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Regular Articles |
From the Department of Pathology,*
Overton Brooks VA
Medical Center, and the Departments of
Pathology,
Cellular Biology and Anatomy and
Medicine,
and Molecular and Cellular
Physiology,§
Louisiana State University Medical
Center, Shreveport, Louisiana
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There has been a view that GA molecules retained in GFVPs may cause calcification. GFVPs are known to calcify in rat subcutis, whereas fresh valves provoke inflammation but do not calcify.7 The amount of GA in bovine pericardium rat implants has also been shown to have a quantitative relationship with calcific deposits.8 However, there have been other reports that question the role of GA in calcification. Fifty percent of GA leaches out of GFVPs in rat subcutis over a period of weeks, and in failed human GFVP xenografts, acid hydrolyzable GA is known to decrease with the graft's duration.9,10 Paradoxically, fixation of porcine aortae in a higher concentration of GA diminishes calcification in rat subcutis.11
The role of the extracellular matrix in calcification has also been extensively studied. Collagen, as in other calcifying tissues, has been implicated as a nucleator of apatite in GFVPs.12 Osteocalcin and osteopontin have been isolated from calcified tissues, including GFVPs, suggesting they may play a role in calcification.7,13 However, a study with isolated matrix demonstrated that osteopontin and osteocalcin did not nucleate apatite in vitro.14 Other noncollagenous proteins, especially phosphoproteins, have been implicated in GFVP calcification.15
In orthotopically grafted GFVPs, calcific deposits have been found selectively in the areas of mechanical stress.16 Insudation of the plasma contents into the stressed areas may have a role in calcification.17 Alternatively, relentless oscillations of the valves may let calcified particles migrate and accumulate in the stressed areas.
Calcification in xenografted GFVPs has been shown to begin intracellularly,18 and several types of cell injury, including GA treatment, result in canine aortic valve (AV) fibroblast calcification.19 Furthermore, removal of cells from GFVPs with detergents and lipid solvents has been shown to prevent calcification.20-22 Nevertheless, the cell's role in GFVP calcification has not been studied in detail. Cell injury has long been known to cause an influx of Ca2+ into the cell.23 Recently, there has been increasing evidence supporting that cell injury also increases cytosolic phosphate ([Pi]i).24-26 Concomitant increases in intracellular Ca2+ ([Ca2+]i) and [Pi]i have been theorized to be the underlying mechanism of calcification.27 In the present study, we have tested the hypothesis that calcification in GFVPs results from GA-induced increases in [Ca2+]i and [Pi]i during the fixation process. Porcine AV fibroblasts were grown in culture and subsequently fixed with GA while measuring cellular and media Ca2+ and Pi changes immediately after fixation and over a period of 7 days. GA was found to cause a massive increase in [Ca2+]i and several-fold increases in [Pi]i followed by calcification of the cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
Porcine aortic valves were obtained from a local slaughterhouse within 30 minutes of sacrifice. Valves for cell culture were transported in ice-chilled RPMI 1640 with 20% fetal bovine serum, 1.25 µg/ml amphotericin B, and 50 µg/ml gentamicin sulfate. The valves were treated with 15% collagenase for 15 minutes, endothelial cells were removed with a scalpel, and the remaining tissue was rinsed twice. The tissue was minced with scalpels, explanted, and subcultured in minimal essential medium supplemented with 10% fetal bovine serum, 100 IU of penicillin, and 100 µg/ml streptomycin under 5% CO2 at 37°C and 90% humidity.
Measurement of [Ca2+]i
Dual-excitation ratiometry of fura-2-loaded cells was performed with a model RM-D Deltascan photometry system (Photon Technology International, Brunswick, NJ). Dual-emission ratiometry of indo-1-loaded cells and single-wavelength fluorescence image analysis of [Ca2+]i were performed with a Bio-Rad MRC-1000 confocal microscope (Bio-Rad Microscience, Herts, UK) with a Diaphot inverted microscope (Nikon, Garden City, NY) with krypton-argon and ultraviolet argon ion laser attachments (Coherent Enterprise, Salt Lake City, UT). LaserSharp 1024 Time Course software (Bio-Rad) and a 40x Fluor oil emulsion objective lens with NA of 1.3 (Nikon) were used.
