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Circulation Research. 1997;81:785-796

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(Circulation Research. 1997;81:785-796.)
© 1997 American Heart Association, Inc.

Articles

Macrophage-Dependent Regulation of Syndecan Gene Expression

Jian Li, Lawrence F. Brown, Roger J. Laham, Rudiger Volk, , Michael Simons

From the Angiogenesis Research Center, Cardiovascular Division, Department of Medicine (J.L., R.J.L., R.V., M.S.), and the Department of Pathology (L.F.B.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.

Correspondence to Michael Simons, MD, Cardiovascular Division, RW-453, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215. E-mail msimons{at}bidmc.harvard.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Heparan sulfates in the extracellular matrix are required for a variety of biological processes, including cellular response to heparin-binding growth factors. However, little is known regarding the regulation of their expression and composition under pathophysiological conditions. In the present study, we have investigated the regulation of expression of two key heparan sulfate chain–carrying core proteins, syndecan-1 and syndecan-4, in a mouse/rat infarct model of tissue injury and repair. Induction of myocardial infarction was associated with a prompt increase in expression of both syndecan genes. Although infiltrating macrophages accounted for a substantial increase in syndecan expression, increased expression was noted in the levels of syndecan-1 mRNA in endothelial cells and syndecan-4 mRNA in cardiac myocytes. This increase in expression was limited to the immediate peri-infarct region and was absent from remote areas of the left or right ventricles. The influx of blood-derived macrophages in the heart correlated with the appearance of PR-39 peptide, which has previously been shown to increase syndecan expression in vitro. Studies in the op/op mice strain (which demonstrates sharply reduced levels of circulating monocytes) showed that myocardial infarction was associated with markedly reduced levels of macrophage influx and corresponding reduction in the expression of PR-39 and both syndecan genes. Pretreatment of op/op mice with granulocyte macrophage colony–stimulating factor restored myocardial macrophage content with corresponding restoration of PR-39/syndecan expression. In summary, myocardial infarction is associated with a distinct spatial and temporal pattern of syndecan-1 and -4 gene expression, which is induced by an influx of blood-derived macrophages.


Key Words: syndecan • macrophage • heparan sulfate • angiogenesis • extracellular matrix


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Complex multistage multicellular processes such as angiogenesis and wound repair require well-orchestrated interactions between numerous effector molecules and different cell types occurring in a rapidly changing extracellular milieu. Although considerable attention has been focused on the role of various cytokines and their receptors in these processes, relatively little attention has been given to changes in matrix and cell surface composition. In particular, changes in heparan sulfate content may be especially significant, given that interactions with cell surface heparan sulfates are required for cellular actions of a number of growth factors, including FGFs, vascular endothelial growth factor, and TGF-ß.1 2 3 Heparan sulfate chains present on the cell surface and in the matrix can be derived from a number of core proteins. These proteins typically possess intracellular, transmembranous, and extracellular domains, with a number of heparan sulfate chains attached to the ectodomains of these cores via a linking tetrasaccharide moiety. Thus, changes in the heparan sulfate content can come about from alterations in the composition or number of heparan sulfate chains attached to core proteins or can be secondary to changes in the expression of the core proteins themselves.

To explore the role played by heparan sulfates in these processes, we examined changes in expression of two key heparan sulfate chain–carrying core proteins, syndecan-1 and syndecan-4 (ryudocan), after myocardial infarction. Both of these proteins belong to a syndecan core protein family and possess a short intracellular domain, a conserved transmembrane sequence, and an extensive extracellular domain, which provides attachment sites for up to 5 (syndecan-1) or 3 (syndecan-4) heparan sulfate or chondroitin sulfate side chains.1 4 Previous studies have shown that expression of both core proteins is increased in arterial smooth muscle cells after balloon injury5 as well as after skin injuries.6 However, relatively little is known about the regulation of heparan sulfate matrix in general, and syndecan gene expression in particular, in vivo and in vitro. Several cytokines, including TGF-ß and IL-1, can affect proteoglycan synthesis in smooth muscle cells,7 and a macrophage-derived secretory product (presumably IL-1) has been shown to induce dermatan sulfate expression.8 Recently, a novel peptide, PR-39, present in skin-wound fluid, has been shown to increase levels of syndecan-1 and -4 in 3T3 fibroblasts.9 However, the origin of this peptide, the cells responsible for its production, its pattern of expression, and its role in vivo have not been well defined.

In the present study, we set out to examine these questions in a mouse/rat model of myocardial infarction. We chose this model because it would allow us to precisely monitor the time course of injury and because it is associated with a well-described course of pathological alterations. In addition, myocardial infarction in rats and mice is associated with easily monitored and well-defined temporal and spatial patterns of expression of a number of heparin-binding growth factors and their receptors, followed by extensive angiogenesis occurring on the periphery of the infarct.10 Thus, the model provides an opportunity to study syndecan expression and function not only in the course of myocardial injury but also during angiogenesis.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Mouse/Rat Infarct Model
Myocardial infarction was induced in male Sprague-Dawley rats (250 to 300 g) and mice (15 to 20 g) homozygous or heterozygous for the op/op mutation. Briefly, animals were anesthetized with sodium pentobarbital, intubated, and mechanically ventilated with a rodent ventilator (model 683, Harvard Apparatus) with room air. Anterolateral thoracotomy was performed, and the heart was rapidly exteriorized. One or two 6-0 silk sutures were blindly snared around the proximal left anterior descending coronary artery by following the procedure of Litwin et al11 and tightly ligated to occlude the vessel. The heart was then replaced in the chest cavity, the lungs were reexpanded, and the chest was closed in three layers with 4-0 silk sutures. Animals were allowed to recover spontaneous respiration and transferred when stable to the animal care facility. Sham animals were treated in the same manner, except that no coronary ligation was carried out.

