Cardiovascular Research

Cardiovascular Research 1999 42(3):794-804; doi:10.1016/S0008-6363(98)00336-8
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Phenotypic changes in rat and guinea pig coronary microvascular endothelium after culture: loss of nitric oxide synthase activity

Derek Lang^a,*, John P Bell^a, Ulvi Bayraktutan^b, Gary R Small^a, Ajay M Shah^b and Malcolm J Lewis^a

^aDepartment of Pharmacology, Therapeutics and Toxicology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK
^bDepartment of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK

^* Corresponding author. Tel.: +44-1222-744824/742-052; fax: +44-1222-747-484. E-mail address: langd@cf.ac.uk (D. Lang)

Received 16 June 1998; accepted 29 October 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objectives: Coronary microvascular endothelial cells (CMVEs) can modulate the contractile performance of the adjacent myocardium by the release of agents such as nitric oxide (NO). Most previous studies using CMVEs have been done in situ, in the intact organ. We set out to study possible differences in NO synthase (NOS) regulation between freshly isolated and cultured rat and guinea pig CMVEs. Methods: CMVEs were isolated from Wistar rats and Dunkin Hartley guinea pigs and then grown in culture for varying times. Fura-2 fluorescence was used to measure agonist-induced changes in CMVE intracellular calcium levels. Agonist-induced changes in CMVE cGMP levels were measured by commercial radioimmunoassay kit. Western blot analysis was used to measure endothelial, constitutive NOS (ecNOS) and soluble guanylate cyclase (sGC) protein levels. Reverse transcription, polymerase chain reactions and Southern blotting were used to measure ecNOS mRNA transcripts. Results: In both fresh (1 h post-isolation) and cultured (14 days with one passage) CMVEs of the rat and guinea pig, bradykinin (BK) and the calcium ionophore A23187 [GenBank] (both 1 µM) elicited significant (P<0.01) increases in the fura-2 340/380 fluorescence ratio. In cultured CMVEs, basal cGMP levels were unaffected by exposure to BK or A23187. [GenBank] Exposure to sodium nitroprusside (SNP) or atrial natriuretic peptide (ANP) (both 1 µM) induced significant (P<0.01) increases in cGMP in guinea pig cells, whereas in rat cells only ANP produced a significant (P<0.01) response. By contrast, freshly isolated CMVEs of both species had higher basal cGMP levels than cultured cells, and on exposure to BK and A23187 [GenBank] , responded with significant (P<0.01) increases in cGMP. Moreover, exposure of both fresh rat and guinea pig CMVEs to SNP or ANP also resulted in significant (P<0.01) increases in cGMP. Western blot analysis demonstrated that ecNOS and sGC protein were lost from the rat CMVEs following culture. Furthermore, there was also a significant loss of ecNOS mRNA from the rat cells following culture. Conclusions: These data demonstrate that freshly isolated rat and guinea pig CMVEs possess ecNOS activity, and that this activity is downregulated following culture. At least for the rat, this effect would seem to lie at both the transcriptional and translational level. Furthermore, rat CMVEs have reduced activity of sGC following culture.

KEYWORDS Endothelial factors; Nitric oxide; Calcium (cellular); Cell culture; Coronary circulation

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
It is now well established that the vascular endothelium plays a pivotal role in the regulation of blood vessel tone through the release of chemical mediators such as endothelium-derived relaxing factor (EDRF) and endothelin [1, 2]. EDRF, now known to be nitric oxide (NO) [3], has been found in all blood vessels, including arteries, veins and microvessels of all species studied.

Throughout the vascular tree, morphological and physiological differences exist between endothelial cells as a result of biological adaptation to local conditions [4, 5]. For example, microvascular endothelial cells from various sources have been shown to differ from macrovascular endothelial cells in many ways including their response to growth factors [6–8], in their expression of cell surface glycoproteins [9]and in changes in their permeability in response to A₂-adenosine agonists [10]. More recently, it has been demonstrated that cultured rat coronary microvascular endothelial cells (CMVEs) do not express endothelins constitutively [11]and demonstrate no significant constitutive NO release [12]. This is in contrast to cultured porcine and bovine aortic endothelial cells that constitutively express both endothelins and NO synthase (NOS) activity [13–16]. It would appear that this is not just a species difference since microvascular endothelial cells from rat epididymal fat pads have been shown in culture to release NO both basally and in response to agonist stimulation [17]. Clearly then, phenotypic differences exist between microvascular and macrovascular endothelial cells and between microvascular endothelial cells from different vascular beds.

