© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Cardiac capillary cells release biologically active nitric oxide at an early stage of in vitro development
aInstitut de Pharmacologie Moléculaire et Cellulaire, CNRS UPR411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
bCNRS ESA8078, Departement de Recherche Médicale, CCML 133 Avenue de la Résistance, 92350 Le Plessis-Robinson, France
* Corresponding author. Tel.: +33-4-9395-7744; fax: +33-4-9395-7708 thuringer{at}ipmc.cnrs.fr
Received 31 January 2000; accepted 15 May 2000
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Abstract |
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Objective: Coronary microvascular endothelial cells (EC) may regulate the myocardial contractile function by releasing cardioactive agents such as nitric oxide (NO). However, understanding of these regulatory mechanisms is complicated by the fact that EC exhibit marked phenotypic changes, such as the loss of endothelial NO synthase (eNOS), when they are placed into culture. Recently, it has been shown that eNOS gene expression is regulated by specific cell–cell interactions with mural cells depending on vascular beds. Since EC and pericytes (PL) are closely associated in capillaries, we have enzymatically isolated these cells from rat hearts to develop a primary culture of capillary cells favoring the re-establishment of cell interactions in vitro. Methods: Expression of transcripts for both eNOS and the inducible isoform (iNOS), was evaluated by using reverse transcription, polymerase chain reaction and Southern blot analysis. Expression of NOS proteins was detected with specific rhodamine-labeled antibodies. Production of NO was assessed (i) from nitrite measurements in culture supernatants by the Griess reaction, and (ii) from its antiproliferative action on cardiac fibroblasts (FIB) in non-contacted cocultures (reporter-cell bioassay) compared to that of sodium nitroprusside in homotypic FIB cultures. Fura-2 fluorescence was used to measure agonist-induced changes in cytosolic free calcium levels. Results: In our heterotypic cultures, EC firstly proliferated to form spots of monolayers (i.e. first phase) before to be covered by PL on the following days (i.e. second phase). The data from RT-PCR analysis demonstrate the presence of mRNAs of both eNOS and iNOS at all developmental stages of the culture. However, eNOS protein was only detected and restricted to EC. During the first phase of cell growth (5–8 days), cells released nitrite and a labile factor, clearly identified as NO, that inhibited the FIB proliferation in reporter-cell bioassay. These effects, not observed during the second phase of cell growth (15–20 days), were prevented by hemoglobin (50 µM) and by N
-nitro-L-arginine methyl ester (L-NAME; 100 µM). At the two periods of culture, EC increased rapidly their cytosolic Ca2+ concentration in response to bradykinin (10 nM). However, this calcium response was associated with an increase in nitrite production only in older cultures. Conclusions: Our data indicate that heterotypic cultures of native capillary cells preserve the eNOS expression by EC. This enzyme is basally active at an early stage of in vitro development, and then becomes activatable by a Ca2+-mobilizing agonist. NO released by growing EC downregulates the proliferation of cardiac FIB, an effect which could be important in the cardiovascular plasticity.
KEYWORDS Angiogenesis; Gene expression; Coronary circulation; Capillaries; Cell culture/isolation; Nitric oxide
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Many reports provide compelling evidence for a direct role of nitric oxide (NO) signaling pathways in regulating the contractile properties of cardiac muscle [1]. NO acts through an alteration in the duration of systole by modulating the onset of ventricular relaxation and rapid refilling of the heart [2]. Although both the endothelial and inducible NO synthase isoforms (eNOS and iNOS, respectively) are found in cardiac muscle, it is generally admitted that eNOS activity in endothelial cells (EC) is the major source of NO that affects subadjacent myocyte function under physiological conditions. In adult rat heart, eNOS is heterogeneously distributed throughout the coronary tree. Higher levels are observed in endothelium of arteries and endocardium as compared to endothelium of veins and capillaries [3].
Endothelial cells in culture have been useful for investigating various aspects of endothelial growth and behavior. In spite of documented similarities between EC in vitro and in vivo, many properties that are lost upon culturing and subsequent dedifferentiation of EC in vitro [4]. For example, unlike large-vessel endothelial cells, confluent homotypic primary cultures of capillary EC exhibit little or no eNOS activity but respond to specific combinations of inflammatory cytokines with increase in iNOS activity [5,6]. In addition, cultured EC do not secrete endothelin-1, although preproendothelin transcripts are detected in EC cocultured with cardiomyocytes [7].