At the end of the experiment, at predetermined time points, the animals were again anesthetized, the heart was rapidly extracted, and myocardial tissue was processed as outlined below. A clear distinction between ischemic and normal tissue was easily visible as early as 6 hours after coronary artery ligation; at 1 hour, ischemic tissue was distinguished by a characteristic pallor. In all cases, samples for tissue collection (see below) were taken from the center of the desired area (ischemic or normal).

Pig Ameroid Constrictor Model
By sterile technique, left thoracotomy was carried out in 2 pigs, and a size-matched ameroid constrictor was placed on a circumflex coronary artery as previously described.12 Approximately 20 days after occluder placement, the pigs were intubated and killed while under general anesthesia. Myocardial samples corresponding to the circumflex (ischemic) and left anterior descending (nonischemic) coronary artery tissues were rapidly extracted and processed for RNA isolation as described below.

All animal experiments were conducted in accordance with American Association for Accreditation of Laboratory Animal Care guidelines under a protocol approved by the Institutional Animal Care and Use Committee at the Beth Israel Deaconess Medical Center.

GM-CSF Treatment
Recombinant human GM-CSF was kindly provided by the Genetics Institute (Cambridge, Mass). GM-CSF injections were initiated immediately after weaning of the mice. Recombinant human GM-CSF (dissolved in 0.1 mL normal saline) or placebo (0.1 mL of 0.9% NaCl solution) was administered for 21 days by intraperitoneal injection as follows: placebo mice received 0.1 mL twice daily, and GM-CSF mice received 10 µg of GM-CSF twice daily for 7 days, 10 µg in the morning and 20 µg in the evening for 7 days, and 20 µg twice daily for 7 days, for a total dose of 630 µg administered over 3 weeks.13

RNA Isolation and Northern Analysis
For RNA analysis of the time course of expression of syndecan-1, syndecan-4, and PR-39, the left ventricular myocardium was dissected free of the atria and the right ventricle, snap-frozen in liquid nitrogen, and then homogenized in 4 mol/L guanidinium isothiocyanate, followed by centrifugation through 5.7 mol/L cesium chloride at 200 000g for 16 to 20 hours. The RNA pellet was dissolved in RNase-free water and ethanol-precipitated. For Northern blots, 10 µg of total RNA were fractionated on 1.3% formaldehyde-agarose gel and transferred to GeneScreen Plus (Du Pont) filter. The syndecan-1 (rat), syndecan-4 (rat), PR-39 (pig), and 18S RNA (human) cDNA probes were labeled with [{alpha}-32P]dCTP (New England Nuclear) by a random-priming labeling kit (Boehringer) and purified of unincorporated nucleotides using G-50 Quick Spin columns (Boehringer). The typical specific activity of the probes used in the experiments was 1 to 2x109 cpm/µg. The blots were hybridized at 68°C for 3 hours in QuikHyb solution (Stratagene), followed by two washes in 2x SSC and 0.1% SDS for 15 minutes at room temperature and then twice in 0.1x SSC and 0.1% SDS for 15 minutes at 60°C. Autoradiography was carried out with Kodak XAR film at -80°C for 16 to 20 hours. For quantitative analysis of expression, the blots were exposed by use of a PhosphorImager (Molecular Dynamics) and analyzed using ImageQuant software (Molecular Dynamics); RNA loading was adjusted by scanning 28S RNA bands on photographs of RNA gels. mRNA levels were then expressed as percentage of baseline values.

cDNA Probes
PR-39 cDNA fragment was obtained by polymerase chain reaction cloning from the total porcine bone marrow RNA using nucleotide sequence primers based on the published PR-39 cDNA sequences.14 15 The identity of the obtained 92-bp fragment of PR-39 (nucleotides 401 to 493 of the porcine sequence corresponding to the fourth exon of the PR-39 gene) was confirmed by sequencing, and the entire fragment was used as a probe. Syndecan-1 and syndecan-4 rat cDNA probes were the kind gift of Dr N. Shworak, MIT, Cambridge, Mass. Mouse lysozyme M probe was a gift of Dr L. Van De Water, Beth Israel Deaconess Medical Center, Boston, Mass.

Generation of Syndecan-4–Overexpressing Cells
A human endothelial cell line (ECV, American Type Culture Collection) culture was maintained in 10% FBS-DMEM. For stable transfection, rat syndecan-4 cDNA construct (courtesy of Dr N. Shworak, MIT, Cambridge, Mass) was cloned distal to a metallothionine-2 promoter. Plasmid DNA was then purified, linearized, and transfected using calcium phosphate.16 Sixteen hours after transfections, cells were harvested, diluted to clonal density, and plated in 24-well plates in 10% FBS-DMEM supplemented with G418. Approximately 2 weeks later, individual colonies were picked, expanded in 100-mm dishes, and analyzed for syndecan-4 expression by Northern and Western blotting. The two clones demonstrating the highest level of 100 µmol/L ZnCl2-inducible syndecan-4 protein expression were used for this study.