Semiconfluent cells on 25-mm round coverslips were mounted on an Attofluor cell chamber (Molecular Probes) and were loaded with 5 µmol/L indo-1/AM in phenol-red-free Hanks' balanced salt solution with 25 mmol/L HEPES, and the total calcium was adjusted to 2.0 mmol/L with CaCl2, pH 7.4 (HBSS2.0), for 30 minutes at 37°C. The cells were allowed to de-esterify for an additional 30 minutes. Alternatively, the cells were loaded with fura-2/AM for 20 minutes and de-esterified for 20 minutes at 37°C or loaded with 5 µmol/L fluo-3, 5 µmol/L CaGr-1/AM, or 5 µmol/L OrGr-1/AM for 30 minutes at room temperature and de-esterified for 30 minutes. The de-esterification step was omitted for fluo-3. Ratiometry of indo-1-loaded cells was performed at an excitation wavelength (WLEX) of 351 nm and emission wavelengths (WLEM) of 460 and 405 nm, and with fura-2, at WLEX of 340 and 380 nm and WLEM of 510 nm. Single-wavelength [Ca2+]i measurements were performed at WLEX of 488 nm and WLEM of 531 nm for CaGr-1 and 523 nm for fluo-3 and OrGr-1.
[Ca2+]i in CaGr-1-loaded cells was measured either in HBSS2.0 or in HBSS with the serum level (2.5 mmol/L) of calcium (HBSS2.5). [Ca2+]i in selected samples was measured in Ca2+-free HBSS (HBSS-0) or in HBSS2.0 with 10 mmol/L EGTA to determine possible intracellular redistribution of Ca2+ caused by GA. To convert fluorescence intensity (FI) to nanomoles per liter, [Ca2+]i measurement with CaGr-1 was calibrated using a calibration kit (Bio-Rad, 170-3141) according to the formula [Ca2+]i = Kd[F - Fmin]/[Fmax - F], where Kd is the dissociation constant of CaGr-1, F is the fluorescence of the indicator at the experimental calcium level, and Fmin is the fluorescence in the absence of extracellular Ca2+ ([Ca2+]o); Fmax was determined by treating CaGr-1-loaded cells with 1.66 µmol/L Ca2+ ionophore 4-Br-A23187.28 The LaserSharp software has a built-in calibration program. To simulate GFVP preparation, [Ca2+]i in cells treated with 0.2% to 0.6% GA was measured at room temperature. Optimal loading of the dyes and the topography of GA-induced changes in [Ca2+]i were assessed with fluorescence images. Cells with mitochondrial loading of the dyes and signs of injury, ie, blebs, were excluded from the study. To illustrate the distribution of [Ca2+]i increase in GA-treated cells, 12-band pseudocolors ranging from blue to white were assigned to 0 to 255 gray scales of captured images with the LaserSharp program.
[Pi]i Measurement
A total of 5 x 105 cells suspended by trypsinization in triplicate were fixed with 0.6% GA in HBSS-0 or HBSS2.5 at room temperature, rinsed three times with 25 mmol/L HEPES in 0.9% saline solution, pH 7.4, by centrifugation, and pelleted in microfuge tubes. The pellets were ground with a motorized pestle, dissolved in 0.25 ml of 0.6 mol/L perchloric acid, neutralized with 1.25 ml of 3 mol/L KOH, and centrifuged at 16,000 x g for 5 minutes. The supernatants were stored at -80°C for up to a week. Pi in the supernatant was measured by an ammonium molybdate color reaction at 720 nm by the method of Ohnishi et al.29 The extraction procedures were carried out on ice in a cold room.
Calcification in Vitro
Confluent cells in 25-cm2 flasks in triplicate were treated with 0.6% GA in HBSS2.5 for 24 hours at room temperature. After rinsing away the GA, cells were incubated in 10 ml of HBSS2.5 without GA for 7 days at 37°C. Live cells incubated in minimal essential medium with 2.5 mmol/L total calcium (MEM2.5) in a CO2 incubator or cells fixed with 0.6% GA in HBSS-0 and incubated in HBSS2.5 served as controls. Samples were harvested on days 1, 2, 3, and 7. Free Ca2+ in the supernatant was measured with an ion-selective electrode (Radiometer, Cleveland, OH). Pi was measured by an ammonium molybdate color reaction at 720 nm.13 The cells were subsequently fixed in 10% formalin in phosphate-buffered saline for 3 minutes and stained with 0.03% calcein for 1 minute. Calcein is a fluorochrome that has been used to fluoresce calcium phosphate deposits in mineralizing cell cultures,30 and fluorescence densitometry of calcein-stained samples has been shown to be a suitable technique for quantification of calcific deposits in fibroblasts.19 Fluorescence densitometry was performed on 10 tandem areas, using a 10x objective lens at WLEX of 488 nm and WLEM of 520 nm. To capture detached cells and blebs, the supernatant was filtered through a polycarbonate membrane filter with pore size of 0.2 µm using a syringe. The surface area of the filter was corrected for 25 cm2, and the FI was added to that of attached cells. Polycarbonate membrane is neither autofluorescent nor stainable with calcein.
