a deeper portion of the fibrous cap than staining of apo(a) and apo B. Bar = 140 µm.

Figure 5. Immunohistochemical staining of granulation tissue from the nasal cavity with a thin fibrous cap. (A) Double immunolabeling for apo(a) (brown, arrowhead) and laminin (gray, arrows). Positive staining of apo(a) is continuously distributed around areas immunostained for laminin, which is localized to the basement membranes of endothelial cells. (B) Immunolocalization of apo B is identical to that of apo(a). Bars = 500 µm.


 
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Table 3. Quantitative analysis of staining scores using antibodies against apo(a), apo B, plasminogen, and fibrinogen in different healing stagesa

Apo(a) and Apo B
Apo(a) and apo B were not present in normal tissue, as determined immunohistochemically. In Stage Ia of healing, only about one fourth of the specimens were reactive against anti-apo(a) and anti-apo B antibodies. More specimens in Stage Ib were positive (Table 3). Apo(a) and apo B in Stage Ib were localized in the necrotic debris (Figure 2A, Figure 2B, and Figure 2E: higher magnification of 2A), inflammatory cell-infiltrated area, the small vessels, and their adjacent extracellular spaces (Figure 2A, Figure 2B, and Figure 2F: higher magnification of 2A). However, immunoreactivities against apo(a) and apo B were focal compared to plasminogen (Figure 2C) and fibrinogen antibodies (Figure 2D) in this stage. At higher magnification, apo(a) staining was somewhat granular (Figure 3A), compared to fibrinogen staining, which was diffuse and/or lace-like (Figure 3B).

In the second stage, apo(a) staining was markedly enhanced on the surface layer in most specimens. Staining for apo(a) on the fibrous cap showed a stratified pattern in many cases and was localized more superficially than that of plasminogen (Figure 4A and Figure 4E). Figure 4B, a higher magnification of the surface portion shown in Figure 4A, shows that the intensity of apo(a) staining of its surface is much enhanced, with a granular pattern. Apo(a) was also localized in the endothelial cells and the extracellular spaces around the small vessels underlying the fibrous cap (Figure 4A and Figure 4C: higher magnification of 4A). Expression of apo B was also found on the fibrous cap, the small vessels, and the adjacent extracellular space, with the same pattern as apo(a) (Figure 4D). Figure 5 shows granulation at a stage when the fibrous cap had been sloughed. Both apo(a) (Figure 5A, brown) and apo B (Figure 5B) were present in endothelial cells and the extracellular space around small vessels in the surface area. Moreover, apo(a)-laminin double staining clearly demonstrated that reactivity for apo(a) was present in the endothelial cells of small vessels and in the outer staining area of laminin (Figure 5A, gray), localized in basement membranes. PCNA-positive endothelial cells also reacted with the apo(a) antibody (not shown).

In the third stage, reactivity for apo(a) and apo B became weaker with re-epithelization (Table 3). It was not detected in tissues resurfaced with epithelium, as shown in Figure 6A and Figure 6C, whereas activity was still observed on the unepithelized surface in the same tissue (Figure 6B and Figure 6D).



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Figure 6. Micrographs of immunohistochemical staining of granulation tissue from the larynx. Immunostaining of apo(a) (A,B) and apo B (C,D). No immunoreactivity for apo(a) and apo B is observed in recovered tissue (A,C), whereas immunoreactivity is observed on the surface of the desquamative epithelial portion of the same tissue (B,D). Pseudo-positive stain is observed at the epithelium of A, and is found frequently in stratified squamous epithelium. Bars = 140 µm.

Figure 7. Apo(a) immunoreactivity in granulation tissue from colon mucosa in the last stage of healing. B and C are higher magnifications of the vessels indicated by arrowheads and arrows, respectively. No reaction for apo(a) antibody is found in the vascular walls and matrix of the organized tissue (A,B), whereas apo(a) immunoreactivity is observed in the vascular walls (A,C) of areas infiltrated by inflammatory cells. Bars: A = 280 µm; B,C = 70 µm.

In the last organized stage, apo(a) immunoreactivity was negative in endothelial cells and the extracellular matrix in completely organized tissue (Figure 7A and Figure 7B: higher magnification of 7A), whereas it was still detectable within vascular walls of the area infiltrated with inflammatory cells (Figure 7A and Figure 7C: higher magnification of 7A). These results were constant when antibody for apo(a) was pretreated with plasminogen.

Basically, there was no difference between apo(a) and apo B in staining intensity and localization pattern (granular) throughout all stages in almost all specimens. In only one tissue sample in Stage Ib and four samples in the second stage, however, the staining area of apo B was approximately 30% greater than that of apo(a). Apo(a) and apo B co-localized with fibrinogen but not with large and dermatan sulfate proteoglycans in any stages.


  Discussion
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The present study provides the first evidence of apo(a) and apo B accumulation during tissue healing. Although there were some differences in staining intensity at different stages of healing, injured tissues were clearly immunoreactive with antibodies against apo(a), apo B, plasminogen, and fibrinogen. Co-localization of apo(a) with apo B may indicate that an apo(a)-apo B complex, or Lp(a), is present in these tissues. In addition, apo B staining areas without apo(a) were recognized in only 5-10% of specimens. This finding suggests that the majority of apo B accumulation represents Lp(a), but not LDL, in the present study.

Although the stages of healing were classified according to histological findings in the present study, the classification was confirmed by the immunohistochemical features stained for PCNA and various components of extracellular matrix.