Evidence is now accumulating to show that CMVEs play an important role in modulating the function of adjacent cardiac myocytes by the release of several factors including NO [18]. Most of the work that has been carried out on CMVEs has been done in situ, in the intact organ. Indeed, work from our own laboratory has shown that myocardial contraction is modified by NO released from the coronary endothelial cells of an ejecting guinea pig heart [19–21]. Guinea pig CMVEs are therefore capable of releasing NO in the intact organ, a direct contrast to the cultured rat CMVEs as outlined above. Is this a species difference or an artifact of the culture process? To investigate this question further, we have recently utilised techniques that allow us to isolate and culture these CMVEs with a view to studying their physiological functions in more detail.

The aims of the present study were to investigate whether freshly prepared (1 h post-isolation) and cultured (for various times) rat and guinea pig CMVEs: (1) express endothelial, constitutive NO synthase (ecNOS) protein, (2) have intact receptor-dependent pathways for the release of NO, (3) respond to their own NO with a rise in intracellular cGMP levels.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Isolation of CMVEs
The method used for isolation of CMVEs [22]was basically the same for both rat and guinea pig. Endothelial cells obtained using this method are a mixture of arteriolar, venular, capillary and endocardial cells, but the statistical average is predominantly microvascular [22].

Male Wistar rats (250–350 g) or male Dunkin Hartley guinea pigs (350–450 g) were anesthetized by intraperitoneal injection of pentobarbitone. Their hearts were then excised and immersed in ice-cold, calcium-free Krebs buffer (buffer 1) of the following composition (mM): NaCl 118, KCl 4.7, NaH₂PO₄ 1.2, MgSO₄.7H₂O 1.2, NaHCO₃ 25, glucose 11. The aorta was cannulated rapidly and the hearts perfused retrogradely on a Langendorff system at 37°C with constantly gassed (O₂–CO₂, 95:5) buffer 1.

Once the hearts had been flushed of all blood and cleaned of any remaining connective tissue, the pericardial surface was fixed with 70% (v/v) ethanol. The hearts were then recirculate/perfused for either 30 (rat) or 15 min (guinea pig) with fresh buffer 1 containing 0.04% (w/v) collagenase (Type II, Sigma, UK) and 0.25 µM Ca²⁺.

At the end of this perfusion period the atria were discarded, the remaining left and right ventricles finely minced, and, together with the collagenase perfusate, were then added to 200 mg of bovine serum albumin (BSA, fraction V, Sigma) and gassed (O₂–CO₂, 95:5) for 10 min at 37°C.

This tissue homogenate was then centrifuged at 150 g for 3 min. The resulting pellet of cardiac myocytes was discarded and the supernatant containing the CMVEs decanted into a centrifuge tube containing 100 mg BSA, 0.001% trypsin (w/v, Type I:250 from porcine pancreas, Sigma) and 0.05 mM Ca²⁺. This solution was warmed and gassed as above for 15 min before being centrifuged at 1000 g for 10 min. The cell pellet was then washed in two further identical centrifugation steps with fresh Buffer 1 containing 0.25 and 0.5 mM Ca²⁺ respectively.

The final cell pellet was resuspended in 40 ml of pre-warmed culture medium 1 (CM1) which consisted of Medium 199 (Gibco, UK), pH 7.4, supplemented with benzylpenicillin (250 U/ml), streptomycin (250 µg/ml), amphotericin B (12.5 µg/ml), gentamycin (50 µg/ml), foetal calf serum (FCS, 10% v/v) and newborn calf serum (NBS, 10% v/v). This cell suspension was then added to a 75 cm² tissue culture flask (Primaria, Falcon, UK) and incubated for 1 h at 37°C in an atmosphere of 5% CO₂ in air. After this time non-adherent cells were washed away with 0.9% (w/v) sterile saline and 20 ml of fresh CM1 added to the flask. Some of these freshly isolated cells were used at this stage for the appropriate experiments.

Instead of being added to a culture flask, some cells were plated directly onto 22-mm diameter fibronectin (20 µg/ml)-coated glass coverslips for [Ca²⁺]_i measurement, or onto standard six-well culture plates for measurement of cGMP levels. Non-adherent cells were washed away after 1 h as above, and experiments carried out as described below.

For the growing cells, some were used 12 h post-isolation, whereas the others were again washed after 24 h and fresh CM1 added. On subsequent days, CM1 was replaced with CM2 (essentially as CM1 except for differing concentrations of benzylpenicillin (100 U/ml), streptomycin (100 µg/ml) and amphotericin B (5 µg/ml), and the omission of gentamycin). Further cells were used 3 days post-isolation, whilst others were left to become confluent, usually after 7 days. Some cells were then used at this stage of growth.