There is an increasing appreciation of the role of cell–cell interactions with mural cells (i.e. cardiac myocytes, smooth muscle cells, pericytes) on regulating EC gene expression [4,7–10]. Recently, environmentally responsive promoter elements which contain information for a specific expression of eNOS have been identified in cardiac capillary EC [11]. In mouse embryonic endothelial progenitor cells, the activity of the eNOS gene promoter is induced by conditioned media from cardiomyocytes, skeletal myocytes and brain astrocytes [11]. Thus, understanding of control mechanisms underlying eNOS expression at the cardiac capillary level will rely on in vitro models that preserve the EC's native microenvironment. Since pericytes are intimately associated with EC in mature capillaries and postcapillary venules [9,12], we have developed a primary culture model of cardiac capillary cells, that comprises both EC and pericyte-like cells (PL). We report here a difference in the expression and activity of eNOS in EC depending on their stage of in vitro development.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The investigation conforms the institutional guidelines (Licence #03387 from the Ministère Français de l’Agriculture).
2.1 Cell isolation and culture
Neonatal fibroblasts (FIB) were isolated as previously described [13]. Cells were plated at a density of 5–10x104 cells/ml in the standard culture medium (M199 containing 10% fetal calf serum (FCS; Bio-Media, France), 200 U/ml streptomycin–penicillin and 100 U/ml kanamycin (Gibco Brl)).
Ventricular myocytes were isolated from adult rat hearts and cultured as previously described [14]. Cells were plated at a density of 9x104 cells/ml in Dulbecco's modified Eagle's medium containing 10% FCS, nonessential amino acids, 1 nM insulin, and antibiotics.
Capillary cells were isolated by collagenase digestion from adult rat hearts [6]. Briefly, excised heart was perfused retrogradely on a Langendorff apparatus for 5 min with Ca2+-free Tyrode solution (in mM: 136 NaCl, 5.4 KCl, 1.1 MgCl2, 5 HEPES, 20 NaHCO3, 0.4 NaH2PO4, 10 glucose; pH: 7.3), for 15 min with Ca2+-free Tyrode solution containing 0.2 mg/ml collagenase (type II, Worthington Corp., NJ), then for 5 min with fresh Ca2+-free Tyrode solution. All perfusates were gassed with 95% O2, 5% CO2 and maintained at 37°C. Left ventricles were briefly immersed in ethanol to devitalize epicardial and endocardial cells. The outer ventricular wall was discarded. The remaining tissue was finely minced and triturated with a pipette in the standard culture medium. The cell mixture was sieved through a nylon filter (80 µm) and plated onto laminin (5 µg/ml)-coated glass coverslips in the standard culture medium.
For coculture experiments, first-passage FIB and freshly isolated capillary cells were plated either on coverslips or on to Transwell inserts (0.4 µm pore-size). Capillary cells were used 5–8 days or 15–20 days post-isolation. Controls consisted of homotypic cultures of FIB or capillary cells. Cells were incubated for 3 days, then fixed or trypsinized to count Trypan blue negative cells.
2.2 Nitrite release in cell-conditioned medium
Culture supernatants were assayed for nitrite, using the Griess reaction [5]. Media were centrifuged at 1500xg for 15 min at 4°C to remove cellular debris, and added to a 1:1 (v/v) Griess reagent (Sigma Chemical Co.). A standard curve was constructed by use of known concentrations of sodium nitrite over the linear range (from 0.003 to 300 µM nitrite).
2.3 Statistical analysis
Results are expressed as means±standard deviation (S.D. mean) from n different experiments. Tests of significance were made by using ANOVA. P values <0.05 were significant.
