ARTICLE |
Correspondence to: Mieczyslaw Marcinkiewicz, Lab. of Biochemical Neuroendocrinology, Clinical Research Inst. of Montreal, 110 Pine Avenue West, Montreal, QC, Canada H2W 1R7. E-mail: Marcinm@ircm.qc.ca
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Summary |
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The aim of this study was to investigate whether transformation of quiescent vascular smooth muscle cells (VSMCs) into proliferating secretory cells is accompanied by an expression of processing enzymes that activate de novo-synthesized growth factors. Three enzymes belonging to the family of the kexin/subtilisin-like mammalian proprotein convertases (PCs), furin, PC5, and PC7, were found to be upregulated after balloon denudation in vivo. To determine their importance in these cell processes, we investigated their gene regulation using a short-term organ culture system. After incubation of rat aorta for 4 and 24 hr in serum-free medium, we demonstrated a significant induction of VSMC proliferation. The affected subset of VSMCs, positive for -smooth muscle actin, also expressed proliferating cell nuclear antigen (PCNA). Our results revealed a parallel upregulation of furin, PC5, and PC7 in PCNA-immunolabeled cells. As a substrate model for comparison with PCs we used nerve growth factor (NGF). NGF is known to be activated by PCs. As shown by Northern blotting analysis, NGF mRNA concentration was significantly increased in cultured explants. NGF was released into the culture medium. In conclusion, both PCs and NGF are coordinately modulated on induction of VSMC proliferation.
(J Histochem Cytohem 49:323331, 2001)
Key Words:
protein processing, aorta organ culture, proliferating cell nuclear antigen, nerve growth factor,, -smooth muscle cell actin, immunocytochemistry, co-localization
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Introduction |
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PROLIFERATION and migration of vascular smooth muscle cells (VSMCs) are key elements in the fibroproliferative phase of atherosclerotic plaque formation, the leading cause of coronary heart disease and cerebral stroke (, where n = 0, 2, 4, or 6 and X is any amino acid, to become biologically active. Thus far, eight mammalian PCs have been identified, including furin, PC1 (also named PC3), PC2, PC4, PACE4, PC5-A and PC5-B (also known as PC6), PC7 (
Each of these enzymes, either alone or in combination with others, is responsible for the tissue-specific processing of multiple polypeptide precursors. This combinatorial mechanism generates a large diversity of bioactive molecules in an exquisitely regulated manner. This type of cleavage occurs in most growth factors and their receptors, polypeptide hormone precursors, surface glycoproteins, including adhesion molecules and viral envelope glycoproteins, and a host of other secretory proteins (
The role of these proprotein convertases in vascular tissue remains largely unknown. We hypothesized that PCs might represent important intermediates in the biosynthesis and activation of growth factors involved in vascular remodeling.
In this study we tested the hypothesis that the induction of VSMC proliferation in vivo and in vitro involves, in addition to the de novo synthesis of growth factors, the expression of their processing enzymes as well. To test our hypothesis we used in vivo and in vitro models. After balloon denudation in rodents in vivo, we examined the regulation of PCs. In vitro we investigated the expression of PCs in VSMCs during early stages of VSMC proliferation using an organ culture system. This in vitro model offers the advantage of maintaining cell/cell and cell/matrix interactions, thus preserving an in vivo-like environment.
In this article we report that the rapid onset of VSMC proliferation in vitro triggers the enzymatic machinery including the upregulation of proprotein convertases and the de novo synthesis of growth factors.
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Materials and Methods |
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Preparation of Arterial Explants
Adult male SpragueDawley rats weighting 300350 g (Charles River; St Constant, QC, Canada) were used in this study. Rats were housed in an air-conditioned animal facility (12:12 hours light:dark cycle), fed a standard diet, and received water ad libidum. Rats were sacrificed by IP injection of 50 mg/kg sodium pentobarbital. The aorta was cannulated at the iliac bifurcation, opened at the region of the aortic arch, and blood carefully washed out with Ringer's lactate solution. Each artery was then opened longitudinally, cut into six pieces (about 1 x 1 cm) and transferred into Petri dishes containing PBS. Excessive fat and connective tissue were removed and explants washed three times in PBS. Single pieces of rat aorta were then transferred to six-well dishes (BectonDickinson; Franklin Lakes, NJ) and kept under 95% relative humidity and 5% CO2 at 37C in Dulbecco's modified Eagles medium (DMEM, catalogue #12100; Life Technologies, Burlington, ON, Canada), supplemented with gentamycin 26 µg/ml, with the endothelial side upwards.
After culturing, explants were either snap-frozen on dry ice at -80C for immunoblotting and RNA extraction or fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) overnight at 4C, then washed in 70% ethanol for 24 hr, embedded in paraffin, and cut into 3-µm slices.