On reaching confluence, suitable flasks of both cell types were sub-cultured following trypsin (0.05%, w/v) digestion onto either fibronectin-coated glass coverslips or standard six-well culture plates as above. Fresh culture medium was then added as above, the cells becoming confluent again after 7 days.

2.2 Characterisation of rat and guinea pig CMVEs
The endothelial nature of the confluent monolayers (14 days post-isolation) and freshly prepared (1 h post-isolation) of both guinea pig and rat cells was determined using a number of different methods. These were the selective uptake of fluorescently labelled acetylated-low density lipoprotein (DiI-ac-LDL) [23], the ability to stain with the microvascular endothelium-specific fluorescently labelled lectin, Lycopersicon esculentum (LEA) [24], the ability to rapidly form tubes when grown on Matrigel [23], the inability to stain for -smooth muscle actin (-sma) [22]and growth in D-valine containing culture medium (endothelial cells, but not fibroblasts, are capable of growth in this medium) [25, 26].

2.3 Fura-2 fluorescence
Changes in fura-2 fluorescence were used as a measure of intracellular calcium release essentially as described previously [27]for cells of macrovascular origin. Briefly, glass coverslip-mounted rat and guinea pig CMVEs (fresh and 14-day cultured) were first washed with Krebs solution of the following composition (mM): NaCl 118, KCl 4.7, NaH₂PO₄ 1.2, MgSO₄·7H₂O 1.2, NaHCO₃ 25, glucose 11 and CaCl₂ 1.5, before being incubated with the pentaacetoxymethyl ester form of fura-2 (1 µM) in Hepes (20 mM) buffered Dulbecco’s modified Eagle’s medium (DMEM) containing 1% BSA (fraction IV, Sigma) and 0.1% (v/v) pluronic f-127 for 45 min at room temperature. They were then transferred to a temperature-controlled cell perfusion chamber (Intracel, UK) on the stage of a Nikon Diaphot inverted fluorescence microscope, fresh Krebs solution added and constantly gassed with (O₂–CO₂, 95:5) at 37°C for [Ca²⁺]_i measurement. Fura-2 fluorescence was excited at 340 and 380 nm and fluorescence emission measured at 509 nm using a Cairn (UK) spectrophotometer. Data acquisition, display and analysis were performed using an IBM-compatible personal computer. The 340/380 fluorescence ratio, R, measured at 509 nm was used as the measure of intracellular calcium.

All cells were exposed to maximal concentrations of either bradykinin (BK, 1 µM) or the calcium ionophore A23187 [GenBank] (1 µM) and measurements of R were made just before the addition of the appropriate agent and then at the peak of the response following their addition.

2.4 Measurement of CMVE cGMP levels
For this part of the study, six-well plates containing the appropriate cell type were first washed with fresh Krebs as described above, before being incubated in a further 2 ml of Krebs at 37°C under an atmosphere of 5% CO₂ in air for 1 h. Drugs were then added at the concentrations and times indicated in Section 3. Concentrations and exposure times were based on our previous experience with these drugs in studies using macrovascular endothelial cells [27, 28].

At the appropriate end point the Krebs was aspirated off and the reaction terminated by the addition of 0.5 ml of ice cold 5% (v/v) perchloric acid (PCA). The cells were scraped from the well and the latter washed with a further 0.5 ml of PCA. The combined volume was then centrifuged at 13 000 g for 2 min. The resulting supernatant was assayed for cGMP content and the cell debris pellet assayed for protein content as previously described [28].

2.5 Measurement of ecNOS protein by Western blotting
Freshly isolated rat and guinea pig CMVEs were plated onto 10-cm diameter Petri dishes. Some were used immediately for ecNOS measurement and some were grown in culture for 12 h, 1, 3 and 7 days (at which time they became confluent). Other cells were grown for 14 days (including one passage) before being used. Cellular protein was extracted by first washing the appropriate cells with phosphate buffered saline (PBS) and then treating with 1.5 ml of lysis buffer (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.05 mg/ml digitonin, 0.1% Triton X-100, 10 µg/ml leupeptin and 10 µg/ml pepstatin A). After 5 min on ice the cells were scraped into Eppendorf tubes, vortexed briefly, and then left on ice for a further 5 min. This suspension was then centrifuged (10 g, 5 min) and the resulting supernatant concentrated using a Centriprep 30 concentrator (Amicon, MA, USA). The concentrated supernatant was resuspended in Laemmli sample buffer, boiled for 5 min and then run on a 7% polyacrylamide, denaturing gel.

