Our in vitro results suggest that HRECs respond to cytokines rather than high glucose for ROS generation, induction of inflammatory pathways, and apoptotic changes. Our studies suggest that in vivo diabetes–related endothelial injury in the retina may be primarily due to glucose-induced cytokine release by neighboring cells rather than a direct effect of high glucose on endothelial cells themselves.
A number of studies have demonstrated that glucose-induced oxidative stress and cytokine production in the retina are associated with diabetic retinopathy (7
). However, before the current study, it was not clear whether endothelial dysfunction is a primary effect of direct glucose toxicity in retinal endothelial cells or a secondary effect mediated by cytokines. In this study, we systematically compared the effects of elevated glucose levels to the effects of two key cytokines on various parameters associated with endothelial cell toxicity using HRECs as a model.
Our first key finding demonstrated that HRECs had a very low level of glucose consumption compared with other retinal cell types, HRPEs and HMCs, from the same donors. Moreover, the rate of glucose consumption by HRECs was independent of glucose concentration in the media. There are a number of parameters that can affect glucose toxicity in different cell types. Among them are expression and regulation of different types of glucose transporters and hexokinase isoforms, the rate of glycolysis versus oxidative phosphorylation, antioxidant protection level, and differential response to insulin. As we and others described before, the predominant glucose transporter in HRECs is GLUT1 with minor GLUT3 expression level (15
). The expression level of these transporters was not controlled by high glucose in HRECs in our studies; moreover, the rate of glucose consumption observed in this study (175.09 nmol · mg protein−1
) was below the Vmax
for glucose uptake observed by us in high glucose in HRECs (56.00 pmol · mg protein−1
= 201.60 nmol · mg protein−1
). Taken together, these data suggest that glucose consumption in HRECs is not likely to be limited by glucose uptake rate but rather by further glucose metabolism steps.
If HRECs do not increase glucose consumption under high glucose conditions, there is no reason to expect glucose-induced radical production by these cells. We performed repeated tests using H2DCFDA ROS indicator dye, MitoTracker Red mitochondrial O2·-specific probe, and three different spin traps for EPR measurements: DMPO, PBN, and TEMPO-AM, all of which were negative for high glucose–induced radical production by HRECs.
Similarly, high glucose had no effect on MAPK protein phosphorylation signaling cascades, namely ERK1/2, p38, and JNK phosphorylation, on tyrosine phosphorylation or on activation of NF-κB pathway. High glucose also did not induce caspase activation or apoptosis for the first 96 h of glucose exposure. Only after 96–144 h of exposure did we detect a modest induction of caspases and a minor amount of apoptosis. Only caspase-8 and caspase-3 were activated in HRECs by elevated glucose levels, indicating a type I caspase signaling pathway, which requires upstream caspase-8 activation followed by direct downstream activation of caspase-3. Caspases and other proapoptotic proteins involved in the mitochondrial death pathway were unaffected by high glucose. This is consistent with the findings that ROS formation and mitochondrial O2·
production were not increased in high glucose because mitochondria-mediated apoptosis requires the production of O2·
within the mitochondria itself to release cytochrome c from the mitochondrial membrane (30
). A major regulator of caspase-8 activation is the protein c-FLIP. c-FLIPs are well-known inhibitors of cell death. To date, three isoforms of the protein have been identified, c-FLIPL
, and c-FLIPR
. All isoforms are structurally similar to caspase-8 but do not have a catalytically active site serving as an alternative substrate for caspase-8 (31
). High glucose–induced caspase-8 activation only occurred after c-FLIPL
levels declined, indicating that apoptosis observed in HRECs was associated with the reduction of c-FLIPL
. Interestingly, although caspase-3 was slightly but significantly activated in HRECs by high glucose at 144 h, this activation of caspase-3 seems to be transient. Long-term exposure of HRECs in high glucose up to 4 weeks did not lead to increased caspase-3 activity or cell death, indicating that HRECs are very capable of adapting to high glucose conditions.
Overall, we found that HRECs were remarkably unresponsive to intermediate, high, and fluctuating glucose conditions in various assays associated with endothelial cell toxicity used in this study.