Western Analysis
Left ventricular myocardial tissues from rat and mice hearts harvested 1 or 24 hours after surgery were homogenized in a lysis buffer containing 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS and centrifuged at 3000g. Protein concentration in the supernatant was determined using a protein assay kit (Bio-Rad). For analysis of syndecan-1 and -4 expression, endogenous proteoglycan cores were purified by DEAE chromatography. Briefly, 500 µg of total protein lysate was loaded on the DEAE column (Pharmacia), and proteoglycan cores were eluted with 1 mL of 1 mol/L NaCl, 0.03% Triton X-100, 30 µg/mL BSA, and 80 ng/mL dextran sulfate used as a carrier. The resulting eluate was applied to ultrafiltration columns (Centrifree, Amicon), centrifuged at 2000g for 30 minutes, and digested with 3 U of heparinase I, II, and III mix (Sigma Chemical Co) and 0.015 U of chondroitinase ABC (Sigma). After ethanol precipitation, the pellets were resuspended in 20 µL of SDS sample buffer, analyzed on 10% SDS-PAGE, and electrotransferred to an Immobilon-P membrane (Millipore). For CD-68, F4/80, and PR-39 protein analysis, 20 µg of total protein lysate from each specimen was subjected to electrophoresis on 10% SDS-PAGE, followed by Immobilon-P transfer. For Western hybridization, membranes were preincubated for 30 minutes in 3% milk–phosphate buffer solution to block nonspecific staining and then incubated with a rabbit anti-rat N-terminal domain syndecan-4 antibody (kind gift of Dr Shworak, MIT, Cambridge, Mass)17 , a mouse anti-rat monoclonal anti–CD-68 antibody (rat tissues, 1:1000 dilution; Synbio MONOSAN), or a rat anti-mouse monoclonal F4/80 antibody (mouse tissues, 1:2000 dilution; Serotec, Inc) for 1 hour at room temperature. After washing with PBS buffer, the membrane was incubated with IgG horseradish peroxidase (1:2000) for 1 hour. The membrane was again washed three times with PBS and then developed using an ECL kit (Amersham), followed by exposure to Kodak XAR film. Equal loading of various samples was confirmed by Coomassie blue staining.

In Situ Hybridization Analysis
Thin (2-mm) sections through the left ventricle were fixed in 4% paraformaldehyde in PBS (pH 7.6) at 4°C for 4 hours and incubated overnight in a solution of 30% sucrose in PBS at 4°C. Tissue was frozen in OCT compound (Miles Diagnostics) and stored at -70°C. Four-micrometer frozen sections were subjected to in situ hybridization as previously described.10 In brief, slides were hybridized overnight at 50°C with 35S-labeled riboprobes (500 000 cpm/slide) in the following buffer: 0.3 mol/L NaCl, 0.01 mol/L Tris (pH 7.6), 5 mmol/L EDTA, 0.02% [wt/vol] Ficoll, 0.02% [wt/vol] polyvinylpyrrolidone, 0.02% [wt/vol] BSA fraction V, 50% formamide, 10% dextran sulfate, 0.1 mg/mL yeast tRNA, and 0.01 mol/L dithiothreitol. Posthybridization washes included 2x SSC/50% formamide/10 mmol/L dithiothreitol at 50°C, 4x SSC/10 mmol/L Tris/1 mmol/L EDTA with 20 µg/mL ribonuclease at 37°C, and 2x SSC/50% formamide/10 mmol/L dithiothreitol at 65°C. Slides were then dehydrated through graded alcohols containing 0.3 mol/L ammonium acetate, dried, coated with Kodak NTB 2 emulsion, and stored in the dark at 4°C for 2 weeks. The emulsion was developed with Kodak D19 developer, and the slides were counterstained with hematoxylin.

In Vitro PR-39 Assays
Freshly isolated adult rat cardiac myocytes (kind gift of Dr N. Yito, Beth Israel Deaconess Medical Center, Boston, Mass) were grown in medium 199 (JRH Biosciences) containing 2 mg/mL BSA, 2 mmol/L l-carnitine, 5 mmol/L creatine, 5 mmol/L taurine, 1.3 mmol/L l-glutamine, and 0.1 µmol/L insulin. Primary human coronary endothelial and coronary smooth muscle cells (Clonetics) were grown in Clonetics-supplied medium with 10% FBS and used in the second passage. A murine fibroblast NIH 3T3 cell line was obtained from American Type Culture Collection and cultured in DMEM supplemented with 10% heat-inactivated FBS, 2 mmol/L glutamine, and 100 µg/mL penicillin/streptomycin. All cultures were incubated at 37°C in 5% CO2. PR-39 peptide (synthesized by Chiron Mimotopes Systems) was dissolved in PBS and diluted with appropriate cell culture medium before use. For peptide stimulation studies, cells were plated at 90% confluence, and 1.0 µmol/L of peptide was added to the culture; in the case of adult rat cardiac myocytes, stimulation was continued for 5 hours; 3T3 cells, human coronary smooth muscle cells, and human coronary endothelial cells were stimulated for 24 hours. At the end of the stimulation period, cells were harvested, and total RNA was extracted as described above. All experiments were carried out in duplicate and repeated three times (twice for cardiac myocytes).