Lp(a) cannot be detected in normal portions of examined tissues. In Stage Ia of tissue healing, Lp(a) was weakly stained in injured tissue, which is covered with coagulated blood and fibrin on the denuded surface, whereas fibrinogen and plasminogen stained more intensively and extensively than Lp(a). On the other hand, Lp(a) staining was positive in parts of plasminogen- and fibrinogen-rich areas in Stage Ib of wound healing, in which inflammatory change was obvious. In the second reparative stage, Lp(a) stained more intensely in a granular pattern on the fibrous cap, endo-thelial cells of small vessels, and the extracellular space underlying the fibrous cap in most specimens. PCNA-positive endothelial cells were also reactive with anti-apo(a) antibody, suggesting that Lp(a) may be associated with proliferating endothelial cells in healing wounds. The results from apo(a)-laminin double staining suggest that Lp(a) is derived from blood and is transferred to the extracellular space through the endothelial cells of small vessels in inflamed tissues. Etingin et al. 1991 have reported in their immunohistochemical study that Lp(a) was present in the microvasculature in inflamed but not in normal tissues. Their result and ours indicate that Lp(a) accumulates in small vessels formed in injured tissue. On the other hand, immunoreactivity for Lp(a) was not detected in the tissue completely covered with epithelium.

In wounded tissue with a denuded surface, the fibrous cap functions to prevent the leakage of protein-rich exudate and to protect against bacterial infections during wound healing. In addition, the fibrous cap is rapidly regenerated, even if it is sloughed. Therefore, it should be also stressed that no Lp(a) is found in the subendothelium and the completely organized wound structures, in which the fibrous cap is unnecessary.

Many studies have demonstrated that Lp(a) may downregulate fibrinolysis in vitro by competing with plasminogen for binding sites on endothelial cells and fibrin (Gonzalez-Gronow et al. 1989 ; Hajjar et al. 1989 ). Rouy et al. 1991 have shown that Lp(a) impairs the binding of plasminogen to fibrin, decreases the generation of plasmin, and inhibits the binding of Glu-plasminogen to fibrin surfaces (Rouy et al. 1992 ). Therefore, Lp(a) may regulate excessive fibrinolysis on the surface of wounds by binding to fibrous structures, which is supported by the staining of Lp(a) found with that of fibrinogen, especially in the fibrous cap. Futhermore, the fact that Lp(a) is present more superficially to the surface of the fibrous cap than plasminogen may support the hypothesis that Lp(a) prevents external fibrinolysis.

It is well known that Lp(a) is able to bind to fibrin or fibrinogen via lysine-binding sites in its kringles, providing a potential explanation for the association between increased Lp(a) concentrations and thrombogenesis and atherogenesis (Loscalzo et al. 1990 ; Harpel et al. 1989 ). Beisiegel et al. (1991) reported that apo(a) and fibrin were found in the same areas of the atheromatous intima of the arterial wall. In this study, Lp(a) and fibrinogen were also observed in the same areas of the fibrous cap in granulation tissues.

As mentioned above, Lp(a) accumulates in injured tissues during various stages of wound healing, with a peak in the second stage, whereas plasminogen and fibrinogen decrease as healing progresses. The immunoreactivities for apo A-I and apo A-II, low molecular weight apolipoproteins of HDL, were found weakly only in necrotic debris in our observation. These findings suggest that Lp(a) accumulation in injured tissues must be specific, not simply leaked from enhanced permeable vessels, although the mechanism by which Lp(a) is attracted to injured tissues is unclear at present. We also speculated from the present findings that Lp(a) may participate in tissue repair through blocking of excessive fibrinolysis and angiogenesis of wounded tissues.

Plasma Lp(a) concentrations and their isoforms could not be measured in the patients from whom tissue specimens were obtained in the present study. It is well documented that plasma Lp(a) concentrations vary widely among individuals in a population. Furthermore, the acute-phase reaction of Lp(a) is independent of both plasma concentrations and apo(a) isoforms (Maeda et al. 1989 ). Hervio et al. 1993 have recently demonstrated that small molecular weight isoforms of apo(a) have a higher affinity for fibrin and show more effective inhibition of plasminogen activation than large molecular weight isoforms. However, the relationship between different apo(a) isoforms and their effects on fibrinolysis has not been defined. Because Lp(a) was consistently found in all specimens in the second stage of inflammation and was never found in normal tissues, Lp(a) lipoproteins accumulate in local inflamed tissues according to the stage of healing, probably independent of plasma Lp(a) concentrations and apo(a) isoforms. There may also be no relationship between plasma Lp(a) levels and the duration of wound healing. Further experiments are required to clarify the relationship between plasma Lp(a) levels and tissue repair.

In summary, the present study provides the first evidence for the presence of Lp(a) on healing tissues, especially on the fibrous cap, endothelial cells of small vessels, and the extracellular space underlying the fibrous cap in the second healing stage. We speculate, on the basis of these data, that Lp(a) participates in tissue repair by preventing external fibrinolysis.


  Acknowledgments

Supported by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (nos. 06672289 and 07457563).

We thank Dr R. L. Boni (National Institutes of Health, Bethesda, MD) for his kind help in preparing the manuscript. We also acknowledge the valuable discussions and suggestions of Drs M. Seishima and K. Saito in our department.

Received for publication June 10, 1996; accepted November 21, 1996.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
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