Proteins were blotted onto a PVDF membrane (0.2 µM pore, Bio-Rad) and non-specific binding blocked by a 1 h incubation at room temperature with 5% Marvel milk powder in Tris-buffered saline (TBS; Tris 10 mM, NaCl 100 mM, pH 7.5) containing 0.1% Tween-20. Membranes were incubated with a 1/1000 dilution of an anti-ecNOS mouse monoclonal antibody (Transduction Laboratories, KY, USA) in TBS Tween with 5% milk powder overnight at 4°C. Following this incubation membranes were washed six times for 5 min each with TBS Tween. Antibody binding was detected by incubation with a second antibody, a 1/4000 dilution of a goat anti-mouse IgG (H&L) horseradish peroxidase-linked species specific antibody (Bio-Rad, USA) in TBS Tween with 5% milk powder for 2 h at room temperature. Following this incubation, membranes were washed six times for 5 min each with TBS Tween. Signals were detected using an ECL Plus detection system (Amersham, UK) and autoradiography films (Eastman Kodak, NY, USA).

2.6 Measurement of soluble guanylate cyclase protein by Western blotting
Soluble guanylate cyclase (sGC) protein levels were measured essentially as above for ecNOS except: the main polyacrylamide, denaturing gel was 10%; TBS Tween buffer was Tris 10 mM, NaCl 500 mM, pH 7.5 and 1% Tween 20; Non-specific blocking was followed by specific blocking using 1/100 dilution of anti-rabbit IgG (H&L)-alkaline phosphatase conjugate (Bio-Rad) in 5% milk powder in TBS Tween for 1 h at room temperature; Primary antibody was a 1/1000 dilution of a polyclonal to the β₁ subunit of sGC (Calbiochem, UK) in 5% milk powder in TBS Tween overnight at 4°C; secondary antibody was a 1/1000 dilution of a goat anti-rabbit IgG (H+L)-horse radish peroxidase conjugate (Bio-Rad) in 5% milk powder in TBS Tween for 2 h at room temperature.

2.7 Reverse transcription and polymerase chain reactions
Rat cells only were used for this part of the study, since polymerase chain reactions (PCR) primers for guinea pig ecNOS were not available. Total cellular RNA was isolated from fresh, and 7 and 14 day cultured rat CMVEs by extraction with guanidinium isothiocyanate as previously described [29]. First strand cDNA synthesis was performed using 10 µg of total RNA, quantified spectrophotometrically at A₂₆₀, in the presence of 500 ng of random hexamers (Promega), 10 mM Tris HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl₂, 33 U of RNasin (Promega). The reaction mixtures were preincubated at 70°C for 3 min followed by cooling on ice prior to the addition of 200 U of Moloney murine leukaemia virus reverse transcriptase (Gibco-BRL). The reverse transcription (RT) reaction was performed at 42°C for 90 min. PCR primers for amplification of rat ecNOS were based on previously published sequences [30]: 5'-GGGCCAGGGTGATGAGCTCTG-3' (sense) and 5'-CCCTCCTGGCTTCCAGTGTCC-3' (antisense). PCR reactions were carried out in 100 µl final volumes containing 2 µl of RT reaction, 48 ng each of sense and antisense primers, 200 nmol of dNTPs, 1.5 mM Mg²⁺ and 2.5 U of Taq polymerase (Promega). Amplification reactions were performed for 35 cycles of denaturation at 94°C (1 min), annealing at 65°C (1 min), extension at 72°C (2 min) followed by an extension reaction at 72°C (10 min). A rat GAPDH cDNA PCR product, as the reference cellular transcript was amplified using the following previously published primers [31]: 5'-GTGAAGGTCGGTGCAAAC-3' (sense) and 5'-CTCCTTGGAGGCCATGT-3' (antisense).

2.8 Southern blotting and quantitation of PCR products
PCR products were separated on 1.2% agarose gels (Bio-Rad) and transferred onto hybond filter (Amersham, UK) by Southern blotting [29]. Filters were prehybridised at 65°C in 6xSSC–1% SDS and 0.5 mg/ml heparin for 2–3 h and then hybridised overnight with either a ³²P dCTP-random-prime-labelled ecNOS cDNA or a rat GAPDH cDNA probe (10 mCi [³²P]dCTP ml^–1 3000 Ci mmol^–1, Amersham, UK) [32]. The filters were washed to a final stringency of 0.1xSSC–0.1% SDS at 65°C and exposed to KODAK X-100 XAT film with intensifying screens at –70°C. Quantification of the autoradiograms was performed by densitometric analyses using an Image Quant Densitometer (Molecular Dynmaics). The densitometric values obtained for ecNOS amplification products were normalised with respect to GAPDH and then values for 7- and 14-day cultured CMVEs converted to a percentage of the levels in freshly isolated CMVEs as previously described for other amplification products [33, 34].