Immunohistochemistry
Blood Sample and Tissue Preparation
Rat peripheral blood sample (5 mL) was collected in a heparinized tube and then immediately transferred to a plastic cell culture dish and kept at 37°C for 6 hours. The floating cells were then washed out with PBS, and the adherent cells were trypsinized and collected for a cytospin, which was then used for immunocytochemical staining with CD-68 antibody. For tissue section staining, freshly collected samples of rat small intestine and spleen were fixed in 4% paraformaldehyde, embedded into O.C.T. compound (Miles), and frozen in liquid nitrogen, after which 6-mm sections were cut using a cryotome.

Immunostaining
The slides were preincubated with 5% goat serum for 20 minutes at room temperature and then incubated with 10 µg/mL mouse-anti human CD-68 antibody for 60 minutes. The slides were then incubated with biotinylated anti-mouse IgM (Vector Labs). Macrophages were visualized using either alkaline phosphatase (for blood sample macrophages) or streptavidin Texas red (Amersham) for tissue sections.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
To study the pattern of syndecan-1 and -4 and PR-39 expression after myocardial infarction, rat hearts (5 per time point) were collected at various times after ligation of a left coronary artery. RNA blot analysis demonstrated a low level of expression of both syndecan-1 and syndecan-4 in normal (not infarcted) rat hearts. Both syndecan genes demonstrated a significant increase in mRNA expression after myocardial infarction, with syndecan-4 mRNA levels rising nearly 8-fold as early as 1 hour after infarct onset (Fig 1Down, left and middle). Expression of both syndecans remained elevated for up to 7 days after infarct, with syndecan-4 demonstrating significantly higher expression than syndecan-1. To demonstrate that the cDNA probes used in the study were selective for syndecan-1 and syndecan-4 messages, human ECV cells were stably transfected with an inducible syndecan-4 expression construct. Northern analysis of syndecan mRNA levels demonstrated distinctly different patterns of syndecan-1 and syndecan-4 cDNA probe hybridization before and after induction of syndecan-4 construct expression (Fig 1Down, right). This increase in syndecan mRNA levels was paralleled by an increase in expression of mature protein as demonstrated by Western blotting of syndecan-4 (Fig 2Down; Western blotting of syndecan-1 could not be performed because of lack of antibody recognizing rat syndecan-1).



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  		Figure 1. Representative RNA blots of syndecan-1, syndecan-4, and PR-39 expression in the rat heart after myocardial infarction (left), quantitative analysis of these changes (middle), and analysis of syndecan-1 and -4 expression in a cloned endothelial cell line (right). Ethidium bromide (Eth Br)–stained gel is shown for comparison of lane loading. The expression of all three genes is indexed as percent control (100% indicates expression in normal myocardium). The data are shown as mean±SD.



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  		Figure 2. Syndecan-4 and CD-68 protein expression in the rat heart. Shown are representative Western blots (left) and quantitative analysis of tissue samples from rat ventricular myocardium before (control) and 1 hour and 24 hours after induction of acute myocardial infarction (right). Molecular weight markers are shown on the left. Solid bars indicate syndecan-4; hatched bars, CD-68. Values are mean±SD.

Examination of the pattern of PR-39 peptide expression demonstrated that although small amounts of PR-39 message were present in the normal heart, there was a rapid early (1-hour) increase in mRNA levels that coincided with an increase in syndecan-4 and preceded an increase in syndecan-1 mRNA expression (Fig 1Up). Furthermore, early (1-hour) appearance of increased PR-39 message expression closely correlated with the appearance of blood-derived macrophages (CD-68– positive cells, Fig 2Up).

To demonstrate the specificity of CD-68 antibody for recognition of rat macrophages, we carried out immunocytochemical analysis of rat intestinal villi and splenic marginal zone (Fig 3Down). In each case, the antibody identified a specific subset of cells in expected locations for blood-derived macrophages. Furthermore, staining of the rat peripheral blood cells demonstrated staining of monocytes but not granulocytes (Fig 3Down).



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  		Figure 3. Tissue analysis of CD-68–positive cells. Blood-derived macrophages in rat tissues (intestinal villi [top] and spleen [middle]) were detected using CD-68 antibody. Left, Immunofluorescent images. Right, Phase-contrast microphotographs. Monocytes/macrophages in the rat peripheral blood (bottom) were detected with CD-68 antibody after cell attachment in tissue culture; note staining of two monocytes (at 1 and 5 o'clock); two granulocytes visible in the field (black arrows) were not stained by the antibody.

To demonstrate that the porcine cDNA probe used for these studies identified correct transcript in rat tissues, we compared PR-39 mRNA detection on an organ blot of porcine tissues to which the rat heart RNA sample was added. Northern analysis showed an expected size ({approx}700 bp) of PR-39 message in pig intestine, esophagus, spleen, kidney, and heart and a similar-sized message in the rat heart (Fig 4Down, top). To further confirm that myocardial PR-39 expression is induced by ischemia, we compared PR-39 mRNA levels in normal and ischemic territories of the porcine heart. RNA blot analysis of these tissues, carried out {approx}2 to 3 days after ameroid constrictor closure, demonstrated a significant increase in PR-39 expression in the ischemic compared with normal myocardium (Fig 4Down, bottom).



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  		Figure 4. PR-39 analysis in porcine tissues. Northern blot analysis of porcine organs for PR-39 mRNA expression using the 92-bp exon IV PR-39 probe cloned from porcine bone marrow demonstrated bands of expected sizes in the intestine, esophagus, spleen, kidney, and heart. Note a similar-sized band in the normal rat heart (top). Studies of PR-39 mRNA expression in the normal (bottom, lanes 1 and 3) and ischemic (bottom, lanes 2 and 4) porcine myocardial tissues demonstrated increased peptide expression in the ischemic myocardium similar to the finding in the rat heart.

