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From the Laboratory of Cell Biology, University Eye Clinic, Geneva, Switzerland.
Abstract
PURPOSE. To document the effect of sustained systemic hypertension on the integrity and ultrastructural morphology of retinal capillaries in diabetic and nondiabetic rats.
METHODS. Normotensive (strain Wistar–Kyoto; WKY) and genetically hypertensive (spontaneously hypertensive; SHR) rats were rendered diabetic by intravenous streptozotocin injection. At 20 weeks of diabetes, endothelial cells, pericytes, and extracellular matrix were evaluated by ultrastructural morphometry. Serum albumin was localized by immunofluorescence microscopy.
RESULTS. The endothelial cell layer was markedly thinner in the diabetic normotensive animals. The number of intercellular junctions was reduced in both the nondiabetic and diabetic hypertensive group but less so in the diabetic normotensive group. No significant endothelial cell loss was noted in either of the experimental groups, whereas the number of pericytes and the number of their cytoplasmic processes were reduced in diabetic and hypertensive animals. Significant thickening of the basement membrane and increased permeability to serum albumin were observed in diabetic and hypertensive rats and were strongly enhanced in the combined diseases.
CONCLUSIONS. Endothelial thinning and shape changes from an elaborate to a simpler form as well as rounding up of the pericytes and loosening of their vascular sheaths indicate remodeling of the vascular wall during chronic diabetes and sustained hypertension, before a characteristic vasculopathy becomes manifest. The combination of diabetes and hypertension enhances these features, as well as basement membrane thickening and breakdown of the blood–retinal barrier.
Essential hypertension, prevailing in persons with diabetes,1 2 increases the risk of nephropathy3 4 and morbidity and death of cardiovascular disease.5 It is also one of the most important risk factors for diabetic retinopathy, influencing both its development and severity,6 7 8 and thereby plays a major role in visual loss in diabetic patients.8 9 10
Because both hypertension and diabetes have an impact on the vascular wall,11 12 they may act in an additive or synergistic manner. Indeed, thickening of the basement membrane, degeneration of pericytes, and rarefaction of vessels are features common to both diseases, whereas constriction of the capillaries in the hypertensive state or their dilation in the diabetic state are divergent traits.13 14 15 16 17 18 19 20 21 The mechanism through which hypertension worsens diabetic retinopathy is still not fully understood. Diabetic patients in whom a controlled rise in systemic blood pressure is performed, show impairment of retinal vascular autoregulation and hyperperfusion.22 This situation may lead to an increase in vascular wall shear stress and, eventually, to lasting endothelial damage. Moreover, in spontaneously hypertensive diabetic rats (SHR strain), the deposition of advanced glycation end products (AGEs) in arterioles and the occurrence of acellular retinal capillaries and microthromboses are strongly enhanced but can be corrected by aminoguanidine treatment that prevents the formation of AGEs.23 These observations suggest that increased nonenzymatic glycation due to hyperglycemia accelerates the development of a hypertensive vasculopathy.
In this study, we documented the effect of diabetes on the structure of retinal capillaries in SHR and normotensive (Wistar–Kyoto; WKY) rats. The model of the SHR rat shares several features with essential hypertension in humans24 and is thus well suited for studying the combined diseases of hypertension and diabetes.23 25 Morphologic changes were assessed by ultrastructural morphometry at 20 weeks of diabetes, a time point when acellular vessels are not observed yet. In addition, we evaluated, by immunofluorescence microscopy, the extent of vascular permeability to serum albumin.
Materials and Methods
Animals
Male rats of the WKY and SHR strains (Iffa Credo, Lyon, France)
weighing 240 g, were rendered diabetic by a single intravenous
injection of 60 mg/kg body weight streptozotocin (Sigma, Buchs,
Switzerland) at 7 weeks of age.