2.9 Drugs and reagents
All drugs and reagents were obtained from Sigma except for fura-2-AM and pluronic f-127 (Calbiochem). All were dissolved in distilled water immediately prior to use except in the case of fura-2-AM (dissolved in dimethyl sulphoxide, Sigma).

Sera, culture medium and trypsin were obtained from Gibco, benzyl penicillin (crystopen) from Glaxo (UK), streptomycin sulphate from Evans Medicals (UK), and fungizone from Bristol-Myers Squibb (UK).

2.10 Statistics
All results are expressed as mean±standard error of the mean (S.E.M.). For comparison of basal fura-2 fluorescence ratios and cGMP data a Student’s two-tailed unpaired t test was used. For analysis of all other within-group data, a one-way analysis of variance was followed by Dunnett’s multiple range test. For analysis of all between-group data, a one-way analysis of variance was followed by Student–Knewman–Keuls multiple range test. All tests identified significant differences at the P<0.05 level.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Characterisation of rat and guinea pig CMVEs
The endothelial nature of the confluent monolayers (14 days post-isolation) and freshly prepared cells (1 h post-isolation) of both rat and guinea pig was determined as described above. CMVEs from both species formed "cobblestone-like" confluent monolayers (Plate Ib) when grown for a number of days, a distinct characteristic of endothelial cells in culture. Both confluent and fresh cells took up the fluorescently labelled Dil-ac-LDL (Plate Ic, confluent cells only), stained positively with the lectin LEA (data not shown), rapidly formed tubes when grown on Matrigel (Plate Ie and f), stained >98% negatively (blue/black) for -sma (Plate Id) and grew normally in D-valine containing culture medium (data not shown).

View larger version (75K): [in this window] [in a new window] [Download PowerPoint slide]   Plate. 1

 
3.2 Fura-2 fluorescence
Very similar baseline fura-2, 340/380 fluorescence ratios were obtained in guinea pig and rat, in both the 14-day cultured [0.21±0.01 and 0.22±0.03 (both n=20), respectively] (Fig. 1a) and fresh CMVEs [0.20±0.02 and 0.13±0.02 (both n=6), respectively] (Fig. 1b). Exposure to BK and A23187 [GenBank] resulted in significant (P<0.01) increases in the baseline ratios in the 14-day cultured (Fig. 1a) and freshly isolated (Fig. 1b) guinea pig and rat cells (all n5).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Graph showing basal (B) and BK and A23187 (both 1 µM)-induced changes in fura-2 340/380 fluorescence ratios (as a measure of intracellular calcium) in (a) 14-day cultured (including one passage) and (b) freshly isolated CMVE from both guinea pig and rat. The BK and A23187 data represent peak responses. **P<0.01 vs. B. Inset graphs show representative fluorescence responses to BK for guinea pig (solid line) and rat (broken line) CMVE. Responses to A23187 are similar, but not shown.

 
3.3 CMVE cGMP levels
In the 14-day cultured guinea pig CMVEs, basal levels of cGMP [0.15±0.06 fmol/µg protein, n=5) were not significantly different from those in the 14-day cultured rat CMVEs (0.16±0.03 fmol/µg protein, n=10]. Exposure of 14-day cultured guinea pig CMVEs to either sodium nitroprusside (SNP) or atrial natriuretic peptide (ANP) (both 1 µM for 2 min) induced significant (P<0.01) increases in cGMP [0.46±0.05 (n=5) and 0.47±0.04 fmol/µg protein (n=4), respectively], whereas in rat cells only ANP (1 µM for 2 min) produced a significant (P<0.01) increase in cGMP (2.24±0.44 fmol/µg protein, n=10) (Fig. 2a).

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 graph showing basal (B) and (a) SNP and ANP (both 1 µM for 2 min)-induced and (b) BK and A23187 (both 1 µM for 90 s)-induced changes in cGMP levels in 14 day cultured (including one passage) CMVE from both guinea pig and rat. **P<0.01 vs. B.

 
Exposure to BK and A23187 [GenBank] (both 1 µM for 90 s) had no effect on the basal cGMP levels in the 14-day cultured CMVEs of either species (Fig. 2b), even following pre-incubation of these cells with the cGMP phosphodiesterase inhibitor zaprinast (1 µM for 1 h).