2.4 Immunofluorescence microscopy
Cells were fixed in 4% formaldehyde then permeabilized with 0.2% Triton X-100 (at 23°C) or with 100% ethanol (at –20°C). Actin microfilaments were stained with 50 µg/ml tetramethyl-rhodamine isothiocyanate (TRITC)-phalloïdin for 40 min at room temperature. For other staining, cells were incubated with the primary antibody for 1 h then with the secondary antibody (F(ab')2 fragment of IgG) conjugated with TRITC, Texas Red or fluorescein (FITC) (Jackson ImmunoResearch Lab.) for another 1-h period. Negative tests were obtained without the primary antibody. The primary antibodies were: polyclonal anti-eNOS (1:100; amino acids 1185–1205) and anti-iNOS (1:100; amino acids 1126–1144), a polyclonal anti-von Willebrand factor (vWF, 1:200; Dako), a monoclonal anti-
smooth muscle actin (1:25), a monoclonal sarcomeric anti--actinin (1:200) and anti-syntaxin-6 (1:500; Transduction Lab.). All chemicals and antibodies were purchased from the Sigma Chemical Co unless otherwise indicated. Specificity of eNOS and iNOS labeling was demonstrated by performing cell labeling with non immune IgG instead of anti-eNOS or anti-iNOS antibodies. Negative controls for eNOS and iNOS labeling were obtained in homotypic cultures of cardiac FIB. Positive control for eNOS and iNOS labeling were obtained in homotypic cultures of cardiomyocytes as well as in capillary cell cultures, activated with IL-1β (5 ng/ml) plus IFN-
(500 U/ml) overnight (in 2% SVF). Confocal observations were performed on a Sarasto-2000 laser confocal (Molecular Dynamics, CA) mounted on a Nikon Optiphot-2 upright microscope. Series of a least 12 optical sections, scanned at 0.49-µm increments, were collected using a standard scanning mode format 512x512 pixels. For each series of optical sections, a look-through projection was calculated.
2.5 Fluorometric measurements
Changes in fura-2 fluorescence were used as a measure of cytosolic free Ca2+ concentration [6]. Briefly, cells grown onto coverslips were incubated with 5 µM of fura-2/AM for 60 min at room temperature after which the incubation medium was replaced with the recording bath solution (Tyrode solution containing 1.8 mM CaCl2). Cells were allowed 30 min to recover. Measurements were done on Leica inverted microscope attached to a dual-excitation spectrofluorometer with excitation wavelengths set at 360 and 380 nm. Emission fluorescence (510 nm band-pass filter) was collected by a photomultiplier tube (model M4314, Hamamatsu) and analysed by using the Argus software.
2.6 RT-PCR analysis of NOS and VEFG receptors
Total RNA was isolated from cells using TRIzol Reagent (Gibco Brl). One µg of RNAs was reverse-transcribed using 200 U of SuperscriptTM II (Gibco Brl). Hot-start PCR for NOS was carried out with 1 U of Taq DNA polymerase (Promega) as following: 2 min denaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C, 2 min 30 s at 72°C. Sense and antisense primers were eNOS: 5'-CCTTCCGGCTGCCACCTGATCCT-3' and 5'-AACATGTGTCCTTGCTCGAGGCA-3'; iNOS: 5'-ATGGCTTGCCCTTGGAAGTTTCTC-3' and 5'-CCTCTGATGGTGCCATCGGGCATCTG-3'. PCR for VEGF receptors was carried out with 5 U of Taq DNA polymerase (Appligene) as following: 3 min denaturation at 94°C, followed by 35 cycles of 50 s at 94°C, 50 s at 52°C, 50 s at 72°C, 7 min at 72°C. Sense and antisense primers were Flk-1: 5'-CAGCGCGAGGTGCAGGAT-3' and 5'-CAGAGGCGATGAATGGTGACC-3'; Flt-1: 5'-TAAACTAGGCAAATCACTCG-3' and 5'-AGGTCGCGATGAATGCACT-3'. Control experiments were performed in the absence of either cDNA or reverse transcriptase.
2.7 Cloning, sequencing and Southern blotting of RT-PCR products
PCR products were separated by 1% agarose gel electrophoresis. The majority of eNOS and iNOS transcripts appeared as 343- and 827-bp cDNA fragments. The eNOS PCR product was purified, subcloned into the SmaI site of a pBluescript SK– plasmid (Stratagene, La Jolla, CA) and sequenced. Prehybridation and hybridation were performed at 65°C according to the instructions of the manufacturer with 32P-labeled iNOS and eNOS PCR fragments.