Electron Microscopy
Cells suspended by trypsinization were fixed with 0.6% GA and incubated in HBSS2.5 in airtight plastic tubes on a rotary drum at 37°C for a week. Cell pellets were embedded in agar, cut into 1-mm3 cubes and fixed in 4% GA in cacodylate buffer, post-fixed in OsO4 in s-collidine buffer, dehydrated in ethanol and propylene oxide, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and examined with a Philips 200 microscope. Selected area electron diffraction was performed at 80 kV. The diffraction pattern was measured according to d = D/K, where d is interplanar spacing, D is diameter of the powder pattern, and K is the camera constant; K is determined by diffraction of ThCl sputtered grids at each diffraction of the sample.31 Crystals were identified with the Inorganic Powder Diffraction Files, Joint Committee on Powder Diffraction Standards (Philadelphia, PA).
Statistical Analysis
Statistical analysis was performed using the SigmaStat software (SPSS, Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When CaGr-1 was used, GA caused a progressive rise in FI for over 1
hour and in a dose-dependent manner. FI of cells treated with 0.6% GA
approached the Fmax obtained from the cells
treated with Ca2+ ionophore (1.66 µmol/L 4-Br-A23187).
Throughout the experiment, the intensity of cell-free background
changed little or none. CaGr-1 FI was converted to
[Ca2+]i using the aforementioned BioRad kit
and fluorescence equation. Photometric data for CaGr-1-loaded cells are
shown in Figure 1
.
[Ca2+]i tended to be higher when measured in
HBSS2.5 than in HBSS2.0. Otherwise, the
[Ca2+]i response to GA was similar in both
solutions (data not shown). Pseudocolored images demonstrated the
increase of [Ca2+]i especially in the
perinuclear area (Figure 2)
.
|
|
. A slight decrease in Ca2+
and an increase in Pi in the supernatant during GA fixation
for over 24 hours was noted.
|
. After an initial decline on day 1,
the slopes of ion deple- tions leveled off for a few days
followed by a precipitous decline in day 7. No ion depletion from the
supernatant of the unfixed live control cells was noted for a week. In
the supernatant of cells fixed in HBSS-0, the precipitous decline did
not occur at day 7 and ion concentrations remained level.
|
. Calcified
cells also tended to detach from the flask surface. A comparison of
media Ca2+ depletion with cellular calcein FI revealed an
inverse linear relationship with a regression coefficient of 0.79
(Figure 6)
. Blebs were scanty to absent
and calcification was not evident in cells fixed in HBSS-0, incubated
in HBSS2.5, and stained with calcein.
|
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. Blebs detached from the cells
frequently contained large amounts of needle-shaped apatite. In heavily
calcified cells, apatite deposited diffusely in the cytoplasm in
apposition to the surface of organelles. In occasional cells, only
nuclei were selectively calcified. Although a limited number of cells
were examined, cells with nuclear calcification did not appear to form
calcifying blebs. Cells fixed in HBSS-0 showed only ruffling of the
plasma membrane resulting from trypsinization but no blebs or calcific
deposits. Electron diffraction of calcified cells yielded the powder
pattern consistent with hydroxyapatite.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our studies, CaGr-1 was used to assess the influx of
Ca2+ into AV fibroblasts during fixation with GA.
[Ca2+]i in resting valvular fibroblasts
ranges between 100 and 150 nmol/L, whereas
[Ca2+]o is approximately 1.5 mmol/L. Thus,
there is a steep electrochemical gradient of Ca2+ across
the plasmalemma. GA fixation led to dose-dependent, progressive
increases in [Ca2+]i over a period of 1 hour
(see Figures 1 and 2
). After an initial immediate increase in
[Ca2+]i, there was a relatively slow and
sustained increase when compared with the transient surges usually
associated with signal transduction. This increase eventually reached a
value comparable to that obtained when unfixed porcine AV fibroblasts
were treated with Ca2+ ionophore 4-Br-A23187 and
subsequently allowed to equilibrate with
[Ca2+]o. These observations suggest that GA
initially permeabilizes the plasmalemma to Ca2+ and allows
leakage into the cell. Intracellular Ca2+ overload exerts
many deleterious effects to the cell and is considered to be the
underlying mechanism of cell death, both apoptosis and
necrosis.23,36
Physiological increases of
[Ca2+]i stimulate mitochondrial ATP
synthesis, but a massive increase in mitochondrial Ca2+
collapses the proton-motive electric potential of the mitochondrial
inner membrane and arrests ATP synthesis.37
In acute cell
injury, mitochondria themselves frequently calcify.