Cultured (14 day) guinea pig and rat CMVEs were also pre-incubated with either the substrate for NO synthesis, L-arginine (1 mM for 5 h) or the NOS co-factor, tetrahydrobiopterin (BH₄, 1 µM for 5 h) alone, or with L-arginine and BH₄ together before exposure to BK and A23187 [GenBank] as above. Again, no increases in cGMP in response to BK or A23187 [GenBank] were detected in the CMVEs of either species (Fig. 3).

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Graph showing the effect of BK and A23187 (both 1 µM for 90 s) on cGMP levels in 14-day cultured (including one passage) CMVE from both (a) guinea pig and (b) rat in the absence of another intervention (B) and in the presence of either L-arginine alone (L-arg, 1 mM for 5 h), tetrahydrobiopterin alone (BH4, 1 µM for 5 h) or L-arg and BH4 together.

 
Basal levels of cGMP were significantly (P<0.05) higher in fresh guinea pig CMVEs (0.82±0.07 fmol/µg protein, n=7) than in fresh rat CMVEs (0.39±0.03 fmol/µg protein, n=7) (Fig. 4a). Exposure of both fresh guinea pig and rat CMVEs to SNP or ANP (both 1 µM for 2 min) resulted in significant (P<0.01) increases in cGMP (4.52±0.54 and 5.79±0.94 fmol/µg protein, respectively, (both n=6) for the guinea pig and 1.34±0.24 and 7.16±0.86 fmol/µg protein, respectively, (both n=6) for the rat) (Fig. 4a).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Graph showing basal (B) and (a) SNP and ANP (both 1 µM for 2 min)-induced and (b) BK and A23187 (both 1 µM for 90 s)-induced changes in cGMP levels in freshly isolated CMVE from both guinea pig and rat. BK and A23187 exposures were repeated in the presence of the ecNOS inhibitor L-nitroarginine benzyl ester (L-NABE, 5 µM for 20 min) **P<0.01 vs. B, +P<0.05 vs. Guinea pig, XP<0.05 vs. BK or A23187 in the absence of L-NABE.

 
Exposure to BK and A23187 [GenBank] (both, 1 µM for 90 s) resulted in significant (P<0.01) increases in cGMP levels in fresh CMVEs of both species (1.63±0.13 and 2.57±0.25 fmol/µg protein (both n=7), respectively, in the guinea pig and 0.72±0.03 and 0.85±0.06 fmol/µg protein (both n=7), respectively in the rat). These increases in cGMP in response to BK and A23187 [GenBank] were completely inhibited by pre-incubation of both cell types with the NOS inhibitor L-nitroarginine benzyl ester (L-NABE, 5 µM for 20 min) (Fig. 4b).

3.4 ecNOS protein activity by Western blotting
Lysates prepared from guinea pig cells, freshly isolated or cultured (all time points), failed to cross-react with any of the commercially available antibodies for ecNOS (data not shown).

Fig. 5a shows that ecNOS protein, present in abundance in the freshly isolated rat CMVEs, is completely absent in the cultured cells at all time points studied, even after only 12 h.

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Blot analysis with (a) anti-ecNOS antibody (100 µg cell lysate/lane) showing ecNOS and (b) anti-sGC antibody (100 µg cell lysate/lane) showing sGC in freshly isolated rat CMVE and the effect of cell culture thereon after 12 h and 1, 3, 7 and 14 (the latter including one passage). Both experiments were performed three times with similar results.

 
3.5 sGC protein by Western blotting
Lysates prepared from guinea pig cells, freshly isolated or cultured (all time points), failed to cross-react with the antibody for sGC used in this study (data not shown).

Fig. 5b shows that sGC protein, present in abundance in the freshly isolated rat CMVEss, gradually disappears with time in culture.

3.6 Southern blotting of RTPCR products
RTPCR Southern blotting analysis revealed a marked decrease in the mRNA for ecNOS after both 7 and 14 (including one passage) days in culture compared to freshly isolated cells (Fig. 6).

