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
nephropathy
3 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 humans
24 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.
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.
All animal care and handling were conducted in accordance with the
Guide for the Care and Use of Laboratory Animals of the National
Institutes of Health, with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research, and with the Swiss Law on Animal
Protection. Anesthesia was induced with a gas mixture containing 2%
isoflurane and 35% oxygen and maintained with an intraperitoneal
injection of thiopental (Pentothal; Abbott, Cham, Switzerland). The
animals were killed by an intracardiac injection of 1 M KCl and the
eyes enucleated immediately.
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 ×14,776 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).
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
method
27 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.
From these primary measurements, other parameters were
calculated: the endothelial cytoplasmic area, measured as the total
endothelial cell area delineated by the IBML minus the luminal and
nuclear areas, and the portion of the capillary circumference (IBML)
covered by pericyte cytoplasmic processes (PL), expressed as the
percentage of total IBML (PL/IBML).
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 junctions
29 ), 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
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, ++.
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.
Caliber.
No significant differences were noted between the four experimental
groups in the caliber of the capillaries measured
(Table 2) .
Basement Membrane.
The SHR-N, SHR-D groups and the WKY-D group showed a significant
increase in basement membrane thickness compared with WKY-N rats
(Table 2) . The increase was most pronounced in the SHR-D group and
significantly higher than in both, the WKY-D and SHR-N groups,
suggesting that both diseases act in an additive or even synergistic
manner. Thickening was focal and confined to segments lining the
perivascular glia limitans
(Figs. 1B 1C 1D) . This local disturbance
was reflected in the increased SD of both diabetic groups
(Table 2) .
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 .
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.
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 reports
13 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 polymers
17 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.
The authors thank Sabina Scarpino, Alain Conti, and Nicole Gilodi
for skillful technical assistance.