The mechanism of GA fixation is complex and incompletely understood. GA is believed to cross-link proteins mainly by piperidine ring formation via a Schiff base reaction.38 Despite its extensive use for fixation of tissues for electron microscopy, relatively little is known about the detailed effects of GA on cell metabolism. As it diffuses into the cell, GA is likely to inactivate many enzymes. The progressive increase of [Ca2+]i in GA-treated cells suggests that Ca2+-Mg2+ ATPase of the plasma membrane no longer functions. GA has been shown to bind specifically to the ATP-binding sites of sarcoplasmic reticulum Ca2+ pumps and to abolish the pump function.38 A similar inactivation of the plasma membrane Ca2+-Mg2+ ATPase seems likely. The cessation of ATP synthesis by overloaded [Ca2+]i and a generalized devitalization of the cell by GA are likely to contribute further to the abolition of the plasma membrane Ca2+ pump activity. On devitalization of the cell, [Ca2+]i will equilibrate with [Ca2+]o. Inhibition of plasma membrane Ca2+ pumps has been shown to induce calcification of canine AV fibroblasts.19
In contrast to Ca2+, Pi is mainly
intracellular. It exists mostly in organic forms. The
[Pi]i in resting cells is estimated to range
between 0.5 and 2 mmol/L by 31P nuclear magnetic resonance
(NMR).39
NMR tends to underestimate
[Pi]i, detecting only one-third of the values
obtained by chemical analysis.24
[Pi]i is closely linked to hydrolysis of ATP,
and in metabolically active cells, eg, rat skeletal muscle, it can be
as high as 8 mmol/L.40
With the recent advent of NMR, it
became apparent that [Pi]i increases during
cell injury, usually in association with a reversal of the
ATP/Pi ratio.24-26
In our GA-treated AV
fibroblasts, GA led to a several-fold increase in
[Pi]i over a period of hours that remained
elevated for at least 24 hours (see Figure 3
). In addition, the
presence of Ca2+ in GA solution was associated with a
greater increase in the [Pi]i than when GA
solution contained no Ca2+. Overloaded
[Ca2+]i has been shown to activate cell
enzymes, ie, phospholipases and endonucleases.36
The
additional increase of [Pi]i in GA-treated
cells can be attributed to the activation of hydrolytic enzymes for
organic phosphates by Ca2+ and the arrest of ATP synthesis
in Ca2+-overloaded mitochondria. The occurrence of
calcification in mitochondria in GA-treated cells concurs with such
Ca2+ increases in mitochondria. Alkaline phosphatase has
been implicated to have a role in calcification, especially of matrix
vesicles.41
The enzyme may hydrolyze phosphoesters and
thereby contribute to the increase in [Pi]i
in GA-treated cells. The enzyme has been shown to persist in cells of
GFVPs.42-44
The gradual decline of
[Pi]i in a week after a several-fold increase
in [Pi]i in hours in GA-treated cells is
evidently due to its leakage into the supernatant. Pi in
the supernatant increases in association with the decrease in
[Pi]i (results not shown). In our study, the
[Ca2+]i x [Pi]i
products in GA-treated cells exceed by several times that of serum,
reaching values sufficient for heterogeneous nucleation of apatite.
Valvular fibroblasts not treated with GA and maintained under otherwise
similar conditions did not calcify. The occurrence of calcification in
GA-treated cells but not in live control cells under similar conditions
attests that the influx of [Ca2+]o into the
Pi-rich cytosol is most likely the mechanism of
calcification. The absence of calcification in cells fixed in HBSS-0 in
comparison with cells fixed in HBSS2.5 is consistent with a necessary
Ca2+ influx during the early stage of GA fixation,
presumably when the [Ca2+]i ·
[Pi]i products are at the peak, leading to
subsequent calcification of the cells. GA-treated cells apparently
remain permeable to Ca2+ as evidenced by the progressive
depletion of Ca2+ from HBSS2.5 (see Figure 4A
). In addition
to the progressive Ca2+ depletion from HBSS2.5, the
Pi content of the buffer also diminished with time,
suggesting intracellular nucleation of Ca2+ and
Pi was occurring in GA-treated fibroblasts.
