University of Groningen
Vasoregression in incipient diabetic retinopathy
Pfister, Frederick
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2011
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Pfister, F. (2011). Vasoregression in incipient diabetic retinopathy: Angiopoietin-2 dependency and the
effect of Erythropoietin and Carnosine treatment Groningen: s.n.
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CHAPTER 10
Discussion and perspectives
185
Discussion and perspectives
Diabetic retinopathy (DR) is primarily a vasoregressive complication of diabetes, which
induces a secondary angiogenic and hyperpermeable response as a result of progressive
capillary occlusion. In order to improve treatment and care of DR, it is important to
understand the molecular and cellular mechanisms initiating the cascade of events finally
leading to retinal vasoproliferation or edema. The earliest morphological changes in the
diabetic retina, i.e. loss of pericytes and endothelial cells and subsequent capillary
occlusion, are long known features of DR, but the mechanisms underlying these changes
and the importance of these events for the progression of vascular damage are still
unclear. Drugs targeting single biochemical abnormalities failed to prevent the
development of DR, suggesting that the pathomechanisms are complex and that other
factors apart from direct hyperglycemia-induced cell toxicity are also involved. Recently,
vascular defects similar to DR could be detected in non-diabetic transgenic animals,
demonstrating that modulation of specific growth factor systems also play an important
role in diabetic vascular damage.
Ang-2 determines pericyte coverage of growing and mature vessels
Proliferative activity of endothelial cells in the vascular stalk of sprouting vessels is
determined by the availability of growth factors such as VEGF and Ang-2 [131, 172, 347].
The transition from a proliferating to a mature quiescent endothelial cell, i.e the transition
from a stalk cell to a so-called phalanx cell, is determined by the firm coverage with
pericytes which render these cells resistant to growth factor depletion [23]. Pericytederived angiopoietin-1 (Ang-1) expression appears to be the initial step of vessel
maturation, causing differentiation, inhibition of proliferation and induction of intercellular
contact proteins of endothelial cells [24, 130, 136, 137]. Furthermore, Ang-1 expression
seems to be involved in the recruitment of pericyte precursors, which stabilize the new
vessels. In contrast, Ang-2 is able to block the stabilizing function of Ang-1 by promoting
smooth muscle cell and pericyte dropout, thereby loosening contacts between endothelial
cells and periendothelial cells [144]. Consistently, overexpression of Ang-2 during retinal
development results in impaired pericyte recruitment and enhanced angiogenesis (chapter
2). Of note, increased density of the deep capillary network in Ang-2 overexpressing mice
is transient, as reported by Oshima et al., suggesting that sensitivity of endothelial cells to
Ang-2 changes during retinal vessel development [143]. In contrast, retinal pericyte
numbers remained reduced in the maturating vasculature of Ang-2 overexpressing mice,
indicating that pericytes are permanently susceptible to changes in Ang-2 (chapter 2 and
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4).
In the mature retinal vasculature, the mechanisms of Ang-2 induced vessel destabilization
are poorly understood and the context-dependent interaction of Ang-2 with other growth
factors in the regulation vascular homeostasis remains to be clarified. Under hypoxic
conditions, VEGF and Ang-2 are upregulated, inducing sprouting angiogenesis [144]. This
concept applies to tumor angiogenesis, wound healing, proliferating DR and other
diseases. The early diabetic retina, however, is not hypoxic and Ang-2 is upregulated due
to biochemical changes. In the absence of hypoxia-driven VEGF, Ang-2 is thought to
destabilize vessels, ultimately leading to vessel regression. In line with this hypothesis,
Ang-2 overexpression induces pericyte loss and vasoregression in non-diabetic retinas,
mimicking the vascular phenotype of incipient DR (chapter 4). Furthermore, the
progressive decline of survival factor VEGF and Ang-1 and subsequent Ang-2
predominance in the aging rodent retina were associated with progressive pericyte loss
and vasoregression. Mice deficient for Ang-2 were protected from these age-induced
vascular changes (chapter 3). The fact that Ang-2 is upregulated in the diabetic retina prior
to pericyte loss and injection of recombinant Ang-2 in healthy rodent eyes induced a dosedependent pericyte loss suggest that pericytes are the primary target of Ang-2
upregulation in the mature retinal vasculature. Accordingly, it has been demonstrated that
Ang-2 deficiency protects from hyperglycemia-induced pericyte loss and vasoregression
was reduced by half in this models [28]. In summary, recent observations and data
generated within this thesis demonstrate that Ang-2 is an important modulator of
physiological and pathological angiogenesis, but also determines pericyte coverage and
the subsequent fate of retinal capillaries in the mature vasculature. Ang-2 induced pericyte
deficiency during retinal development results in an enhanced angiogenic activity of
endothelial cells, whereas in the mature retinal vasculature, reduced pericyte numbers
precede vasoregression. In the diabetic retina, loss of capillary pericytes is induced by
upregulation of Ang-2 and consequently, susceptibility of the denuded endothelium to
hyperglycemic toxicity and regressive or angiogenic signals generated by surrounding
cells might be increased.