The animals were kept on a 14-hour-on–10-hour-off light cycle and fed a normal diet with water ad libitum. At monthly intervals, body weight, blood glucose levels after an overnight fast, and basal glucose levels were determined (glucose analyzer; Beckman Instruments, Geneva, Switzerland; Table 1 ). Under the same experimental conditions, systolic blood pressure was measured by indirect tail-cuff plethysmography in 12-week-old animals and at the end of the study (i.e., at 27 weeks) before the animals were killed.
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Tissue Processing
The morphometric study was performed on seven animals in
each of the following four experimental groups: WKY-N (normotensive
nondiabetic), WKY-D (normotensive diabetic), SHR-N (hypertensive
nondiabetic), and SHR-D (hypertensive diabetic). Eyes were enucleated
and fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate
buffer (pH 7.4) containing 0.2% tannic acid. Tissue pieces were washed
overnight in several changes of cacodylate buffer and postfixed with
0.5% osmium tetroxide in the presence of 0.8% potassium ferrocyanide
in 50 mM cacodylate buffer. After block staining in 2% uranyl acetate
in 50% ethanol, the tissue was dehydrated through a graded series of
ethanol and embedded in Epon. Electron micrographs of thin sections
were taken with a video camera at a magnification of x14,776 and
transferred to a computer (AcerPower Pentium 133; Hewlett Packard,
Geneva, Switzerland). Images were acquired by means of a frame grabber
board (Matrox PCI; Matrox Electronic Systems Ltd, Dorval, Quebec,
Canada).
Computer-Assisted Morphometric Measurements and Statistical
Analysis
Because structural differences exist among the capillaries
of the individual layers of the retina,26
we evaluated
only the capillaries of the outer plexiform layer, which harbors half
the capillary bed. Electron micrographs of 10 to 12 randomly selected
capillaries were taken from four different tissue blocks corresponding
to up to 84 capillaries per experimental group. Only cross-sectioned
capillaries were considered. A software program (KS 300; Kontron,
Zurich, Switzerland) was used for morphometric analysis.
On each capillary, the following primary measurements were taken (Fig. 1 A): 1) inner basement membrane length (IBML, segment surrounding the endothelial cells) and outer basement membrane length (OBML, surrounding both pericytes and endothelial cells). Correspondingly, the area circumscribed by the IBML was the one occupied by the capillary lumen plus the endothelial cell cytoplasm, and the area circumscribed by the OBML was the total area of the vessel including both endothelial cell and pericyte; 2) the area of the capillary lumen; 3) the width of the basement membrane, using the two-point minimum method27 that avoids an overestimation of its thickness.28 These measurements were obtained exclusively in segments that were lined by perivascular glial cells; 4) pericyte area and pericyte length (PL); 5) number of endothelial cell nuclei and pseudopod pairs at intercellular junctions per cross-section through the capillary; and 6) number of pericyte nuclei and processes per cross-section.
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The inner and outer basement membrane areas calculated from the values of IBML and OBML were used to evaluate the caliber of the capillaries.14 The measurements of the endothelial cytoplasmic area and of the number of pseudopod pairs (derived from the number of intercellular junctions29 ), taken together with the number of endothelial nuclei, were used as the parameter of endothelial cell shape. Pericyte loss was evaluated using the number of pericyte nuclei and processes and cell adhesion by the proportion of the IBML covered by pericytes. Pericyte hyperplasia was evaluated by the number of pericyte nuclei, pericytic processes, and cytoplasmic area.
All measurements were performed in a masked manner by investigators without prior knowledge of the capillary origin. All parameters are shown as mean ± SD. The significance of differences between groups was tested by analysis of variance and the Student–Newman–Keuls test.30
Assessment of Vascular Permeability
Retinas were snap frozen and embedded (Tissue-Tek, Miles, Elkhart,
IN). Five cryostat sections of each retina were immunofluorescently
stained with a rabbit antiserum to rat serum albumin (dilution ,1:50;
Anawa, Wangen, Switzerland) and then in goat anti-rabbit Igs coupled to
Texas red (dilution 1:200; Jackson Immunoresearch–Milan Analytica; La
Roche, Switzerland). The specimens were examined in a masked manner for
the presence of extravasated serum albumin. The sections were
semiquantitatively graded for intensity of extravascular fluorescence
using the following subjective grading scale: no staining, -; moderate
staining, +; intense staining, ++.
