Correlated Endothelial Caveolin Overexpression and Increased Transcytosis in Experimental Diabetes
Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montreál, Quebec, Canada
Correspondence to: Lucian Ghitescu, Département de Pathologie et Biologie Cellulaire, Université de Montréal, CP6128 Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada. E-mail: ghitescd{at}patho.umontreal.ca
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Summary |
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Key Words: endothelium lung caveolae caveolin transcytosis diabetes
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Introduction |
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The morbidity and mortality caused by diabetes mellitus are mainly related to vascular complications (Turner 1998). Whereas at the level of large vessels the disease appears to be expressed as an acceleration of an atherosclerotic process (LopezVirella 2001
), the microangiopathy shows a more diverse picture, with specific facets for each affected organ (Libby 1997
; Naruse and King 2001
). Because of their dominant clinical incidence and pattern, the diabetes-induced alterations in the microvasculature of three vascular beds (retina, renal glomerulus, and nerves) have been the focus of a vast number of studies. From these we have learned that the direction in which the diabetic microangiopathy develops and the sequence of events vary among these vascular beds but that several elements, such as an increase in capillary permeability and the thickness of the endothelial basement membrane, are common to all three (Marre 1997
). A continuously increasing number of investigations show that, even in the absence of a significant clinical impact, diabetes does affect the microvasculature of other organs, such as the mesentery (Arshi et al. 2000
), the skin (Oomen et al. 1999
), the skeletal muscle (Beals et al. 1993
), and the brain (Bouchard et al. 2002
). In these vascular beds, changes in vascular permeability appear to represent a hallmark of the diabetic endothelial dysfunction.
The mechanism by which such alterations take place is not precisely known. Our study aims to investigate this aspect by correlating the modifications induced by diabetes in the transendothelial transport of macromolecules to the alterations of the subcellular endothelial vesicular compartment as revealed by the biochemical marker for the latter, i.e., caveolin. This was done according to the following experimental strategy. The amount of caveolin 1 present in the alveolar endothelium of normal and diabetic rats was comparatively assessed by immunoblotting performed on the purified luminal aspect of lung endothelial plasma membranes. The presence of the same protein was assessed independently by quantitative immunocytochemistry performed either before embedding on purified membranes or after embedding on the entire endothelial cells. The correlation between the expression of caveolin 1 and the population of plasmalemmal vesicles was investigated morphometrically by counting the number of endothelial caveolae and measuring the partial cell volume occupied by them. The functional implication of the recorded variation in the numerical density of the plasmalemmal vesicles was further tested in relation to the endothelial permeability to macromolecules. This was achieved by comparatively measuring, in diabetic and normoglycemic animals, the in situ distribution of either endogenous albumin or its exogenous variant, a hapten-labeled bovine serum albumin, between the vascular lumen and the interstitium.
The results presented below show that, in the early stages of experimentally induced diabetes, the permeability of the lung microvascular bed to albumin increases. This modification is due to a significant hypertrophy of the plasmalemmal vesicle compartment of the alveolar endothelium, accompanied by a net overexpression of caveolin 1.
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Materials and Methods |
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Purification of the Lung Endothelial Plasma Membrane
The luminal aspect of the endothelial plasma membrane was isolated from the microvascular pulmonary bed of normal and diabetic rats by the cationic colloidal silica method (Jacobson et al. 1992; Ghitescu et al. 1997
). This procedure involves perfusion of the lung microvasculature with a suspension of cationic silica nanoparticles, which adhere to the accessible surface (blood front only) of the endothelium. The high density imparted by the colloidal silica to the labeled plasmalemma facilitates purification of the latter by simple centrifugation. The purity of the isolated membrane fractions, operationally labeled P2, was assessed by electron microscopy as previously described (Ghitescu et al. 1997
). Each preparation was derived from a single animal and was treated as an independent sample.
Immunochemical Evaluation of Caveolin in Purified Lung Endothelial Plasmalemma
To make a precise appraisal of the protein concentration, the measurements were made in quadruplicate by the Amido black method (Schaffner and Weissmann 1973) in the supernatants of the plasma membrane fractions solubilized in 1% SDS, sonicated, boiled, and microfuged to remove the interfering silica beads. Equal amounts (5 µg) of P2 fractions from diabetic and normal rats were loaded in Laemmli buffer on 10% acrylamide Minigels (BioRad; Mississauga, Ontario, Canada), resolved by electrophoresis, and electro-transferred to nitrocellulose membranes. The membranes were probed overnight with 1:4000 anti-caveolin 1
rabbit polyclonal antibody (Santa Cruz Biotechnology; Santa Cruz, CA), followed for 1 hr by a 1:5000 dilution of anti-rabbit IgHRP (Jackson ImmunoResearch; West Grove, PA). The quencher, washing solutions, and enhanced chemiluminescence (ECL) substrate, as well as the detailed immunoblotting protocol, were those of the Roche Diagnostics BM chemiluminescence blotting substrate kit. The ECL signal was recorded at various exposures on X-ray films and the intensity of the individual bands was measured by densitometry in a BioRad GS800 calibrated densitometer.