To better define cell populations responsible for syndecan and PR-39 expression, we used in situ hybridization and immunocytochemical analysis. In control (normal) hearts, syndecan-1 message was present predominantly in endothelial cells of larger vascular spaces and was most prominent in arteries and arterioles (Fig 5ADown and 5BDown). No strong expression of syndecan-4 was identified, but possible low-level labeling of myocytes was noted (not shown). Likewise, no unequivocal expression of PR-39 mRNA in any cell type could be identified, and hybridization with a lysozyme-specific probe demonstrated only a few scattered lysozyme-positive cells (not shown).



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  		Figure 5. In situ hybridization analysis of syndecan-1 and syndecan-4 gene expression. In situ hybridization of syndecan-1 expression in normal myocardium (A and B) and of syndecan-4 gene expression in rat ventricular myocardium 6 hours after induction of acute myocardial infarction (C and D) is shown. Notice the predominantly endothelial cell hybridization in the vessel wall in panels A and B and the diffuse hybridization throughout the vessel wall in panels C and D. Bars=25 µm.

By 6 hours after infarction, syndecan-1 message was detectable in the same pattern as seen in normal heart, whereas syndecan-4 mRNA was now expressed throughout the vessel wall (Fig 5CUp and 5DUp). Also at 6 hours, increased numbers of lysozyme-positive macrophages were noted in the myocardium and were most numerous near blood vessels (Fig 6ADown and 6BDown). Cells in the same pattern of distribution as the macrophages and having the same histological features also demonstrated strong expression of PR-39 message (Fig 6CDown and 6DDown). No PR-39 mRNA expression was seen in the invading neutrophils (not shown).



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  		Figure 6. Macrophage expression of PR-39 peptide. In situ hybridization analysis of the rat ventricular myocardium 6 hours after acute myocardial infarction is shown. Lysozyme M probe (A and B) demonstrates the presence of perivascular lysozyme-positive cells (macrophages). The PR-39 probe demonstrates hybridization with similarly situated cells (C and D). Bars=25 µm.

At 1 day after infarction, large numbers of lysozyme-positive macrophages were present in the myocardium (Fig 7ADown and 7BDown), concentrating at the edges of areas containing necrotic myocytes (not shown). Cells in the same pattern of distribution and with the same histological features as the lysozyme-positive cells were also strongly expressing mRNA for syndecan-1 (Fig 7CDown and 7DDown) and syndecan-4 (Fig 7EDown and 7FDown), whereas prominent expression of syndecan-4 in vessel walls seen at 6 hours after infarction was no longer present. Expression of syndecan-1 by endothelial cells and low-level expression of syndecan-4 by cardiac myocytes was noted as before.



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  		Figure 7. Syndecan-1 and -4 gene expression after myocardial infarction. In situ hybridization analysis of rat ventricular myocardium 1 day after acute myocardial infarction is shown. A and B, Lysozyme-positive cells are present along the infarct periphery. C and D, Syndecan-1 expression is present in the same areas, predominantly in macrophages (note similar appearance to areas in Fig 5AUp and 5BUp). E and F, Syndecan-4 expression is noted in macrophages and in myocytes on the infarct periphery. Bars=25 µm.

By 3 days after infarction, granulation tissue consisting of macrophages, other inflammatory cells, fibroblasts, and new blood vessels had begun to replace the infarcted areas of myocardium. Large numbers of lysozyme-positive macrophages were present within the granulation tissue. Strong expression of syndecan-1 and -4 was also noted by cells in these areas. Although many of these cells had the histological features of macrophages, the diffuse distribution of macrophages throughout the cellular granulation tissue made the study of expression of syndecan by other mononuclear cell types (eg, fibroblasts) impossible. Outside the infarcted areas, expression of syndecan-1 and -4 was similar to the pattern seen in normal heart. Findings at 7 days after infarction were very similar to those at 3 days. At 6 weeks after infarction, granulation tissue was more mature, with a higher proportion of fibroblasts and fewer inflammatory cells. A number of lysozyme-positive macrophages were still present in the granulation tissue, but their number had markedly decreased. The number of cells strongly expressing syndecan-1 and -4 had also decreased markedly in the infarcted areas.

To further examine the pattern of PR-39 and syndecan gene induction and the cell-type specificity of their expression seen in vivo, we analyzed the effect of PR-39 peptide treatment on various cell lines in vitro. Addition of 1.0 µmol/L of synthesized PR-39 to cell cultures resulted in a prompt and significant increase in both syndecan-4 and syndecan-1 expression in 3T3 fibroblasts similar to that described by Gallo et al9 and to that seen in smooth muscle cells in vivo (Fig 8Down). However, in primary cardiac myocytes, PR-39 induced a significant increase in expression of syndecan-4 but not syndecan-1 message levels (Fig 8Down), whereas there was no statistically significant increase in the expression of either gene in endothelial cells. Thus, in vitro studies have demonstrated that PR-39 is able to induce syndecan-1 and -4 gene expression in a cell type–specific pattern similar to the pattern observed in vivo.