Mechanisms of diabetic pericyte loss and vasoregression
At present, the mechanisms by which diabetes induces loss of pericytes and endothelial
cells remain uncertain. Oxidative stress, formation of advanced glycation endproducts
(AGE), upregulation of protein kinase C (PKC) and increased polyol pathway flux are
187
Discussion and perspectives
possibly involved [55]. In fact, chronic hyperglycemia activates multiple cell damaging
pathways and a number of downstream mechanisms that induce apoptosis in vascular
cells [90, 99, 101-103, 109, 113]. However, the number of apoptotic cells in diabetic retinas
is low and many apoptotic cells detected by TdT-mediated dUTP-biotin nick end labeling
(TUNEL) staining and active caspase-3 immunoreactivity in streptozotocin (STZ)-diabetic
rat retina are distinct from the vasculature, indicating a neural identity [265, 348].
Furthermore, GLUT-1 transporters, which promote glucose flux into the cell and which are
thus the prerequisite for the aforementioned biochemical abnormalities are expressed by
endothelial cells and Müller cells, but not by pericytes [349]. Consequently, focusing on the
pericyte as target for hyperglycemic damage, the role of intracellular ROS, AGE and other
bioactive intermediates of glycolysis should be carefully recapitulated.
Keeping in mind that in the healthy mature vasculature the Ang-1/Tie-2 signaling ensures
the interaction and attachment of pericytes and endothelial cells, it is conceivable that the
upregulation of Ang-2 in diabetes interferes with these functions. In chapter 5, we provide
evidence that favor pericyte detachment and migration as a new mechanism contributing
to diabetic pericyte loss. We show that this process is Ang-2-dependent. Although retinal
digest preparations used in our studies represent snapshots of ongoing remodeling of the
vasculature, and since we did not monitor the dynamics of pericyte migration in vivo and
thus failed to determine the ultimate fate of detached pericytes, our conclusions are
supported by work on brain pericytes that suggests that they migrate away from injured
capillaries or in response to hypoxia [219, 220, 350]. As demonstrated in the traumatic
brain injury model, pericytes are capable of migrating away from injured capillaries thereby
ensuring their survival. It was demonstrated that pericytes which migrated away from
capillaries survived while those which stayed attached to capillaries were more likely to
undergo apoptosis. Since pericytes that have migrated away from the capillary were able
to survive in the perivascular compartment, it is likely that they still participate in some sort
of cell-cell communication. These functional adaptations may include differentiation along
multiple lineages reflecting the pluripotency of this enigmatic cell population. However, this
remains to be investigated in detail. Apart from pericyte migration, anoikis represents an
alternative mechanism of diabetic pericyte loss. It has been reported that hyperglycemia
induces upregulation of Cyr61 and CTGF as anti-adhesive molecules leading to retinal
pericyte anoikis, a form of apoptosis resulting from loss of cell adhesion and/or celladhesion dependent signaling [351]. Still, anoikis is debated since the factors initiating cell
detachment and subsequent apoptosis are not well understood. Furthermore, a role for
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Ang-2 in this process has yet to become established. It is also not known why some retinal
pericytes are not affected by hyperglycemia, while others are lost, as analyzed in chapter
5. Different developmental origins may dispose subsets of retinal pericytes to variable
responses. Alternatively, the final spatial position of pericytes within the capillary tree may
cause a localization specific adaptation and thereby a differential susceptibility to
hyperglycemia. Retinal pericytes are heterogeneous with regard to their origin and
phenotype [195, 206]. However, functional differences of retinal capillary pericytes in
response to hyperglycemia have not been tested before. We show that in the diabetic
retina the localization of pericytes at vessel branches is protective against hyperglycemic
stimuli, whereas pericytes in midcapillary positions are susceptible. Although unproven at
present, the position at vessel branches might be beneficial because of the multiple
interactions with endothelial cells, or because pericytes at capillary branches have a
specialized phenotype with an impact on the resistance to hyperglycemic cell stress.