In Vivo Balloon Denudation of Rat Aorta
Balloon injury of rat aorta was performed according to
Rats were sacrificed in groups of four to six at Day 2, 7, or 28 after the operation. The aorta was fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 hr, washed for 24 hr in 70% ethanol, and then embedded in paraffin.
Immunocytochemistry
The antibodies used are indicated in Table 1, providing information about the antigen, host, protein sequence, and dilution for both Western blotting and immunocytochemistry.
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Immunocytochemical stainings were performed with the Zymed Histostain-Plus kit (Zymed Laboratories; San Francisco, CA) as recommended by the manufacturer. The immunoreactions were revealed with horseradish peroxidase activity producing brown staining and/or alkaline phosphatase activity producing blue and/or red staining. When necessary, double immunocytochemical stainings were performed by combining alkaline phosphatase and horseradish peroxidase activity as described (-SMA. Statistical analysis was by Student's t-test.
Western Blotting Analysis
Immunoblotting was performed as described previously (-mercaptoethanol 2 mM. Up to 50 µg protein was mixed with sample buffer and applied on 8% or 12% TrisTricine SDS PAGE. Serum-free media were also used after 10-fold concentration by centrifugation through a #3 Centricon concentrator (Amicon; Beverly, MA). After migration, proteins were transferred onto PVDF membranes (Schleicher & Schuell; Keene, NH). The membranes were first incubated overnight at 4C with the primary antibody, followed by incubation with donkey anti-rabbit IgGperoxidase (Jackson Laboratories; West Grove, PA) diluted 1:10,000. Peroxidase activity product was revealed with the BM Chemiluminescence blotting substrate POD (BoehringerMannheim; Mannheim, Germany). For controls, antisera were preadsorbed with synthetic peptides. Densitometry was done with the Northern eclipse program.
Northern Blotting Analysis
Northern blotting analysis was conducted as described elsewhere (
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Results |
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Immunocytochemical Localization of PCs after Balloon Denudation In Vivo
Immunocytochemical localization of the proprotein convertases furin, PC5, and PC7 was studied in intact rat aorta and in rat aorta after balloon injury at various time points (Fig 1). In intact rat aorta, PC7 (Fig 1A), furin (Fig 1E), and PC5 (Fig 1G) immunoreactivity was found in rare cells throughout the media. Depicted as an example, immunocytochemical analysis revealed upregulation of PC7 on Day 2 in the media (Fig 1B) and on Day 7 in the beginning neointima (Fig 1C). After establishment of a mature neointima on Day 28, PC7 was found throughout the neointima (Fig 1D). Furin (Fig 1E and Fig 1F) and PC5 (Fig 1G and Fig 1H) paralleled the time course of PC7 upregulation (Days 2 and 7 not depicted). On the basis of staining of consecutive sections with -SMA, medial and neointimal cells expressing PC immunoreactivity were identified as VSMCs (Fig 1I and Fig 1J). Studied for comparison, the proprotein convertases PC1 and PC2 were not detected in VSMCs (data not shown).
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As noted by
Western Blotting Analysis of Cell Proliferation in Explant of Rat Aorta: Evidence for Upregulation of PCs
To detect cell proliferation in rat aorta cultured for 24 hr in serum-free medium, we performed Western blotting analysis with proliferating cell nuclear antigen (PCNA) using an appropriate mouse monoclonal antibody (Table 1). PCNA is associated with DNA synthesis (
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Immunocytochemical Detection of Proliferating VSMCs in Aorta Explants
Counting of the total cell number in the media revealed no statistically significant difference between intact aorta and explants cultured for 24 hr (Table 2). PCNA immunocytochemistry demonstrated that cell proliferation occurred in clusters in the media of cultured arteries. Quantitative analysis demonstrated a statistically significant increase in PCNA-labeled nuclei density from 1% in intact vessels to
35% after 24 hr of culturing. As demonstrated by stainings on consecutive sections (Fig 3C and Fig 3D), PCNA-labeled cells were further identified as VSMCs by their positive immunoreactivity to the cytosolic
-SMA antigen (
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Immunocytochemical Localization of PCs in Cultured Rat Aorta and Co-localization with PCNA
To address the question of whether the upregulation of furin, PC5, and PC7 demonstrated by immunoblotting parallels the pattern of PCNA distribution on immunocytochemistry, we performed immunocytochemical studies using rabbit antisera to furin, PC5, and PC7 (Table 1). Fig 4 shows that, in intact aorta, the expression of all three PCs was mostly confined to the adventitia, whereas after incubation for 24 hr, the distribution pattern of furin, PC5, and PC7 matched that of PCNA labeling observed. Subsequent co-localization studies demonstrated that all PCs were upregulated in proliferating PCNA-positive VSMCs (Fig 5). Quantitative analysis of furin, PC5, and PC7 revealed no statistical difference from PCNA-labeled nuclei after 24-hr incubation of explants (Table 2).