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  		Figure 8. In vitro effect of PR-39 administration in various cell lines. Primary cardiomyocytes (CM), coronary endothelial cells (EC), coronary smooth muscle cells (SMC), and NIH-3T3 cells (3T3) were cultured for 24 hours (5 hours for cardiac myocytes) in the presence of 1.0 µmol/L PR-39 peptide. Shown is quantitative Northern blot analysis of syndecan-1 and syndecan-4 mRNA levels expressed as percentage of control (mean±SD). *P<.05 vs baseline.

To confirm these observations and to establish a direct link between the presence of myocardial macrophages, PR-39, and increased syndecan gene expression in vivo, we studied PR-39 and syndecan-1 and -4 mRNA expression in a similar infarct model in an op/op mouse strain, characterized by markedly decreased numbers of circulating monocytes with consequent reduction in tissue levels of blood-derived macrophages.13 18 As in the rat model, infarct induction in mice heterozygous for the op/op mutation (n=4) was characterized by a prompt increase in syndecan-1 and -4 gene expression (Fig 9Down). However, homozygous op/op mice (n=4) showed a markedly reduced increase in syndecan message expression after myocardial injury (Fig 9Down). Similarly, homozygous op/op mice showed a significantly reduced amount of PR-39 message, which increased only minimally after infarction.



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  		Figure 9. Syndecan gene expression in homozygous and heterozygous op/op mice. Shown are representative RNA blots (top) and quantitative analysis (bottom) of syndecan-1, syndecan-4, and PR-39 gene expression in ventricular myocardial tissues from normal (control) hearts of mice heterozygous for the op/op locus (+/-), heterozygous mice 24 hours after myocardial infarction (+/- MI), homozygous op/op mice before (-/-) and 24 hours after (-/- MI) infarction, and homozygous op/op mice treated with GM-CSF before (-/- GM-CSF1) and 24 hours after (-/- GM-CSF1 MI) myocardial infarction. Note the absence of increased syndecan-1 and syndecan-4 expression in homozygous compared with heterozygous op/op mice (also compare with expression in the rat; Fig 1Up) and restoration of PR-39 and syndecan-1 and 4-expression after GM-CSF treatment. Eth Br indicates ethidium bromide.

Immunocytochemical staining with a rat anti-mouse F4/80 anti-macrophage antibody showed a marked reduction in the number of myocardial macrophages in homozygous but not in heterozygous op/op mice (not shown). Western blot analysis confirmed that although the macrophage population in the heart increased after myocardial infarction in rats (Fig 2Up, CD-68 antibody) and in heterozygous op/op mice (Fig 10Down, F4/80 antibody), there was no significant increase in the amount of F4/80-positive antigen in the op/op homozygotes (Fig 10Down).



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  		Figure 10. Macrophage populations in homozygous and heterozygous op/op mice. Shown are representative Western blot and quantitative analysis of macrophage-specific F4/80 antigen expression in the myocardium of op/op heterozygous (+/-), homozygous (-/-), and homozygous GM-CSF–treated (-/- GM-CSF1) mice before and 24 hours after induction of myocardial infarction. Note markedly reduced macrophage content in the myocardium of op/op homozygous mice and restoration of baseline and postinfarct macrophage contents after GM-CSF administration (compare lanes 3 and 5 and lanes 4 and 6).

To demonstrate a direct link between the presence of blood-derived macrophages and the expression of PR-39 and syndecan-1 and -4 after myocardial infarction, op/op homozygous mice were treated for 3 weeks with increasing doses of GM-CSF by following the protocol of Wiktor-Jedrzejczak et al.13 Myocardial infarction was induced at the end of the 3-week period, and tissues were harvested 24 hours later. Western analysis demonstrated a significant increase in the number of F4/80-positive myocardial macrophages in treated mice before the infarct to the level seen in heterozygous mice before the infarct (Fig 10Up; compare lanes 1 and 5), which further increased 24 hours after myocardial infarction (Fig 10Up, lanes 5 and 6). At the same time, Northern analysis demonstrated induction of PR-39 message after myocardial infarction (Fig 9Up; compare lanes 5 and 6) that was associated with restoration of syndecan-1 and -4 expression to a level comparable to that seen in heterozygous op/op mice (Fig 9Up; compare lanes 2, 4, and 6). Importantly, GM-CSF treatment in op/op homozygous mice by itself was not associated with an increase in syndecan-1 and -4 expression (Fig 9Up; compare lanes 3 and 5). Thus, GM-CSF–dependent restoration of tissue macrophage levels resulted in increased PR-39 mRNA levels, which was paralleled by an increase in syndecan-1 and -4 expression.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Increased expression of core proteins capable of carrying heparan sulfate chains likely translates into increased numbers of these chains on the cell surface and/or in the extracellular space. This increase in turn may affect the biological activity of various growth factors and cytokines that require heparan sulfate binding for their interaction with specific high-affinity receptors. Thus, modulation of the heparan sulfate matrix composition may play an important role in various processes, including wound healing and angiogenesis. The rat myocardial infarction model provides a convenient way of examining both of these processes, since a sudden onset of ischemia leads to a well-defined sequence of events involving removal of damaged tissue, formation of connective scar tissue, and intense angiogenesis at the periphery of the infarct. However, little is known regarding the alterations in heparan sulfate matrix during these processes.