The major function of pericytes in the mature vasculature is maintenance of capillary
integrity and survival. Pericytes promote endothelial cell survival by secretion of VEGF,
regulate endothelial cell proliferation and differentiation via TGF-β signaling and promote
vessel integrity and tightness of blood-retinal barrier by secretion of Ang-1. Furthermore,
proper pericyte coverage renders endothelial cells refractory to shifts in oxygen tension
and growth factor levels and pericyte-derived PGI2 protects endothelial cells against
oxidative injury [25, 136, 137, 352-354]. Regarding these important functions of pericytes
for capillary integrity and survival, it is obvious that their preferential loss in DR has
profound consequences for the underlying endothelium. If pericytes are not recruited to
the developing capillaries, endothelial cells are more susceptible to vasoregression. This
has been demonstrated in mice with a heterozygous deletion of PDGF-B in which pericyte
numbers recruited to the capillaries were reduced by 28% [179]. As a result, a moderate,
but significant increase in acellular capillaries developed with time, emphasizing that
pericytes themselves can function as survival factors for endothelial cells in the mature
vasculature. Correspondingly, reduced pericyte coverage in Ang-2 overexpressing mice
used in this thesis led to an increased formation of acellular capillaries at 3 months of age.
Comparable to diabetic PDGF-B deficient animals, superimposition of hyperglycemia in
Ang-2 overexpressing mice exacerbated vascular damage, suggesting that proper pericyte
coverage supports endothelial cells survival under diabetic conditions (chapter 4).
However, capillaries could regress even in the presents of attached pericytes and
preserved pericyte coverage does not necessarily protect against diabetic vasoregression
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Discussion and perspectives
[30, 68, 353]. Moreover, acellular capillary formation occurs in numerous vascular
pathologies, like Alzheimer disease, tumors and retinal degeneration without previous or
concomitant pericyte depletion and despite the presents of hyperglycemia [164, 355-358],
indicating that other factors than hyperglycemia-induced pericyte loss and glycotoxicity
might also contribute to diabetic vasoregression. This is further illustrated by our recent
findings in transgenic rats, in which photoreceptor cells are destroyed due to a defect in a
cilia gene (PKD-2-mut-rat), leading to pericyte loss and vasoregression in the absence of
hyperglycemia [356]. In this model, disturbed integrity of the neurovascular unit is
responsible for pericyte loss to an extend comparable to diabetic animals and exorbitant
vasoregression. The data emphasize the importance of neuroglial and vascular cross-talk
for capillary integrity. Of note, retinal neuroglial damage can precede vascular changes
under diabetic conditions, and survival of retinal vessels appears partially dependent on
the supportive function of neuroglial cells in diabetic retinas [359]. Therefore, elucidating
crosstalk of neuroglial and vascular compartments in the pathophysiological of DR might
offer new therapeutic approach for the prevention of DR.
Novel approaches for the prevention of DR
Current evidence-based treatment for DR includes strict glycemic control, blood pressure
and lipid control, and the general reduction/modification of risk factors such as smoking.
Destructive surgical approaches such as laser photocoagulation and vitrectomy are
implemented during late stage disease and can only prevent progressive loss of vision [7,
360-364]. Impaired visual acuity, once established cannot be restored by available
therapies and primary prevention requires near-normal glycemic control that is difficult to
achieve in clinical practice. Therefore, novel therapeutic and preventive strategies are
needed.
Since, hyperglycemia-induced mitochondrial overproduction of ROS seems to be the
instigator of biochemical, functional and structural abnormalities in DR, the use of
appropriate antioxidants may have potential in the treatment of DR [55]. Yet, a number of
treatments that reduced oxidative stress, such as benfotiamine, nicarnatine and vitamine C
and E have been tested and only some of them show promising effects in vitro and
experimental diabetic retinopathy [31, 60, 68, 117, 234, 365, 366]. However, antioxidants
failed to improve diabetic vascular disease in clinical trials [234, 367, 368]. One
explanation for the failure of these drugs in humans might be that the antioxidant treatment
was not initiated before non-proliferative DR developed. Contrarily, antioxidants in
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experimental DR have already been administered before or soon after the onset of
experimental diabetes. It is also possible, that the antioxidants did not reach sufficient
levels in the retina to counteract hyperglycemia-induced oxidative stress or the tested
agents did not penetrate the blood-retinal barrier to reach target sites. Over and above,
there is evidence that classical antioxidants are unsuitable to counteract hyperglycemiainduced ROS production, because they act stoichiometrically, whereas catalytic
antioxidants might be efficient in preventing diabetic vascular damage [31].