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3 Results |
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3.1 In vitro development of cardiac capillary cells
Fig. 1 shows the main developmental stages of cardiac capillary cells in vitro. Primary cultures were heterogenous. They consisted of clusters of polygonal endothelial cells (EC) and of irregularly shaped cells, namely pericyte-like cells (PL). For a better visualization, we have stained actin microfilaments of cells with TRITC-phalloïdin.
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On the first days of the culture, few capillary cells were spread onto the coverslips. Clusters of EC tended to grow as colonies while PL extended neurite-like processes to contact surrounding EC. Actin filaments were dispersed throughout the cytosol in isolated EC clusters. When the contact was established between the two cell types, filaments reorganized in distinct dense bands at the EC periphery.
In the following days, EC growth continued to form spots of coherent monolayers covering larger area while sparse PL connected to EC did not proliferate. One week later, EC stopped growing and PL progressively overlayed EC. Thus, two distinct phases of capillary cell growth in vitro can be distinguished: an initial phase of EC growth (during the first week) followed by a late phase of PL growth (during the second week).
3.2 Characterization of cardiac capillary cells at the two stages of in vitro development
EC in clusters (5–8 days) and in ‘cobblestone-like’ monolayers (15–18 days) were stained for von Willebrand's factor (vWF) but not for -smooth muscle actin (Fig. 2a). The vWF labeling was homogeneously distributed within EC in clusters as well as in monolayers partially covered by PL.
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The nature of irregularly shaped cells (PL) could not be clearly defined because of the lack of appropriate cell markers [12,15,16]. It has been suggested that pericytes are generated by in situ differentiation of mesenchymal precursors at the time of endothelial sprouting, and are formed in vitro by migration and dedifferentiation of arterial smooth muscle cells or fibroblasts from the surrounding tissue [12,15,16]. Whatever may be their lineage in our culture, their irregular morphology differentiates them from EC (cobblestones), fibroblasts (long spindle-shaped) or smooth muscle cells (fusiform and compact). Their growth in an ‘umbrella’ of actin filaments covering spots of EC, the lack of ‘hill and valley’ growth pattern (characteristic of confluent smooth muscle cells) and the presence of
-smooth muscle actin (Fig. 2a) allow us to consider that most non-EC cells were pericyte-like (PL).
Moreover, contamination by cardiomyocytes cannot be considered because of the cell isolation procedure used and the absence of staining of the sarcomeric apparatus with sarcomeric -actinin. No growing myocytes was detected in our capillary cell cultures in contrast to those typically observed in primary cultures from adult rat ventricles at the same periods of in vitro development (Fig. 2b).
Expression of the VEGF receptors in capillary cell cultures was evaluated in RT-PCR experiments. Using primers derived from the nucleotide sequence of rat Flk-1 cDNA, we amplified the expected 400-bp cDNA fragment from reverse-transcribed total RNA extracted from capillary cell cultures being 8- or 15-days old. No 588-bp cDNA fragment was amplified with primers derived from rat Flt-1 cDNA (data not shown). Thus, capillary EC expressed vWF and Flk-1 mRNAs.
3.3 Expression, distribution and activity of eNOS in capillary cells
Expressions of eNOS and iNOS were evaluated by RT-PCR and Southern blotting (see Methods). Fig. 3a shows that capillary cells expressed the two species of mRNAs. The eNOS amplication product displayed 99% identity with rat eNOS gene (Genbank database no. AJ 011116). Expressions were observed in 8-day old as well as in 15-day old capillary cell cultures. No detectable eNOS or iNOS mRNA was observed in Northern blot experiments suggesting low amounts of eNOS or iNOS transcripts. Moreover, our culture conditions did not permit us to extract enough proteins to perform a Western blot analysis.
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We next used antibodies to document expression of eNOS or iNOS proteins. No specific iNOS labeling was detected in capillary cell cultures under basal conditions (i.e. in absence of IL-1β (5 ng/ml); data not shown). In contrast, eNOS labeling was clearly observed in EC but not in PL at all stages of the culture (Fig. 3b). Labeling was mainly perinuclear in 5-day old cultures. At 8 days of culture, it extended to the entire cell and included some regions of the cell membrane. At 15 days of culture, larger regions of the plasma membrane and the nuclear area showed immunoreactivity. It is worthy to note that eNOS labeling was observed in absence of mechanical or chemical stimulus. Moreover, identical patterns of labeling were observed in cultured EC with 2 or 10% FCS in media (data not shown).