Similar detections were performed for two other plasma membrane-associated proteins by using antibodies to a C-terminal epitope common to all three isoforms of actin (product A2066; Sigma Aldrich, St Louis, MO) and to annexin II (clone C-16; Santa Cruz Biotechnology), respectively.
Pre-embedding immunocytochemistry for caveolin was performed on lung endothelial plasma membranes (P2 fractions) from three diabetic and three healthy rats. Freshly purified membranes were lightly fixed for 1 hr in 1% paraformaldehyde in 0.1 M phosphate buffer (PB), then washed and quenched in PB containing 0.1 M glycine. Given the high density of the silica-decorated membranes, low-speed centrifugation (400 x g/min) sufficed to separate them from the different fluids in which they were sequentially dispersed. The membranes were incubated overnight at 4C in a 1:100 dilution of anti-caveolin antibody and washed three times in PB, followed by 1-hr incubation in the cold in 10 nm protein Agold complexes at a concentration corresponding to A520 = 0.5. After several cycles of resuspension in PB and sedimentation at low speed to remove unattached protein Agold conjugates, the membranes were tightly packed by microcentrifugation at 12,000 x g for 10 min, fixed again overnight at 4C in a mixture of 2.5% paraformaldehyde and 1.5% glutaraldehyde, postfixed in 1% OsO4, dehydrated, and embedded in Epon. In this experiment and in all ICC approaches further described, the specificity of the labeling was tested in control samples that were not exposed to the primary antibody but only to the protein Agold conjugate. Three ultrathin sections for each animal, normal or diabetic, provided the 119 images (x31,000 final magnification) used for counting the number of gold particles decorating the unit length of the plasma membrane cytoplasmic face. The morphometry was performed on a Zeiss Videoplan 2 Image Analysis System (Carl Zeiss Canada; Don Mills, Ontario, Canada). The statistical significance of the difference found between the two lots was validated by the unpaired Student's t-test here, as well as in all other subsequent assays.
Postembedding ICC Detection of Caveolin
Six animals (three normal and three diabetic) served as donors for lung tissue samples. Under deep anesthesia, the thorax was opened and the lung was fixed in situ for 10 min with warm 4% paraformaldehyde. Small blocks of tissue were harvested from the same anatomic site (periphery of the lower left lobe), and further fixed for another hour at 4C. The tissue was rinsed in PB and embedded at low temperature in Lowicryl K4M as previously described (Bendayan 1984). For each animal, three ultrathin sections, each from a different block, mounted on Parlodion-coated nickel grids were sequentially incubated in 1% ovalbumin in PBS for 10 min, a 1:100 dilution of anti-caveolin 1
antibody (2 hr) followed by 10 nm protein Agold conjugated (30 min) with extensive PBS washing in between. The intensity of the labeling was measured over the entire profile of endothelial cell cytoplasm and was expressed as number of gold particles per unit area. The alveolar endothelium of the rodent lung exhibits zones of extremely attenuated cell body (down to 0.1 µm) that do not contain any vesicles or organelles (Simionescu and Simionescu 1983
). These "avesicular zones," as well as those containing the nuclei, were excluded from the morphometrical evaluation.
Morphometry of the caveolar compartment of the alveolar endothelial cells was also performed on three diabetic and three age-matched normoglycemic rats. The lung tissue was prepared as described above, except that, to improve the visibility of the vesicular compartment, a mixture of 1.5% glutaraldehyde and 2.5% paraformaldehyde was used for the primary fixation and a postfixation step in 1% OsO4 was added. The lung specimens were embedded in Epon and three ultrathin sections from different blocks were examined for each animal. The numbers of plasmalemmal vesicles (caveolae) per unit surface of endothelial cell profile, as well as the total area of these vesicles, were measured by planimetry for each of the 188 images (x55,000 final magnification) taken into consideration. The first parameter represents a measure of the numerical density of the endothelial plasmalemmal vesicles. For each micrograph, the ratio between the aggregated surface of sectioned caveolae and the corresponding endothelial profile was calculated. This parameter gives an estimate of the partial volume occupied by the caveolae in endothelial cells.