Results
Weight and Blood Glucose
At 27 weeks of age, WKY-N and SHR-N animals did not differ
significantly in their body weight or in their blood glucose levels
(Table 1) . Both the WKY-D and the SHR-D groups had a significantly
lower body weight and higher blood glucose levels (fasting and basal)
than the WKY-N, and SHR-N animals. The mean systolic blood pressure was
significantly higher in all SHR animals, whether diabetic or not. No
increase in systolic blood pressure was induced by the diabetic state
in normotensive or hypertensive animals.
Morphometric Measurements of Retinal Capillaries
Caliber.
No significant differences were noted between the four experimental
groups in the caliber of the capillaries measured (Table 2)
.
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Endothelial Cells.
In the WKY-D group, a significant decrease of the cytoplasmic
area and a concomitant significant increase in the lumen area (Table 2) reflect a marked thinning of the endothelial cell layer. A similar,
though less pronounced tendency was observed in the SHR strain. The
number of endothelial cells, estimated by the number of nuclei per
cross-section, was similar in all four experimental groups. An earlier
estimation of the endothelial cell number was based on the number of
cytoplasmic pseudopod pairs that are regularly associated with
intercellular junctions (cf. Fig. 1
; ref. 29
). Because their number may
also vary with the complexity of cell shape (sections through
rectangular or roundish cells display less pseudopod pairs than cells
of a more elaborate form), we took the significant decrease in the
number of pseudopod pairs in the SHR-N and SHR-D groups as an
indication of a less intricate cell shape rather than as a sign of loss
of cells.
Pericytes.
The number of pericytes, as reflected by the nuclear number per
cross-section, was significantly decreased in the WKY-D and SHR-D
groups compared with the nondiabetic groups. This cell loss was
particularly significant in the SHR-D animals and was also noted,
although not statistically significant, in the SHR-N group. The
percentage of IBML covered by the pericytes was decreased in WKY-D,
SHR-N, and SHR-D animals indicating a marked diminution of
cell-wrapping around the outer vessel surface associated with both the
diabetic and hypertensive diseases. This feature was accompanied by a
significant decrease in the number of pericyte processes. It is
noteworthy, however, that the overall cytoplasmic area remained
unchanged, except in the SHR-N animals, which showed a higher value.
Besides cell loss and a decreased number of cytoplasmic processes, the
results reflect hypertrophic modification and a change in the pericyte
shape, from flattened to roundish, as exemplified in Figures 1B
1C
and 1D .
Immunohistochemical Assessment of Blood–Retinal Barrier
Permeability
In WKY-N retinas, fluorescent staining for serum albumin was
strictly confined to the vessel lumen, resulting in a relatively weak
yet distinct outlining of the vascular profiles (Fig. 2
A). In the SHR-N tissue (Fig. 2B)
, staining was enhanced and the
definition of the vascular profile blurred because of leakage of serum
albumin into the perivascular tissue. In SHR-D retinas (Figs. 2C
2D)
,
extravascular staining was observed in a fraction only of the vascular
profiles (C), whereas other profiles, even within the same tissue
section, showed no sign of leakage (D). A semiquantitative evaluation
of serum albumin extravasation revealed an increase in vascular
permeability in both WKY-D and SHR-N retinas (Table 3)
This increase was most marked in SHR-D animals, and all the examined
tissue sections displayed serum albumin extravasation.
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In the present study, we showed that chronic diabetes in normotensive (WKY-D) and sustained systemic hypertension in nondiabetic (SHR-N) rats induced microvascular remodeling characterized by a thickened extracellular matrix (basement membrane) along the glia limitans, loss of pericytes, shape changes in both pericytes and endothelial cells, and by an increased permeability to serum albumin. The combination of both diseases markedly enhanced most of these features.