Another possible way to counteract hyperglycemia-induced ROS production is stimulating
the antioxidant defense mechanisms of vascular and non-vascular cells in order to
maintain redox homeostasis. Important intracellular defense system against oxidative
stress are superoxide dismutase (SOD), glutathione reductase and catalase. Recently, it
has been demonstrated that overexpression of mitochondrial SOD reduces oxidative
stress, thereby preventing diabetes-induced vascular pathology, indicating that SOD
mimics, such as desferrioxamine-Mn(III) chelate could provide an attractive
pharmacological approach to inhibit the development of diabetic retinopathy [369]. Another
widely existing enzyme belonging to the antioxidant defense mechanisms is Heme
oxygenase-1 (HO-1), which can be induced in glial cells by NO donors, such as S-nitrosoN-acetylpenicillamine [370, 371]. In line with previous observations, we show in chapter 8,
that in the retina HO-1 can be highly induced by hyperglycemia [372] and increased HO-1
expression prevents excessive cellular apoptosis of pericytes due to oxidative stress [373].
Whether delivery of HO-1 protein as a protective mechanism against oxidative stress could
have potential in the treatment of DR has not been tested yet. In chapter 6, we show that
treatment with Epo reduces oxidative stress in diabetic rat retinas, thereby inhibiting glial
cell activation, pericyte loss and ultimately vasoregression. Epo is not a classical
antioxidant. However, antioxidative effects of Epo have been described [257, 374, 375]. In
line with previous observations, our data favor the assumption that the protective effect of
Epo on diabetic retinas is rather receptor mediated, although a final proof is still missing.
The activation of Epo receptor, which is expressed on retinal vascular cells, Müller cells
and retinal neurons, induces a number of signaling pathways known to be involved in cell
protection and survival. We show that Epo treatment normalizes the activation of the
serine/threonine protein kinase AKT in diabetic retinas, a known stimulator of antioxidants
and anti-apoptotic factors. Together, recent findings and data generated in this thesis
indicate that activation of pro-survival systems, including antioxidative defense enzymes
and factors appears to be promising in inhibiting the development of diabetic retinopathy.
191
Discussion and perspectives
Besides, hyperglycemia-induced overproduction of ROS activates alternative biochemical
pathways, which are themselves implicated in the pathogenesis of DR [55]. Hence,
inhibitors of the biochemical pathways have been explored and tested in preclinical animal
models. However, none of the agents targeting biochemical abnormalities, such as
increased AGE formation or activation of polyol pathway have led to effective therapy of
DR, suggesting that it might not be sufficient to block a single biochemical pathway to treat
DR. In chapter 8, we show that it is possible to protect retinal capillaries from
hyperglycemic damage even without correcting biochemical abnormalities, indicating the
importance of other factors than biochemical dysregulation in the pathogenesis of diabetic
retinopathy. On one side, the protective effect of carnosine on retinal capillaries could be
archived by a direct effect on retinal endothelial cells and pericytes, implicating that retinal
vascular cells provide potent defense systems against hyperglycemic damage, which can
be activated pharmacologically. On the other side, vascular protection by carnosine might
be mediated indirectly via activation of retinal glial and neuronal cells. A common intricate
system of all cells to protect from environmental and physiologic insults are heat shock
proteins (Hsp), also known as stress proteins and molecular chaperons. We show that
carnosine induces the expression of Hsp27 in retinal glial cells, but we also found
increased expression of Hsp27 in retinal and kidney endothelial cells (data not shown in
this thesis). The accumulation of Hsp, whether induced physiologically, pharmacologically,
or by direct administration of the proteins, is known to protect cells from a great variety of
pathological conditions. In preclinical studies, the non-toxic Hsp-coinducer bimoclomol
diminished diabetic neuropathy, retinopathy, and nephropathy [334, 376]. Comparably,
carnosine might induce Hsp expression in the retina, thereby protecting against
hyperglycemic tissue damage. In fact, it has been reported that carnosine increases the
expression of Hsp in a number of different cells and organs [331, 332, 377-379]. However,
we failed to establish a causal interconnection of Hsp-induction by carnosine and vascular
protection in this thesis.