The eNOS activity was assessed by measuring nitrite contents of conditioned culture media. Fig. 3c shows that capillary cells produced nitrite and that this production was maximum at 8 days of culture. Taken together, these results indicated that EC in capillary cell cultures expressed eNOS. This enzyme is probably active under basal conditions as evidenced from nitrite measurements.
3.4 NO produced by EC is biologically active
We next attempted to define whether NO produced by EC was biologically active by using a reporter-cell bioassay with cardiac fibroblasts (FIB). Fig. 4 shows that NO, exogenously released by sodium nitroprusside (SNP) inhibited the proliferation of cardiac FIB in dose dependent manner. The antiproliferative action of SNP concentrations <300 µM, was not due to cytotoxicity (assessed by Trypan blue exclusion) or apoptosis (assessed by the TUNEL reaction and the absence of specific DNA fragmentation pattern; data not shown).
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The reporter-cell bioassay was devised in the following manner. Capillary cells were first grown for 5–8 days or 12–15 days onto Transwell inserts. Inserts were then transferred to clusters above a layer of FIB. After 3 days, FIB were fixed and stained with TRITC-phalloïdin. Fig. 5 compares typical photomicrographs of FIB cultured alone (left panels) or in the presence of 8-day old capillary cells (right panels). Clearly, the presence of capillary cells limited the growth of FIB and altered their morphology. In the presence of EC, FIB adopted a more asymmetric shape and extended processes in various directions. The distribution of actin filaments that was seen generally in a closely parallel pattern in FIB grown alone, was reorganized in actin bundles tangled into the entire cell. In contrast, when FIB grown onto Transwell inserts were added above the 5-day old culture of capillary cells for 3 days, no inhibition of EC growth was observed. EC were polygonal in shape, clearly defined by the dense peripheral band of actin, and closely packed in colonies.
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The antiproliferative action of EC on FIB was quantitated by cell counts. The initial density of FIB was 105 cells per ml. It increased two fold to reach 2.2x105 cells per ml following 4 days of culture. Fig. 6 shows that the FIB proliferation was slowed down in the presence of 8-day old capillary cell cultures. In contrast, transwell inserts containing 15-day old capillary cells or FIB after 1 day of attachment, did not reduce their proliferation. Taken together, these suggest that, during the initial phase of EC growth, capillary cells produced a diffusible factor that inhibited FIB proliferation.
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Nitrite levels were also determined under these culture conditions (Fig. 6, right scale). They were below the detection limit of the Griess reaction in homotypic cultures and cocultures of FIB. They were 1.47±0.11 and 0.66±0.07 µM (n=6) with 8- and 15-day old capillary cells, respectively.
Contribution of NO released by 8-day old cultures to the FIB-growth inhibition was evaluated by using hemoglobin (HbO2), a NO scavenger, and L-NAME, an inhibitor of endogenous NO production. Fig. 5 (lower right panel) and Fig. 6 show that 50 µM HbO2 or 100 µM L-NAME suppressed the inhibitory effect of capillary cells. The mean cell numbers (x103 per ml±S.D.; n=6) were 209.3±6.4 (FIB alone), 150.7±9.4 (P<0.05; coculture), 209.2±4.5 (coculture plus HbO2) and 215±8.7 (coculture plus L-NAME). Nitrite production was drastically reduced in the presence of L-NAME as compared to 8-day old capillary cells (Fig. 6). Furthermore, media conditioned for 3 days by cultured capillary cells were inactive on growing FIB (209.3±9.4x103 per ml, n=6; Fig. 5 lower left panel) as expected for a labile inhibitor as NO is. Taken together, our results indicate that, at an early stage of development in vitro, capillary EC produce bioactive NO in the absence of physical or hormonal stimulus.