Transendothelial Distribution of Endogenous Albumin
The same mildly fixed, low-temperature Lowicryl K4M-embedded lung tissue blocks, previously used for the postembedding ICC detection of caveolin, also served for the experiment in which the steady-state distribution of the endogenous albumin across the endothelium was monitored with an anti-rat albumin antibody and protein Agold. The ultrathin sections, mounted on grids, were sequentially incubated (30 min, room temperature) in PBS containing 150 mM glycine and 1% fish gelatin, a 1:100 dilution of anti-rat serum albumin antibody (Cappel; West Chester, PA) in PBS (overnight at 4C), followed by 10-nm protein Agold conjugates (30 min, RT) with thorough washing in PBS in between. The intensity of labeling (number of gold particles per unit surface) was counted by planimetry (Ghitescu and Bendayan 1992) over the endothelial cell and basement membrane profiles present in the 215 images taken at x38,000 final magnification. The specificity of the approach was assessed by omitting the anti-albumin antibody in the labeling protocol and incubating the grids in a suspension of protein Agold only.
Evaluation of the Transcytosis of Dinitrophenylated Albumin (DNPBSA)
An exogenous tracer for transcytosis, bovine serum albumin (BSA), was tagged with dinitrophenol (Ghitescu and Bendayan 1992); 150 mg of this marker in 1.5 ml PBS at 37C was injected into the bloodstream of three diabetic and three normal rats. Before the injection, a volume of blood identical to that of the tracer was withdrawn from the jugular vein. After a delay of 30 min, the animals were sacrificed by cervical dislocation and a warm solution of 1.5% glutaraldehyde, 2.5% paraformaldehyde in 0.1 M PB was injected into the thoracic cavity to rapidly fix the lung in situ. After 5 min the lung was harvested, and small pieces from the periphery of the lower left lobe were further fixed overnight in the same mixture of aldehydes. Postfixation in 1% OsO4, dehydration in graded ethanol, and embedding in Epon followed. For each animal, three grids were prepared and immunolabeled with anti-DNP antibody followed by protein Agold, as described elsewhere (Ghitescu and Bendayan 1992
). The intensity of labeling for the exogenous tracer was measured over the capillary lumen, within the endothelial cell contour, and over the endothelial basement membrane, and was expressed as number of gold particles per surface unit. The "porosity" of the endothelial basement membrane was evaluated according to an established test (Bendayan et al. 1986
), in which the position of each gold particle detected over this structure was defined by the ratio between the distance separating the particle from the abluminal front of the endothelium and the whole thickness of the basal lamina measured at that location.
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Results |
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To strengthen these data, a pre-embedding quantitative ICC detection of caveolin was performed on endothelial membranes purified from healthy and diabetic rats. Only the exocytoplasmic aspect of these membranes carries the silica particles. This distribution leaves the cytoplasmic face freely accessible to all elements used for immunodetection, in this case anti-caveolin 1 antibody and protein Agold conjugates. The vast majority of the gold particles, reporting the presence of caveolin, were observed as clusters around the caveolae associated with the membrane sheets (Figure 3) . Membranes that, as a control for the specificity of the immunolabeling, were incubated in protein Agold only were virtually devoid of gold particles (0.17 ± 0.06 particles/µm; n=26; 101.5 µm examined membrane length). The density of the labeling for caveolin sharply differed between the experimental lots (Table 2). On average, 1.73-fold more gold particles were found to decorate the cytoplasmic face of the membranes derived from the diabetic animals than those of normoglycemic rats.
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Discussion |
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The morphometrical evaluation of the presence of endogenous albumin in the endothelial cells and in their basement membrane suggests that, at equilibrium, approximately 1.6-fold more albumin is found in the abluminal compartment of the alveolar capillaries of diabetic rats. This appears to represent not an increase in the "available space" for this protein in the alveolar wall interstitium but rather an active, intensified transcytosis, as indicated by the fact that, in the diabetic lot, the endothelial cells themselves appear to take up on average 1.4-fold more albumin molecules than in the control animals. This scenario is further strengthened by the data obtained with the exogenous, hapten-tagged variant of serum albumin (DNPBSA) injected into the circulation. We have previously demonstrated (Ghitescu and Bendayan 1992) that such an approach preserves the normal physiological conditions in the vascular system and that the DNPBSA is handled by the endothelium in exactly the same way as its endogenous untagged counterpart. Only 15 min after administration of the tracer, the density of labeling for DNPBSA over the endothelial profiles amounted to 13.5% of that over the pulmonary capillary lumen in normoglycemic rats and 18.6% in diabetic animals (Table 6). The amount of DNPBSA detected in the space between the alveolar endothelial and epithelial cells also increased rapidly at levels approximately twofold in diabetic than in normal rats. These results prove that, during the early stages of experimental diabetes, the lung capillaries, like other vascular beds endowed with a continuous type of endothelium, become hyperpermeable to plasma macromolecules.