In general, the tight association between retinal vascular and glial cells implies a close
functional relationship between these two cell types and suggests that changes in one has
profound consequences for the other. Like the brain, the retina contains glial cells, which
are normally quiescent but adopt a reactive state during infection, injury and also diabetes.
In diabetic rats, activation of the principle glia cell, the Müller, induces the expression of a
variety of angiogenic growth and trophic factors, such as Ang-2, VEGF, FGF2, CNTF and
NGF and cytokines, acute-phase response proteins and other inflammation-related genes,
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which could have an impact on retinal vasculature [45, 380-382]. Of importance, one third
of the genes differentially expressed in diabetic Müller cells are associated with
inflammation. Among the most significantly upregulated genes are clusters of acute phase
response proteins, such as β-2-macroglobulin, ceruloplasmin, complement components,
lipocalin-2, metallothionein, serine protease inhibitor-2, transferrin, tissue inhibitor of
metalloproteases-1, transthyretin, and the transcription factor C/EBP. Interestingly, there is
a considerable overlap of these genes with genes upregulated in animal models of retinal
neurodegeneration which are characterized by progressive vasoregression without
concomitant hyperglycemia, suggesting these factors to be causally involved in diabetic
vasoregression [383-385] (Feng et al. unpublished data). Identifying common mechanisms
of hyperglycemia-induced vasoregression and vasoregression in animal models like the
PKD-rat or the rd/rd-mouse could help to invent new strategies to prevent vasoregression
in DR.
Another important factor, secreted by retinal Müller cells under diabetic conditions is
Ang-2. As demonstrated in this thesis, Ang-2 is important in the development of early
vascular damage of DR. Therefore, targeting Ang-2 signaling might be a strategy
correcting diabetic pericyte loss and thereby supporting vessel stabilization. The inhibition
of Ang-2 in tumor vessels leeds to normalization of blood vessels, as evidenced by
increased junctional proteins in endothelial cells, increased pericyte coverage and reduced
endothelial sprouting [386]. It has also been demonstrated that Ang-1 prevents and
reverses diabetic retinal vascular changes in both new and established diabetes [132] and
in the absence of mural cells, recombinant Ang-1 restored a hierarchical order of the larger
vessels, and rescued edema and hemorrhages, in the growing retinal vasculature of
mouse neonates [123]. Thus, delivery of recombinant Ang-1 or soluble Tie-2 or Ang-2
inhibition might be an efficient therapeutic approach counteracting Ang-2 predominance in
early diabetic retinas to restore pericyte numbers. Keeping in mind that reduced pericyte
coverage in the mature vasculature renders the underlying endothelium vulnerable to
destructive and regressive signals, preservation of retinal pericyte coverage may provide
additional endothelial protection and endothelial cell protection is known to be a primary
therapeutic target to prevent vasoregression in DR.
Conclusion
Similar to all other vascular complications of diabetes, endothelial dysfunction and
degeneration in early stages of DR are the consequence of hyperglycemia-induced cell
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Discussion and perspectives
damage. Beyond that, other constituents of the retina, such as pericytes, retinal glial cells
and neurons are affected as well, leading to disturbed cross-talk with the retinal
vasculature. The interactions between retinal neurons, glial cells and the vasculature
under diabetic conditions are complex and the precise mechanisms between the different
systems remain poorly understood. Nonetheless, it is obvious that enhancing or preventing
the expression of factors released by retinal glial cells might be an efficient therapeutic
approach to protect the vasculature from diabetic damage. Elucidating these mechanisms
and identifying key players in this context may provide new therapeutic options to treat DR.
Thus, beside strict glycemic control and regulation of the diabetic biochemical dysfunction
by antioxidants and AGE-inhibitors, adjunct therapies targeting mechanisms on the cell
biological level could help to prevent advanced DR. Activators of cell survival systems,
agents protecting from Ang-2-driven pericyte loss, neuroprotective and immunomodulatory
substances are promising targets that should be investigated in future studies.
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