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Blot analysis of ecNOS (a, top panel) and GAPDH mRNA transcripts (a, bottom panel) showing the effect of cell culture for 0 (fresh, lane 1), and 7 (lane 2) and 14 (including one passage, lane 3) days on rat CMVE. The densitometric values obtained for ecNOS amplification products were normalised with respect to GAPDH and values for 7- and 14-day cultured CMVE converted to a percentage of those in freshly isolated CMVE. Plate IPhotographs (rat cells only) obtained using a Nikon Diaphot inverted phase contrast/fluorescence microscope (all using x40 objective). Plates show freshly isolated (a) and confluent (b) CMVE, positive staining for fluorescently labelled Dil-ac-LDL (c, confluent CMVE only), negative staining (blue/black) for -sma (d, fresh cells only) and tube formation on Matrigel 15 min (e) and 3 h (f) after initial seeding.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Most previous studies involving CMVEs have been done in situ, in the intact organ. The main aim of the present study was to isolate and culture CMVEs from the rat and the guinea pig to study their physiological functions, in particular NO production, in more detail. The endothelial nature of the confluent monolayers (14 days post-isolation) and freshly prepared (1 h post-isolation) guinea pig and rat cells was confirmed using a number of different methods. These cells were positive for the selective uptake of fluorescently labelled DiI-ac-LDL [23], their ability to stain with the microvascular endothelium-specific lectin LEA [24]and their ability to quickly form tubes when grown on Matrigel [23]. They were also negative (>98%) for the ability to stain for -sma [22]and grew normally in D-valine containing culture medium [25, 26].

It is becoming increasingly evident that CMVEs play a major role in the control of cardiac function. Work in our own laboratory has already shown that the CMVEs of the ejecting guinea pig heart release NO in situ, and that this CMVE-derived NO has significant effects on myocardial contraction [19–21]. The data presented in the present study show that these cells still produce NO when freshly isolated from the heart, but lose this ability when grown in culture. Other workers have reported that cultured rat CMVEs demonstrate no significant constitutive NO release [12]. This would seem to be an artifact of the culture process, since our data show that freshly isolated rat CMVEs are indeed capable of releasing NO.

It is well known that endothelial cells can lose the ability to express some cell surface receptors, or at least lose the activity of these receptors with time in culture. The failure of the cultured cells in this study to release NO in response to BK is clearly not due to the loss of BK receptors, since the same cells are capable of responding to this agonist with an increase in intracellular calcium. Furthermore, these cells also respond to the calcium ionophore A23187 [GenBank] with a significant increase in intracellular calcium. It is well known that the rise in intracellular calcium precedes the release of NO [35], with the resulting increase in NO release outlasting the calcium transient [35]. Our data (see inset Fig. 1) demonstrate that the rise in intracellular calcium in both cultured and fresh CMVEs is indeed transient. These observations would therefore seem to suggest that the ecNOS, if present in these cultured CMVEs, is not activated by the BK/A23187-induced increase in intracellular calcium.

The question remains as to whether these cultured CMVEs actually express ecNOS or, if expressed, whether this ecNOS is inactive. A lack of ecNOS substrate or suitable co-factors may account for the apparent inactivity of the enzyme. The BK/A23187/cGMP experiments included in this study were repeated in the presence of excess (1 mM) L-arginine, but this had no effect on the cGMP levels described earlier. The absence of NO release is therefore unlikely to be due to a lack of ecNOS substrate, though a dysfunction in the L-arginine transport mechanism in the cultured cells cannot be ruled out. However, the evidence provided by the molecular studies below does not support this latter hypothesis.

The supplementation of CMVE culture medium with the ecNOS co-factor BH₄ [36]also failed to have any effect on the cGMP responses to BK or A23187. [GenBank] This observation would suggest that a lack of this co-factor at least, is not responsible for the ecNOS inactivity.

Measurement of the cGMP levels of CMVEs is only an index of the ability of these cells to produce NO. Western blotting analysis however, is a more direct method for assessing the ecNOS levels in these cells, albeit semi-quantitatively. Our data clearly show that in the freshly isolated cells of the rat, ecNOS protein is indeed present in significant amounts. However, after only 12 h in culture, there is a significant reduction in these protein levels in the rat cells, to the extent that they are almost undetectable. We are unable to say whether this is also the case for the guinea pig, since all the commercially available antibodies for ecNOS failed to cross-react with the guinea pig samples.

The data from the RTPCR analysis demonstrates, at least for the rat, that the absence of ecNOS protein in the cultured cells is accompanied by a lack of ecNOS mRNA. The fact that there is some residual ecNOS mRNA in the 14-day- (including one passage) old cells suggests that the fault in ecNOS protein production may lie at both the transcriptional and translational level. The results from the cGMP experiments suggest that the same is also true for the guinea pig CMVEs, though we cannot say for certain from the data described here. The inability of the cultured guinea pig and rat CMVEs to respond to BK or A23187 [GenBank] with an increase in cGMP would therefore seem to be due to a lack of ecNOS activity.