The signal for endogenous albumin or for DNPBSA, localized over the endothelial cell profile, was almost exclusively associated with the plasmalemmal vesicles, either open to one of the cell fronts or apparently closed, within the cytoplasm. We interpret this as a proof that the transendothelial transport of albumin takes place via the caveolar system and that, as a corollary, the diabetes-induced increase in permeability is due either to hypertrophy of this system or to an acceleration in the vesicular dynamics. As we will further discuss, our results support the first case. The involvement of caveolae in the transcytosis of macromolecules was recently questioned by the model of caveolin knockout mouse in which, despite the capillary endothelium lacking caveolin and caveolae, normal (Drab et al. 2001) or even higher concentrations of albumin were found in the interstitial fluid (Schubert et al. 2002
). This challenge was only apparent, however, because the caveolin-null animals reportedly show caveolin-independent vesicles in the venular part of the microvasculature (Drab et al. 2001
) and significant alterations at the level of the interendothelial junctions (Schubert et al. 2002
).
The interendothelial clefts represent an alternative pathway, frequently considered, for the egress of molecules from the circulation. This is particularly attractive in relation to the so-called hemodynamic hypothesis, which regards the documented increase in capillary pressure as one of the main causes of diabetic microangiopathy (Tooke 1996). Videodensitometry has shown that capillary leakage of Nafluorescein in the skin is significantly higher in long-term diabetic patients than in healthy ones (Oomen et al. 1999
). The small (376 Da) negatively charged dye is likely to evaluate the patency of the nonspecific passageways for water and small hydrophilic molecules (small endothelial pores in the terminology of vascular physiologists), most probably the interendothelial junctions. Incubation of endothelial cells in vitro with advanced glycation end products (AGE)-modified BSA or vascular endothelial growth factor (VEGF), conditions presumed to mimic those of diabetic patient blood vessels, was found to trigger a reduction in the number of molecules responsible for the adherens and tight junctions such as VE-cadherin, its associated ß- and
-cathenins (Otero et al. 2001
) and occludin, respectively (Antonnetti et al. 1998
). However, we did not observe in the lung alveolar capillaries of hyperglycemic or normal rats any interendothelial junctions permeated by either endogenous albumin or DNPBSA. This result agrees with previous studies also reporting the absence of open intercellular clefts in various vascular beds of diabetic animals (McCuskey and McCuskey 1984
; Caldwell and Slapnick 1992
; Yamaji et al. 1993
; Vinores et al. 1998
), except when diabetes was combined for the long term with aggravating conditions such as hyperlipidemia (Popov et al. 1996
). Taken together, these facts rule out the possibility of explaining the diabetes-induced hyperpermeability to macromolecules through more permissive interendothelial clefts. The latter appear to account only for the abnormal transendothelial passage of small-sized molecules (Bendayan 2002
; Simionescu et al. 2002
).
Our data show that diabetes produces in its early stage a net expansion of the compartment of plasmalemmal vesicles, which correlates with the documented hyperpermeability of lung alveolar endothelium to albumin. Immunoblotting assays and quantitative ICC performed on purified plasma membrane preparations have shown that, in diabetic animals, the luminal surface of the alveolar endothelium contains a significantly higher amount of caveolin 1 but not of other plasmalemma-asociated proteins, such as actin and annexin II. The existence of a direct relationship between the expression of caveolin and the number of caveolae has been previously demonstrated (Scherer et al. 1994) and confirmed by our results. Therefore, the natural conclusion is that, in diabetes, more caveolae come into direct contact and fuse with the apical plasmalemma of the pulmonary endothelium.
We are not considering at present what proportion of these organelles are connected to the plasma membrane and how many might be free in the cytoplasm as free-floating plasmalemmal vesicles. This was the reason for performing in parallel the ICC detection and quantification of caveolin in the purified plasma membrane preparations as well as in the whole endothelial cell profile fixed in situ. Both assays indicated a significant (1.73-fold and 1.24-fold, respectively) increase in the amount of caveolin 1 in diabetes.