After an initial relatively rapid depletion rate, HBSS2.5 ion
depletions lagged for a few days, then progressed rapidly once again.
These observations suggest that the intracellular apatite mass grows to
a critical size and levels off before additional, more rapid extraction
occurs from the buffer. There was also observed a relative delay in
HBSS2.5 Pi depletion, when compared with Ca2+.
This delay was most likely attributable to a higher
[Pi]i than [Ca2+]i
found initially in cells. Cells fixed in Ca2+-free media
did not deplete Pi (see Figure 4B
). Calcein fluorescence
densitometry studies of cells and blebs revealed that the earliest
calcification preceded the aforementioned lag phase and occurred in
blebs and mitochondria. This was followed by intense FI in the
fibroblast soma (see Figure 5B
). HBSS2.5 Ca2+ depletion
significantly and inversely correlated with cellular calcein FI,
further indicating that intracellular nucleation occurred concomitantly
with media ion depletion (see Figure 6
). Interestingly, the ion
depletion lag phase is not observed in other types of cell injury such
as anoxia or repeated freezing and thawing,19
presumably
because membranes are damaged. Membranes are well preserved after GA
treatment (see Figure 7
). The mechanism of Pi entry into
GA-treated cells was not addressed in the present study, but in
functioning cells, Pi is co-transported along with
Na+.39
In this study, HBSS2.5 is tailored for
the short-term experiments of cellular calcification, and the
[Ca2+] was higher, whereas [Pi] was lower
than in serum. This medium, however, allowed the identification of
calcified cells within a week.
We also observed that the nuclei of control valvular fibroblasts exhibited a stronger calcein fluorescence than the cytosol. The nuclear concentration of Ca2+ is higher or lower than the cytosol depending on the type and the physiological status of the cells and is believed to have a role in cell proliferation. The nuclear membrane, which is continuous with the endoplasmic reticulum membrane has its own Ca2+ transport mechanisms, including inward Ca2+ pumps and inositol 1,4,5-trisphosphate receptors.45 The observation of nuclear calcification indicates that nuclear Ca2+ and Pi can reach sufficiently high levels for apatite nucleation. Nuclear calcification has been described in calcification of GFVPs in vivo.18
In addition to nuclear calcification, calcification may be promoted in injured cells through blebbing. Blebbing has long been known to occur in cell injury.46 Blebs have been shown to be associated with Ca2+ overload in injured cells and to contain higher concentrations of Ca2+ than the remaining cytosol.47,48 Cells apparently have a capacity to condense overloaded Ca2+ into blebs. Blebbing may be a cellular defense mechanism to protect against Ca2+ overload. Isolated blebs formed by senescent canine AV fibroblasts have been shown to readily calcify in MEM2.5,19 and calcification in membranous cell debris has been observed in GFVPs removed from human hearts.49 In a variety of human calcinoses, including aging aortic valves, bleb-like membranous vesicles are the predominant foci of calcification regardless of the type of tissues involved.27 It has been proposed that matrix vesicles are blebs formed by apoptotic cells. For example, hypertrophic chondrocytes of the epiphysis, the major producer of matrix vesicles, undergo apoptosis.see 27
Our in vitro data are consistent with the hypothesis that
blebbing may contribute to calcification in GFVPs. Calcein fluorescence
of fibroblasts fixed in HBSS2.5 containing GA frequently revealed
cellular blebbing (Figure 5)
, and the earliest calcific deposits were
observed in these blebs. Electron microscopy showed these deposits were
usually found either in close apposition to the inner surface of the
plasma membrane or in swollen mitochondria. Cells and blebs that became
detached often contained large amounts of needle-shaped crystals that
were confirmed by electron diffraction to be hydroxyapatite (Figure 7)
.
By the 7th day after GA exposure, calcification of entire cells was
observed. In heavily calcified cells, apatite was deposited throughout
the cytoplasm but usually in apposition to organelle surfaces. Blebs
were scanty to absent, and calcification was not evident in cells fixed
in HBSS-0 containing GA.