Our results confirm previous reports13 31 32 33 34 35 36 showing that both diabetes and hypertension, regardless of their dissimilar triggering mechanisms, lead to hyperpermeability of the blood–retinal barrier. Leakage is dramatically enhanced in the superimposed diseases, as was also noted in previous studies,37 38 although it maintains its focal character typical of the chronic diabetic state. Uncontrolled passage of glucose and serum factors through a compromised blood–retinal barrier are likely to stimulate abluminal cells to overproduce extracellular matrix components. The resultant thickening of the basement membrane and deposition of unusual collagen polymers17 18 19 20 21 29 in turn could lead to qualitative and quantitative changes in the extracellular molecular sieve properties. The most likely candidate cells to upregulate the synthesis of extracellular matrix components are, besides endothelium and pericytes,39 the glial cells, specifically the Müller cells which sheathe capillaries of the outer plexiform layer.
The conspicuous tendency of pericytes toward reduction of the number of processes, toward becoming round, and toward decreasing contact with the underlying matrix indicates impaired cell adhesion. Although our results do not provide an explanation regarding the underlying mechanisms, they point out that the decrease in vessel sheathing is an early sign of pericyte loss resulting not only from cell death but also from detachment. In diabetic and hypertensive arteries of the human retina, we in fact observed single smooth muscle cells detaching from the vascular wall.40
By contrast, we could not document any significant loss of endothelial cells at 20 weeks of diabetes or hypertension. The endothelial layer is significantly thinner in the diabetic rat, however, and, to a lesser extent, in the hypertensive rat, as deduced from a decrease in the endothelial surface area and a concomitant increase in the capillary lumen. In addition, our numeric evaluation of endothelial pseudopod pairs as a measure of endothelial junctions indicates that endothelial cells in both diabetic and hypertensive animals displayed a less elaborated, simpler shape than in the normotensive nondiabetic control animals. Superposition of both diseases renders this feature most conspicuous. Acquisition of a simpler cell morphology may well represent a transitory stage in the progression of endothelial disease before cell loss, acellular capillaries, and microaneurysms are noted.
In our study, the vascular caliber did not differ significantly in the four experimental groups, although we noted a slight tendency of diabetic capillaries to be enlarged. Because the capillary diameter in the outer plexiform layer is likely to be regulated by the pericytes, this would imply that the regulatory activity of the remaining pericytes in capillaries of diabetic rats is sufficient to maintain a vascular tone within normal range. Moreover, in none of the experimental groups studied did we note vasoconstriction as was previously reported in older (44 weeks) hypertensive rats.21 This feature may manifest itself only in an advanced state.
In conclusion, the observed alterations in the vascular wall during an intermediate stage of diabetes and/or hypertension showed, besides thickening of the basement membrane and hyperpermeability of the blood–retinal barrier, changes in endothelial and pericyte morphology and weakening of the pericyte sheath. These features document a loss of vascular integrity, which may well be the origin of massive vessel leakage and vascular ischemia. They occurred indiscriminately in diabetic and hypertensive animals. However, the enhancement of the pathologic traits in animals with both diseases underscores the importance of a tight control of hypertension in diabetic patients.
Acknowledgements
The authors thank Sabina Scarpino, Alain Conti, and Nicole Gilodi for skillful technical assistance.
Footnotes
Supported by Swiss National Science Foundation Grant 32-39372.93.
Submitted for publication December 19, 1998; revised April 4, 1999; accepted April 21, 1999.
Proprietary interest category: N.
Corresponding author: André A. Dosso, Laboratory of Cell Biology, University Eye Clinic, 22, rue Alcide–Jentzer, 1205 Geneva, Switzerland. E-mail: andre.dosso@hcuge.ch
References
This article has been cited by other articles:
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  T. Oshitari, P. Polewski, M. Chadda, A.-F. Li, T. Sato, and S. Roy Effect of Combined Antisense Oligonucleotides Against High-Glucose-and Diabetes-Induced Overexpression of Extracellular Matrix Components and Increased Vascular Permeability Diabetes, January 1, 2006; 55(1): 86 - 92. [Abstract] [Full Text] [PDF]
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