A prominent feature of diabetic retinopathy is increased levels of proinflammatory cytokines in the retina, and several inflammatory pathways are activated at the early stage of diabetic retinopathy. Diabetes leads to the activation of caspase-1, the enzyme responsible for the production of the proinflammatory cytokines IL-1β and IL-18 in the retinas of diabetic animals and diabetic patients (20
). IL-1β can induce IL-6, TNF-α, and itself (32
). IL-1β and TNF-α have many overlapping physiological functions. Both have also been associated with the induction of oxidative stress and the mitochondrial death pathway (33
). Cytokines, such as TNF-α, its soluble receptor, and IL-1β, are increased in serum of diabetic patients, and increased TNF-α levels have been associated with the development of diabetes complications (34
). Furthermore, increases in TNF-α and IL-1β levels have been shown in vitreous fluid of diabetic patients and in retinas of streptozotocin-induced diabetic rats and mice (26
Endothelial cells are extremely susceptible to cytokines, such as IL-1β, TNF-α, and IFN-γ, which induce production of endothelial cell–derived cytokines (e.g., IL-8, MCP-1, and RANTES), recruitment of leukocytes to the cell surface, and cell death (37
). They are also very responsive to cytokine stimulation, leading to ICAM-1 and VCAM-1 upregulation, as we have previously shown (19
). In the current study, we demonstrated that high glucose did not induce caspase-1 activation, the enzyme responsible for IL-1β production, and did not lead to the production and release of IL-1β and TNF-α by HRECs. In contrast, we have shown that IL-1β induced more than a twofold increase in glucose consumption and mitochondrial O2·
generation in HRECs, whereas glucose alone did not. We also show that IL-1β is a potent inducer of caspase-3 activity and apoptosis in HRECs. After 12 h of IL-1β stimulation, we detected a 234% increase in caspase-3 activity compared with control, an activation never reached with high glucose alone at any given time point in these cells. In contrast to high glucose treatment, IL-1β led to the activation of caspases involved in the mitochondrial type II death pathway and was preceded by the formation of mitochondrial oxidative stress required for mitochondrial-mediated apoptosis. Anti-apoptotic c-FLIPL
levels were dramatically decreased by IL-1β, indicating that HRECs might be very well protected against hyperglycemia but seem to be less able to protect themselves against proinflammatory stress.
In addition to retinal endothelial cells, almost all other cell types in the retina are known to be adversely affected in diabetes. These include pericytes, microglia, astrocytes, HMCs, and HRPEs cells, as well as retinal neural element, such as ganglion cells, bipolar cells, and photoreceptors (rev. in 39
). Any of these cells could be the actual source of the glucose-induced cytokine production. In this study, we used two examples, HRPEs and HMCs, to demonstrate the principle of potential involvement of other retinal cell types in glucose-induced toxicity. In agreement with other studies, our results demonstrate that HMCs and HRPEs produce proinflammatory cytokines, such as IL-1β, in response to elevated glucose (21
). HRPEs and HMCs are known to produce growth factors and neuroactive peptides (41
) that maintain endothelial health; however, pathologically high levels of growth factors and cytokines can exist during diabetes and during retinal ischemia (42
). Because HMCs are known to encircle the microvasculature, protecting the integrity of the microvasculature, the likelihood of cytokines produced by HMCs reaching endothelial cells is very possible (45
). Our study indicates that high glucose–induced stimulation of HMCs might have detrimental effects on the vasculature. But again, most other retinal cell types surrounding the vasculature can possibly affect endothelial cell viability in the similar fashion.
Although glucose-induced ROS expression by endothelial cells has been reported by others (18
), outcomes are highly dependent on experimental conditions and the vascular bed and the species of origin of the endothelial cells (49
). Perhaps more important is that cell types typically contaminating retinal endothelial cultures, such as HMCs, HRPEs, pericytes, microglia, and astrocytes, may be the actual source of the glucose-induced cytokine production measured previously in retinal endothelial cell cultures. HRPEs and HMCs exposed to high glucose produced 2.5 and 12.3 ng/ml IL-1β, respectively, which is 2.5- to 12.3-fold higher than the 1 ng/ml IL-1β required for stimulation of endothelial cells. Thus, cytokines secreted by these cells could in turn stimulate endothelial cell ROS production, apoptosis, activation of MAPK signaling cascades, tyrosine phosphorylation, NF-κB pathway activation, and adhesion molecules expression. We have extensively published studies using primary HREC cultures and only HRECs of 99% or higher purity, as were used in the current study.
In conclusion, our in vitro studies show that high glucose conditions do not increase endogenous ROS generation by HRECs but rather that these cells respond to cytokines that upregulate ROS. Furthermore, our in vitro studies also suggest that in vivo retinal endothelial injury resulting in capillary drop-out and the retinal pathology associated with diabetic retinopathy may be due primarily to glucose-induced cytokine release by neighboring cells rather than the direct effect of high glucose on endothelial cells.