The comparative morphometrical analysis of the caveolar compartment in the alveolar endothelium of diabetic and normoglycemic rats has shown that the overexpression of caveolin in diabetes is accompanied by a notable increase in the number of plasmalemmal vesicles and in the partial volume occupied by them in the endothelial cell. Consequently, the data presented here define these vesicles as the main endothelial structural elements involved in transendothelial transport of plasma proteins and the ones responsible for the diabetes-induced increase in endothelial permeability. It is likely that this mechanism is not limited to the alveolar capillaries but may also account for the hyperpermeability of other microvascular beds endowed with a continuous type of endothelium, such as retina and nerves, in which diabetic microangiopathy is significantly more spectacular.
An attractive hypothesis regarding the mechanism by which diabetes induces caveolin overexpression involves the altered VEGF production in the tissue surrounding the blood vessels. VEGF is a family of potent mitogens with relative specificity for endothelial cells, playing a key role in angiogenesis and also able to increase vascular permeability, hence its alternative name of vascular permeability factor. The implication of VEGF in diabetic microangiopathy draws its support from studies that have already documented that vascular complications, particularly those in the retina and kidney, are associated with transcriptional activation of the VEGF gene (Hammes et al. 1998; Cooper et al. 1999
). An upsurge in the amount of VEGF (more than fourfold) was also demonstrated in the lung of spontaneously diabetic BB rats at the onset of the disease. The increase is identical to that measured in the kidneys of the same animals (Braun et al. 2001
). Possible VEGF producers in the lung are the vascular and airway smooth muscle cells (MataGreenwood et al. 2003
) and the endothelial cells themselves (Favre et al. in press
). VEGF gene transcription is stimulated by a host of factors such as hypoxia, other growth factors, and cytokines, and also by several particular conditions of diabetes, such as hyperglycemia (Williams et al. 1997
), advanced glycation end products (AGEs) (Treins et al. 2001
; Yamagishi et al. 2002
), and oxidative stress (Beinert et al. 2000
; Sasaki et al. 2000
). Moreover, when various types of endothelial cells were treated in vitro with VEGF, a rapid increase in the number and diameter of caveolae was evident (Vasile et al. 1999
; Chen et al. 2002
). However, even if proved true, this scenario of VEGF-dependent microvascular hyperpermeability in diabetes must actually be considerably more complex. Several experimental data do not entirely fit to it. For example, VEGF was proved to induce, over the long term, a change in endothelial cell phenotype from continuous to fenestrated (Roberts and Palade 1995
; Chen et al. 2002
). None of these fenestrations was observed in the microvasculature of our diabetic rats. In addition, the oxidative stress present in diabetes and known to activate the production of VEGF (Chua et al. 1998
) was reported to inhibit caveolin palmitoylation and therefore its association with the membrane (Parat et al. 2002
).
Caveolin overexpression by endothelial cells in diabetes is certainly not a singular example. Aside from the very well-demonstrated increase in the production and secretion of extracellular matrix components, several other proteins, such as endothelin and vascular cell adhesion molecule-1, key to other important endothelial functions such as the regulation of vascular tone and leukocyte diapedesis, respectively, are affected by diabetes (Ribau et al. 1999; Apostolova et al. 2001
).
Our results also show that the basement membrane of alveolar endothelium, like that in other vascular beds, such as skeletal muscle (Ghitescu and Bendayan 1992) and mesentery (Arshi et al. 2000
), does not represent a barrier to the bloodtissue transport of macromolecules, at least of the serum albumin size. A restricted diffusion of the endogenous albumin or DNPBSA across the thickness of the basement membrane should manifest itself through an accumulation of the endogenous albumin or DNPBSA in the lamina rara, the region of the basement membrane adjacent to the endothelium (Bendayan et al. 1986
). No such distribution was observed. In diabetic rats the alveolar epithelial and endothelial basement membranes thicken to an extent (1.3-fold) comparable to that detected in the kidney glomerulus at the same early stage of the disease (Weynand et al. 1999
; Doucet et al. 2000
). However, unlike the glomerular basement membrane, which becomes leaky in diabetes, the porosity of the alveolar basement membrane does not change. The same relative distribution of the albumin molecules across the thickness of this structure is encountered in normal and diabetic rats.
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Acknowledgments |
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This article represents part of the work required for fulfillment of the MSc program by M. Pascariu.
We thank Dr Irene Londoño, Ms Diane Gingras, and Ms Elisabeth Gervais for their precious help.
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Footnotes |
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