The syndecan family of cell surface proteoglycans has been implicated in a number of biological processes, including regulation of blood coagulation, cell adhesion, and signal transduction.1 2 3 Originally found on epithelial cells,19 syndecans were later shown to be present in several other mesenchyma-derived cell types, including fibroblast and smooth muscle cells.20 21 22 The family consists of four closely related genes termed syndecan-1 through syndecan-4. Syndecan-1 and syndecan-4 (ryudocan) are the most widely studied members of this family and show expression in a variety of adult cell types, including epithelial cells and fibroblasts, although expression in quiescent tissues is fairly low.23 24 Syndecan-2 (fibroglycan) is expressed at high levels in cultured lung and skin fibroblasts, yet it is barely detectable in most adult tissues, and it is present in very low amounts in most epithelial tissues.23 Syndecan-3 (N-syndecan) demonstrates a much more limited pattern of expression, being largely restricted to peripheral nerves and central nervous system tissues, although high levels are noted in neonatal heart.25 Biological activity of these molecules is thought to be largely due to the presence of attached heparan sulfate chains capable of binding growth factors, such as FGFs, cell adhesion receptors, and other biologically active molecules.2 3 26 27 28 29 30 Although the biochemistry and structure of syndecan core proteins is well understood, relatively little is known regarding their function, regulation, and cell and tissue specificity of expression. Several observations suggest that syndecan expression may be related to cell proliferation, including transient induction of syndecan-1 gene expression in mesenchymal cells during tooth organogenesis31 and increased levels of syndecan-1 and syndecan-4 mRNAs in arterial smooth muscle cells after balloon catheter injury5 and in the skin after wounding.6

Macrophages are involved in a number of key processes associated with injury, repair, and angiogenesis and have an ability to affect the synthesis and/or degradation of a wide variety of extracellular matrix proteins, including fibronectins,32 thrombospondin 1,33 and proteoglycans.8 Both monocytes and tissue-resident macrophages express syndecan-134 and syndecan-435 core proteins and secrete a number of cytokines, including IL-1, TGF-ß, and TNF-{alpha}, known to affect expression of these proteins. Finally, macrophage activation is associated with changes in expression of sulfated proteoglycans8 36 37 38 .

Recently, a peptide isolated from pig wound fluid has been shown to increase syndecan-1 and -4 gene expression when added to mesenchymal cell cultures in vitro.9 The peptide was identical to an antibacterial peptide known as PR-39, which was originally isolated from the pig intestine and thought to be involved in nonimmune defense of intestinal integrity14 by playing a role similar to that of magainins,39 cecropins,40 and ß-defensins.41 PR-39 is a member of the cathelin family of proteins42 and is synthesized as a precursor molecule, which shares high sequence homology with all other members of the cathelin family in its N-terminal end while possessing a unique proline-arginine–rich carboxy-terminal domain encoded by a separate exon.42 This latter domain encodes a 39–amino acid peptide that appears to be responsible for induction of syndecan expression. Although the exact cellular origin of this peptide is not fully known, both circulating granulocytes9 43 and macrophages42 appear to be capable of producing the PR-39 protein.

We set out to investigate in the present study the pattern of syndecan expression in the normal myocardium as well as during myocardial injury and repair after an acute infarction and the role played by macrophages in this process. In the normal heart, syndecan-1 expression was largely restricted to endothelial cells lining intramyocardial blood vessels, whereas syndecan-4 expression was noted predominantly in cardiac myocytes. No PR-39–expressing cells appeared to be present, and there were very few macrophages. This paucity of tissue-resident macrophages in the normal myocardium is in accord with other studies.44 45 The onset of myocardial ischemia was accompanied by an immediate increase in the expression of syndecan-4 mRNA, with a substantial increase in syndecan-1 mRNA levels almost 24 hours later. Using Western blotting, we were able to confirm a comparable rise in syndecan-4 protein level; however, similar studies of syndecan-1 expression could not be carried out because of the lack of antibody recognizing syndecan-1 in rat tissues.

At the earliest time point examined by in situ hybridization (6 hours), there was an increase in syndecan-4 expression in the vessel wall that coincided with the appearance of perivascular cells expressing the PR-39 message. Morphologically, these cells appeared to be macrophages. This observation suggests that PR-39–expressing macrophages migrating through the blood vessel wall may have been responsible for the increased syndecan-4 mRNA levels seen in the arterial vessel wall. In vitro studies have confirmed the ability of PR-39 peptide to induce syndecan-4 expression in coronary smooth muscle cells. We could not directly confirm the presence of PR-39 peptide in macrophages (or tissues) because previously reported monoclonal anti-porcine PR-39 antibody9 is not PR-39 specific in rat and mouse tissues (data not shown). However, demonstration of PR-39 mRNA expression in macrophages in vivo by in situ hybridization (Fig 5Up) and a strong association between macrophage levels and PR-39 mRNA expression (Figs 8Up and 9Up) strongly argue for macrophages as the source of this peptide. In addition, examination of the time course of PR-39 mRNA expression and the appearance of blood-derived macrophages demonstrate that the early (1-hour) rise in PR-39 mRNA levels (Fig 1Up) closely correlates with the influx of CD-68–positive macrophages at the same time (Fig 2Up). However, although the number of macrophages continues to increase (Fig 2Up, 24-hour time point), the expression of PR-39 mRNA declines, suggesting that the expression of the peptide is rather transient. Whereas there was a diffuse increase in the expression of both syndecans throughout the infarct region, with some of the increase in syndecan-4 mRNA noted in cardiac myocytes and some of the increase in syndecan-1 mRNA noted in endothelial cells, most of it appeared to be due to the influx of mononuclear lysozyme-positive cells. At no time point was PR-39 expression noted in granulocytes, contrary to the pig skin-wound studies of Gallo et al.9