In previous studies in our laboratory we have used Western blot analysis to measure ecNOS protein levels in cultured porcine aortic endothelial cells. These cells were grown under exactly the same conditions as the CMVEs in the present study, but retained the ability to express ecNOS (unpublished observations). It would therefore seem that this culture-induced phenotypic change is particular to the CMVEs, highlighting the difference between these endothelial cells and those of macrovascular origin.

The data presented in this study also demonstrate a significant loss of sGC protein from the rat CMVEs with time in culture. This would explain the lack of a cGMP response to SNP in these cells. The loss of sGC protein is not as dramatic as the loss of ecNOS protein, but there is a possible link between these two events. Since there would be no NO produced as the cells grow in culture, the stimulus for sGC is lost. Does this then lead to a gradual down regulation of sGC activity? If the latter is true, then it would seem peculiar to the rat cells, since the cultured guinea pig CMVEs were still capable of responding to SNP with a rise in cGMP levels. Though we have no direct evidence, it would seem that the guinea pig CMVEs do not lose sGC activity with time in culture, at least not to the extent seen in the rat. These observations suggest that not only are there phenotypic differences between endothelial cells of different vascular beds, but also between species for endothelial cells of the same vascular bed.

As indicated above, the cause of the loss of ecNOS following culture would seem to lie at both the transcriptional and translational level. However, it is not yet known what could cause this culture-induced defect. Endothelial cells in vivo are continuously exposed to certain stimuli, the removal of which may lead to a change in phenotype. It has been demonstrated that endothelial cells grown under conditions of dynamic flow display much greater differentiation compared to those cells grown in more conventional static cultures [37, 38]. This differentiation is characterised by cytoskeletal reorganisation and increased expression of typical endothelial cell markers such as Weibel Palade bodies [37]. Furthermore, it has also been demonstrated that ecNOS expression can be modulated by shear stress [39–42]. These latter studies demonstrated that the exposure of cultured endothelial cells to various levels of continuous shear stress for 3–24 h markedly elevated the ecNOS mRNA content of these cells. It is therefore possible that removal of flow and shear stress may contribute to the de-differentiation of the CMVEs used in the present study as indicated by a decline in ecNOS levels.

This effect caused by the removal of shear stress may not be universal amongst microvascular endothelial cells. It has been demonstrated that conventional two-dimensional cultures of brain microvascular endothelial cells are still capable of releasing significant amounts of NO [43]. Throughout the vascular tree, morphological and physiological differences exist between endothelial cells as a result of biological adaptation to local conditions [4, 5]. It is possible that these local conditions control the phenotype of endothelial cells also, and that the observed differences between brain and coronary microvascular endothelial cells reflect the differing natural environments in which these cells exist.

The growth of endothelial cells in isolation in culture is obviously a very artificial environment compared to that experienced by these cells in situ, in the intact blood vessels. Cell–cell contact/signaling between endothelial cells and other cell types such as cardiac myocytes is a major, and important, difference between these two situations. It is well known that substances produced by one cell type can have substantial effects on the functioning of the recipient cell type. It has been reported [11]that the production of endothelin precursor transcripts by cardiac microvascular endothelial cells could not be detected unless the latter were co-cultured with cardiac myocytes. Cell–cell contact, or at least close apposition would seem to be necessary, since exposure of cardiac microvascular endothelial cells to ventricular myocyte-conditioned medium failed to increase proendothelin mRNA in the endothelial cells. It is therefore possible that the removal of such cell–cell contact may have contributed to the loss of ecNOS activity in the CMVEs in the present study. The effect on proendothelin mRNA mentioned above was thought to be mediated by transforming growth factor β [11]. It may be that the CMVEs in the present study require this or other growth factors, alone or in combination, to express ecNOS.

The data presented in this study demonstrate that both guinea pig and rat CMVEs lose their ecNOS activity when grown in culture whilst retaining the ability to respond to receptor activation. The cause of this loss appears to lie at both the transcriptional and translational level since, at least for the rat, low levels of ecNOS mRNA transcripts are accompanied by the absence of ecNOS protein following culture. Furthermore, the rat CMVEs lose their sGC protein with time in culture. The exact mechanism(s) of these culture-induced artifacts is unknown, but may involve the absence of shear stress and cell–cell interactions present in the in vivo situation. The findings of the present study indicate that in culturing endothelial cells, great care must be taken when extrapolating from the in vitro to the in vivo situation, particularly with reference to the CMVEs.

Time for primary review 30 days.

	Acknowledgements

 
This work was supported by the UK Medical Research Council and the British Heart Foundation.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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