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PCs mRNA Regulation During VSMC Proliferation
Northern blotting analysis demonstrated the mRNA upregulation of furin, PC5, and PC7 (Fig 1). Furin mRNA of 4 kb and PC7 mRNA of 3.0 and 3.7 kb increased maximally after 24 hr of incubation. In contrast, PC5 mRNA of 3.5 kb (PC5-A) (
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De Novo Synthesis of NGF in Cultured Rat Aorta: Evidence for Release of Growth Factors
To test our hypothesis that the upregulation of PCs in proliferating VSMCs reflects a de novo synthesis of growth factors, we investigated the mRNA and protein regulation of nerve growth factor (NGF) as a model. Among a large number of putative substrates for PCs, NGF is activated by PCs (
Northern blotting analysis demonstrated upregulation of two NGF mRNA isoforms (1.3 and 1.7 kb). The 1.3-kb NGF mRNA isoform was found to increase markedly after incubation for 4 hr and declined somewhat thereafter (Fig 7). A second NGF mRNA isoform with a molecular weight of 1.7 kb displayed much less regulation.
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Attempts were made to co-localize NGF with PCs and/or PCNA by immunocytochemistry. Surprisingly, no NGF immunoreactivity could be produced in aorta explants. The same was true for Western blotting analysis with extracts from control and incubated explants, which were negative as well (Fig 7). Assuming that the absence of NGF in the tissue might result from its release, culture media were subjected to immunoblotting. Our data demonstrate that the upregulation of NGF mRNA on Northern blotting analysis is paralleled by the presence of immunoreactive NGF in explant culture media, because a 28-kD immunoreactive NGF was detected after 4 and 24 hr of incubation (Fig 7B).
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Discussion |
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In this report we demonstrate that the proliferation of VSMCs in vivo and in vitro is accompanied by an upregulation of several proprotein convertases. In vivo, we demonstrate the strong upregulation of furin, PC5, and PC7 in VSMCs after balloon denudation of rodent arteries. The increase in PCs immunoreactivity paralleled the time course of PCNA staining until a neointima was established, whereas later on furin-, PC5-, and PC7-positive cells outnumbered PCNA-positive cells. This finding suggests that PCs might participate in the very early phase of VSMC proliferation and in later stages of vascular remodeling. To define these events more closely, we investigated the regulation of PCs using an organ culture model of intact rat aorta explants, known to have low levels of VSMC cell divisions (
Here, we demonstrate a rapid onset of VSMC proliferation in intact arteries incubated over a brief time period (24 hr) without external trophic support. Proliferating VSMCs were identified by PCNA immunostaining, which has been demonstrated to be equivalent to BrdU and [3H]-thymidine labeling in vascular tissue (
Furthermore, co-localization studies demonstrated that increases in furin, PC5, and PC7 immunoreactivity were mostly localized to PCNA-positive VSMCs after 24-hr incubation. Our results indicate that PCs might play an important role in the proliferation of VSMCs. The upregulation of furin, PC5, and PC7 in PCNA-labeled VSMCs indicates that these enzymes may constitute a novel set of gene markers for proliferating VSMCs. The finding of focal areas of PCNA-labeled VSMCs after incubation in our study supports the notion that subsets of VSMCs might initiate proliferation and subsequent changes without trophic support (
Other groups have reported similar results using serum-free organ culture systems of arteries.
To test our hypothesis that VSMC proliferation involves the upregulation of growth factors, we attempted to compare their expression with that of nerve growth factor (NGF). Among a large number of putative substrates ( (
Although strong upregulation of NGF mRNA is evident in rat aorta cultured for 4 and 24 hr (Fig 7), to our surprise no detectable NGF could be found by immunocytochemistry or immunoblotting in aorta explants. Assuming that the strong upregulation of NGF mRNA found on Northern blotting might be paralleled by its release into the culture medium, we examined proteins released from aorta explants. Immunoblot results revealed a release of NGF into the medium after 4 and 24 hr of incubation.
In conclusion, this study demonstrates that short-term incubation of intact rat arteries induces significant histomorphological changes. One of these is the rapid onset of VSMC proliferation, paralleled by a selective upregulation of proprotein convertases in these cells. The importance of these enzymes is further supported by our in vivo data. The finding that VSMCs that are immunopositive for PCs outnumber VSMCs that are immunopositive for PCNA in the mature neointima during late stages of vascular remodeling in vivo indicates that they might be involved in the processing of extracellular matrix proteins as well (
Although the exact role of PCs in proliferating VSMCs has yet to be determined, these enzymes may well be implicated in the development of vascular pathologies.
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Acknowledgments |
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Supported by a grant from the Medical Research Council of Canada (MT-14466). Dr Philipp Stawowy was a fellow of the Charité, Humboldt-University Berlin and German Heart Institute Berlin.
We thank Mr Andrew Chen for technical skills, Dr James Cromlish for review of the manuscript and Ms Sylvie Emond for secretarial assistance.
Received for publication July 6, 2000; accepted October 12, 2000.
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