An important point in these experiments is a conclusive identification of tissue-resident blood-derived macrophages in rat/mice hearts. We used two different techniques to achieve this goal: in situ hybridization with a lysozyme M probe and immunocytochemistry using anti–CD-68 antibody in rats and F4/80 antibody in mice. Use of the lysozyme M probe for macrophage identification has been tested previously,45 and there are extensive data regarding the specificity of rat anti-mouse F4/80 antibody.44 46 However, no such data exist with regard to CD-68 antibody. To ensure correct cell-type recognition, we tested CD-68 antibody on a rat peripheral blood sample and tissues known to possess large numbers of blood-derived macrophages, namely, intestinal villi and the splenic marginal zone.44 Using a peripheral blood sample, we observed selective antibody binding to adhered monocytes/macrophages but not other mononuclear cells or granulocytes, whereas in both intestinal and spleen tissues, the antibody also correctly identified macrophages in the expected locations.

To further confirm the role of blood-derived macrophages in the regulation of PR-39 and syndecan-1 and -4 expression, we examined the same process in mice heterozygous and homozygous for the op/op phenotype. The op/op strain is characterized by a spontaneous CSF-1 mutation that results in severe reduction of circulating monocytes and a number of skeletal abnormalities, including osteopetrosis and abnormal dental development.13 47 This reduction in the number of circulating monocytes is thought to be responsible for a decrease in some macrophage populations, including markedly reduced numbers of peritoneal macrophages and decreased numbers of Kupffer and Langerhans cells.18 However, other, presumably not CSF-1–dependent, macrophage populations remain present in normal numbers.18

As in rats, myocardial infarction in op/op heterozygous mice (50% reduction in monocyte level) resulted in the prompt appearance of PR-39 and syndecan-1 and -4 messages. However, op/op homozygous animals (>95% reduction in circulating monocyte level) showed no significant increase in either PR-39 or syndecan gene expression. Immunocytochemical studies using mouse blood–derived macrophage-specific F4/80 antibody44 confirmed the marked reduction of this macrophage subset in op/op homozygous animals. As expected, PR-39–expressing cells also were not present. Thus, the absence of blood (monocyte)-derived macrophages in the op/op homozygous animals likely translated into reduced production of PR-39, with subsequent failure of augmentation of syndecan gene expression.

To further link the absence of blood-derived macrophages to the reduction in syndecan gene expression, we restored the monocyte population in homozygous op/op mice by a prolonged infusion of GM-CSF, which in turn resulted in the reappearance of tissue-resident myocardial macrophages (presumably of monocyte origin). In the absence of injury, we did not observe any effect of GM-CSF infusion on baseline syndecan-1, syndecan-4, or PR-39 message levels. However, after myocardial infarction, GM-CSF–treated op/op mice demonstrated full expression of both syndecan genes and the reappearance of PR-39 message. Thus, restoration of tissue macrophage content had a marked effect on postinjury expression of syndecan genes.

Although we did not directly demonstrate that reintroduction of myocardial tissue–resident macrophages affected syndecan expression secondary to the reappearance of PR-39, indirect evidence points toward this mechanism. In particular, we observed a tight temporal correlation between the appearance of PR-39 expression and an increase in syndecan message levels in both rats and GM-CSF–treated op/op mice. In addition, PR-39 studies in vitro demonstrated a pattern of peptide-induced activation of syndecan expression that was very similar (with regard to cell-type specificity and temporal patterns) to that seen in vivo. Thus, although confirmation of the role of PR-39 in the regulation of syndecan expression in vivo remains to be demonstrated, it appears likely that this peptide is at least partially responsible for this effect.

The macrophage origin of PR-39 is not surprising, given its recent cloning from porcine bone marrow and myeloid cells.15 48 49 Our findings in the op/op mice further underscore the importance of monocyte (blood)-derived macrophages as the source of this peptide. Interestingly, we did not detect any PR-39 mRNA expression in other cell types, suggesting that in rats and mice macrophages may be the only cell type capable of producing significant amounts of this peptide. The peptide induced a prompt and significant increase in syndecan gene expression in several mesenchymal cell types present in the myocardium, including endothelial cells, smooth muscle cells, cardiac myocytes, and macrophages themselves, which demonstrated by far the most significant increase in syndecan expression. This hitherto unappreciated aspect of macrophage biology combined with their well-documented presence in healing wounds and around forming blood vessels adds another dimension to the role these cells play in reparative and angiogenic processes.

In summary, we have shown a rapid increase in syndecan gene expression after myocardial injury in a rat/mouse model. This increase was predominantly due to the influx of blood-derived monocytes/macrophages secreting PR-39 peptide. The functional significance of increased syndecan synthesis and the role of PR-39 in tissue repair and angiogenesis after myocardial ischemia are the subjects of ongoing studies.


*    Selected Abbreviations and Acronyms
 
IL-1 = interleukin-1
TGF-ß = transforming growth factor-ß
TNF-{alpha} = tumor necrosis factor-{alpha}
FGF = fibroblast growth factor
GM-CSF = granulocyte macrophage colony–stimulating factor


*    Acknowledgments
 
This study was supported in part by a National Institutes of Health grant R01-HL-53793 (Dr Simons) and an NIH training grant (Dr Li).

Received February 10, 1997; accepted July 31, 1997.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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