3.5 Agonist-induced responses of capillary cell cultures
To see whether eNOS activity could be stimulated by a Ca2+-mobilizing agonist such as bradykinin (BK), we determined the 360/380 fluorescence ratios in fura 2-loaded capillary EC of 8- and 15-day old cultures (Fig. 7a). No significant difference was observed at the resting fluorescent level between the two groups of EC studied (P<0.001; n=14). The mean resting ratio was 0.15±0.03 (n=14) and 0.16±0.03 (n=14) in young and old cultures, respectively. Thus, the larger basal production of bioactive NO by 8-day old cultures cannot be attributed to a higher resting Ca2+ level as compared to 15-day old cultures. Cell exposure to 10 nM BK produced a biphasic increase in ratios, characterized by an initial rapid rise (peak) followed by a slowly decaying phase (see insets, Fig. 7a). Although the mean peak ratio was smaller in young cultures (0.77±0.09; n=14) than in older (1.06±0.24; n=14), it was statistically different from the resting ratio.
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To determine whether BK induced the eNOS activity, nitrite levels were determined after a 20-h incubation in low serum (2%) culture medium containing 10 nM BK and compared to that produced by the inflammatory cytokine IL-1β (5 ng/ml) via an increase in iNOS activity [5]. As shown in Fig. 7b, a significant increase in nitrite was produced in response to BK by old cultures (to 1.29±0.40 µM; n=4, P<0.01) but not by younger (to 0.49±0.08 µM; n=4 P>0.01). By contrast, IL-1β always induced a significant (P<0.01) increase in nitrite release by cultured capillary cells of both ages. A larger increase was produced by 15-day old cultures (from 0.23±0.02 to 1.34±0.09 µM; n=4) than by 8-day old cultures (from 0.40±0.07 to 0.73±0.04 µM; n=4).
To gain insight into the mechanisms underlying the age-related response of capillary EC to BK, cultures were treated with BK (10 nM) for 10 min, a time period sufficient to induce NO release [17], then double stained with antibodies against eNOS and against syntaxin-6, a 255-amino acid protein localized in the Golgi apparatus [18]. In 5-day old cultures, eNOS and syntaxin-6 were co-localized in the juxtanuclear region in EC, forming a ‘croissant’ shape pattern of yellow immunofluorescence (not shown). Following cell stimulation by BK, no apparent translocation of eNOS could be detected. In 15-day old cultures, eNOS was mainly stained at the plasmic membrane and weakly in the nucleus. It was not observed in the Golgi area stained by syntaxin-6 (upper panels, Fig. 7c). In response to BK, the eNOS labeling appeared in cytosolic granules, different from the Golgi apparatus (lower panels, Fig. 7c). Following a more prolonged incubation with BK (30 min), most of the cytosolic enzyme subsequently translocated back to the cell membrane (not shown).
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4 Discussion |
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This study describes the age-related expression and activity of eNOS in cultured capillary EC isolated from adult rat hearts. In contrast to previous studies [5,6], we did not use a pure population of EC. We conserved the cell heterogeneity obtained from the isolation procedure in order to restore, at least partially, their local microenvironment in vitro. Heterotypic cultures were mainly composed of two cell types: EC (positive for vWF) and PL (expressing
-smooth muscle actin), with the former being progressively covered by the latter.