Thus, our studies of porcine AV fibroblasts suggest that the GA fixation process itself may be an important contributor to the calcification that occurs following after with GFVP. The data suggest that GA fixation leads to an early, sustained influx of Ca2+ into the valvular cell followed by apatite nucleation. In addition to the fixation process, others have provided evidence that tissue injury before GA fixation may affect the subsequent calcification of GFVPs. A delay in GA fixation of bovine pericardium and porcine aorta intensifies calcification of GFVPs in rat subcutis.11,50 Furthermore, anoxic exposure at room temperature before GA fixation resulted in a heavy calcification of GFVPs, whereas the exposure at 4°C significantly reduced calcification in rat subcutis.51 It has been theorized that GA preserves certain structures that promote calcification during the early stage of fixation, thus resulting in delayed calcification.52 The major difficulty of the Ca2+ influx theory is linking GA-induced cell calcification, or any short-term calcification, with the slow progression of calcification of GFVPs that occurs in vivo. Calcification in GFVPs xenografted in human hearts progresses over years4,5 and, in aging human aortic valves, over decades.1
Several explanations can be offered for the delayed calcification in vivo. First, a variety of calcification inhibitors have been identified in tissue fluid.53 Fibroblasts inhibit calcification of bone cells cultured in the same dish.54 Cryopreserved AV allografts with viable cells survive twice as long as GFVPs in human hearts.55 Thus, calcification of GFVPs in vivo appears to be delayed by cellular inhibitors. Second, GFVPs are usually fixed in a Ca2+-free solution. Therefore, GFVP extracellular Ca2+ is likely diluted by the fixative. This could lead to less Ca2+ influx into valvular cells during fixation and delayed calcification. Third, nucleated apatite during GA fixation of GFVPs is likely to be dissolved during the storage in Ca2+-free solutions. It has been shown that when calcified rat aortae were decalcified with EDTA, they calcify more readily than the aorta without previous calcification (unpublished data). Once tissues have been calcified in GA, they appear to retain a template for heterogeneous nucleation of apatite. If such a template exists in GFVPs, it could predispose the valves to delayed calcification after grafting. Fourth, cellular membranes may cause delayed calcification. Liposomes made of phosphatidylserine and red cell ghosts readily calcify in serum, suggesting the phospholipid bilayer may serve as a nidus for heterogeneous nucleation.56 This may be one of the reasons that removal of cells from GFVPs prevents calcification.19-21 In addition, a complex and poorly understood interaction exists between plasma membrane and the extracellular matrix.57 The extracellular matrix may play the role of diffusion barrier for both fixative and ions and thus delay calcification. At the same time, the matrix may predispose valvular grafts to this delayed calcification. For example, collagen is known to stimulate calcification in matrix vesicles isolated from epiphyseal growth plates.58
Along with tetracycline, calcein has been used for the study of new bone deposition. In view of its relative inertness, acetoxymethyl ester of calcein has also been used as a vital dye. Although calcein has a strong affinity for Ca,2+ it also reacts with a variety of divalent metals. However, as opposed to Ca2+ and Mg2+, binding of other metal ions quenches the fluorescence of calcein.59 This property of the dye has allowed the measurement of labile iron pools in certain cells.60 Calcific deposits in GFVPs are usually quantified by atomic absorption spectrophotometry of charred samples. The method is destructive and ignores topography of the deposits. Findings in this study further support that calcein can be useful for monitoring the progress of calcification, especially in cell cultures. Plasma membrane is impermeable to calcein. Fixation of the cells, eg, with formalin, is usually required for staining of cells with calcein. GA-treated cells obviates the need for the fixation. A small amount of calcein (0.15 µmol/L) added to HBSS2.5 allows a continuous monitoring of the progress of calcification in the same cells (results not shown).
In conclusion, cultured porcine AV fibroblasts appear to be a useful model for studies of the Ca2+ influx theory of GFVP calcification. GA treatment of cultured fibroblasts led to increases in [Ca2+]i and [Pi]i and intracellular heterogeneous nucleation of apatite. Early cellular blebbing appeared to play a significant role in the calcification process.
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Acknowledgements |
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Footnotes |
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Supported in part by the American Heart Association, Louisiana Affiliate, grant LA97GS08.
Accepted for publication December 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-carboxyglutamic acid and calcium in explanted bioprosthetic heart valves. J Heart Valve Dis 1994, 3:111-116[Medline]
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) and in HBSS-0
(
). HBSS2.5
Ca2+ leveled off for a few days after an initial drop. A
precipitous decline of Ca2+ occurred with cells previously
fixed in HBSS2.5, whereas Ca2+ remained level with cells
previously fixed in HBSS-0. B: A progressive depletion of
Pi from HBSS2.5 was caused by cells fixed with 0.6% GA in
HBSS2.5. Cells fixed in HBSS-0 and incubated in HBSS2.5 did not deplete
Pi.