In this model, we show the presence of low amounts of eNOS and iNOS transcripts in RT-PCR experiments. By using specific antibodies, we further showed that only the eNOS protein was expressed and that its expression was limited to EC. The enzyme was active under basal conditions, as evidenced by nitrite measurements and a reporter-cell bioassay, and could be activated by bradykinin, a known inducer of eNOS activity [17]. These properties are lost in pure cultures of cardiac capillary EC [5,6]. However, the expression of eNOS gene in EC can be specifically induced by conditioned media from cardiomyocytes but not from fibroblasts [11]. Since no contamination by cardiomyocytes was detected in our culture, we believe that PL exerts a similar effect on EC. In this way, Martin et al. [19] have reported that retinal pericyte-conditioned medium down regulates expression of iNOS by capillary EC without affecting eNOS expression. By using a sensitive reporter-cell (FIB) bioassay for NO, we observed that the FIB proliferation was reduced more efficiently by capillary cells than by exogenous NO (Figs. 4 and 6
). A 30% reduction of FIB proliferation was observed with 30 µM SNP and was associated to an accumulation of 10 µM nitrite. A similar inhibition was produced by 8-day old cultures of capillary cells but with 10-fold lower nitrite levels. This suggests that capillary cells produce additional factor(s) for potentiating the antiproliferative action of NO on FIB. Among the possible factors released by capillary cells are transforming growth factor-beta [7], platelet derived growth factor [11] and VEGF [12,20]. Interestingly, VEGF (also produced by pericytes) does not cause proliferation of mural cells in vitro [21] and up-regulates eNOS expression in EC [22] via the stimulation of Flk-1 receptor [23]. Since RT-PCR analysis revealed the presence of Flk-1 mRNA at all developmental stages of our heterotypic culture, this factor may take an active part in the processes described here. Whatever the factor(s) is, the antiproliferative action of growing capillary EC on neighboring cardiac FIB was not produced by the capillary cell-conditioned media. This observation supports the important role of NO in both formation and function of cardiac capillaries which is consistent with observation that long-term blockade of NO synthesis causes coronary microvascular remodeling, cardiac hypertrophy and myocardial fibrosis in rats [24,25] and with the notion that NO limits the extent of interstitial and perivascular fibrosis in adult hearts.
Two distinct phases of capillary cell growth were distinguished: an initial phase of EC growth during the first week, followed by a later phase of PL growth on the following days. Observations are consistent with ultrastructural studies with an in vivo wound healing model showing that newly formed capillaries stop growing when pericytes migrate into the basement membrane [12]. The eNOS activity also changes during these two phases, as attested by the basal nitrite production and the inhibition of FIB growth (Figs. 3 and 6). It increased during the first phase to reach a peak value at the end of EC growth then decreased during the second phase. In the heart, eNOS activity is generally related to increases in cytosolic Ca2+ elicited by fluid shear stress or by activation of G-protein coupled receptors [26,27]. This is also observed in 15-day old cultures (Fig. 7). A nitrite production and a subcellular translocation of eNOS from the cell membrane to cytosolic compartments followed the BK-induced Ca2+ increase, as reported in other EC types [28–31]. Following a 10-min period of cell stimulation by BK, the eNOS immunoreactivity was primarily found in cytosolic vesicles clearly distinct from the juxtanuclear pattern, stained by anti-syntaxin-6 and suggestive of the Golgi apparatus [18]. By contrast, no nitrite production and no detectable translocation of eNOS followed the BK-induced Ca2+ increase in 5–8-day old cultures while they basally produced NO (Fig. 7). Thus, eNOS in the juxtanuclear area (identified as the Golgi apparatus with syntaxin-6) seems to be basally active and unresponsive to BK. There is accumulating evidence from the literature that eNOS activity is controlled by Ca2+-dependent and/or by Ca2+-independent mechanisms leading to a sustained formation of NO, for instance, induced by VEGF [22,31]. The differential NO production may reflect a difference in Ca2+ activation in the juxtanuclear region versus the membrane (caveoli) region. It is also possible that the cytosolic Ca2+ increase measured in Fura-2 experiments does not reach the juxtanuclear region in young cultures (Ca2+ is a non-diffusible cytosolic ion). Whatever it is, our experiments do not allow us to correlate a specific Ca2+ modulation of the enzyme with the stage of cell differentiation.
Heterotypic cultures of cardiac capillary cells reveal important cell–cell interactions between EC and PL. We report here that EC rapidly develop direct contacts with PL that lead to a marked reorganization of their actin cytoskeleton. These preliminary data suggest that cardiac PL are involved in the maintenance of the capillary EC phenotype. A precedent for an important role of pericytes in regulating EC phenotype and stabilizing the microvasculature, is provided in the well-characterized system of retina [12]. Our culture system may be a valuable tool for estimating the contribution of cell–cell contacts in establishing and/or maintaining capillary functions and capillary-mediated diseases in the myocardium.
Time for primary review 29 days.
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Acknowledgements |
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This work was supported by CNRS. We are grateful to Dr E. Benoit for advice in confocal microscopy (CNRS-UPR9040, Gif-sur-Yvette), Dr A. Ladoux and Dr F. Presse for advice in PCR experiments and F. Aguila for photomicrographs.
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