In vitro experiments demonstrated that apoptosis is increased in retinal pericytes with increased concentrations of TNF-α in a dose-dependent manner (). TNF-α at 20 ng/ml increased apoptosis in pericytes sevenfold compared with the control, which was statistically significant (p<0.05). Similarly, CML-collagen induced a dose-dependent increase in pericyte apoptosis, while control collagen did not (). CML-collagen compared with unmodified collagen induced a 2.9-fold increase in apoptosis (p<0.05; ). When caspase-3 activity was measured, a similar increase was observed (). TNF-α (20 ng/ml) increased caspase-3 activity 7.8-fold compared to unstimulated cells, while CML-collagen, compared with control collagen at a concentration of 200 μg/ml, increased caspase activity 3.2-fold (p<0.05). In subsequent studies a concentration of 20 ng/ml TNF-α and 200 µg/ml CML-collagen or control collagen was used, unless stated otherwise.
Figure 1 Tumor necrosis factor-alpha (TNF-α) and Advanced glycation endproduct (AGE) induce apoptosis in pericytes in vitro. Primary bovine retinal pericytes were incubated with the indicated concentration of (A) TNF-α (0–20 ng/ml) or ( (more ...)
Studies were undertaken to determine whether TNF-α or CML-collagen stimulated DNA-binding activity of FOXO1 by EMSA. In the absence of TNF-α or CML-collagen stimulation, insignificant amounts of FOXO1 activation were detected in retinal pericytes (). Incubation with TNF-α (20 ng/ml) or CML-Collagen (200 μg/ml) induced FOXO1 DNA-binding activity. This was shown to be specific by competitive inhibition with excess unlabeled FOXO1 probe ().
Figure 2 Forkhead box O1 (FOXO1) was activated in response to Tumor necrosis factor alpha (TNF-α) and advanced glycation endproduct (AGE) in retinal pericytes. Primary bovine retinal pericytes were stimulated with TNF-α (20 ng/ml), carboxymethyllysine (more ...)
siRNA studies were next performed to establish the functional role of FOXO1 activation as an essential component in TNF-α-stimulated retinal pericyte apoptosis (). Cells were pre-incubated for 48 h with siRNA, which was previously shown to be a strong FOXO1 silencer (siRNA-A), a partial silencer (siRNA-B), or scrambled siRNA, as discussed in the Methods section. In the presence of siRNA-A, binding activity of FOXO1 in response to TNF-α (20 ng/ml) stimulation decreased significantly, while siRNA-B had a partial effect and scrambled siRNA was not able to decrease FOXO1 DNA-binding activity (). To further investigate the functional role of FOXO1 activity on TNF-α- or CML-collagen-induced apoptosis, we measured pericyte apoptosis in the presence of different siRNAs. In the absence of TNF-α stimulation, there was little apoptosis, which was not affected by transfection with siRNA-A (). TNF-α induced an eightfold increase in the level of apoptosis (p<0.05). When transfected with strong silencing siRNA-A, apoptosis was reduced approximately 71% and was reduced by approximately 33% with moderately silencing siRNA-B (). CML-collagen stimulated a threefold increase in apoptosis (). Preincubation with FOXO1 siRNA-A reduced the level of apoptosis in response to CML-collagen by 60% (p<0.05). Transfection per se was not responsible for the reduced apoptosis since scrambled siRNA had no effect ().
Figure 3 Forkhead box O1 (FOXO1) plays a major role in tumor necrosis factor-alpha (TNF-α)- or advanced glycation endproduct (AGE)-induced apoptosis in retinal pericytes. A: Primary cultures of retinal pericytes were transfected with different small interfering (more ...)
To gain insight into how TNF-α or an AGE might regulate FOXO1 activation and apoptosis, specific inhibitors of the MAP kinase signaling pathway were used. When retinal pericytes were preincubated with p38 or JNK inhibitors, TNF-α-induced FOXO1 DNA-binding activity was substantially reduced, as determined by EMSA (). A similar result was obtained in response to AGE-induced FOXO1 activation. Specificity was demonstrated by competitive inhibition with excess unlabeled probe (). The same inhibitors were then used to determine the functional role of p38 and JNK in TNF-α- or AGE-induced apoptosis (). TNF-α stimulated an eightfold increase in apoptosis. Apoptosis was reduced approximately 63% and 38% when cells were incubated with JNK and p38 inhibitors, respectively. However, when both inhibitors were used simultaneously, TNF-α-stimulated apoptosis was reduced by 77%, which was greater than either inhibitor alone (p<0.05; ).
Figure 4 Jun N-terminal kinase (JNK) and P38 inhibitors block tumor necrosis factor (TNF)-induced Forkhead box O1 (FOXO1) activation and apoptosis. Primary bovine retinal pericytes were preincubated with or without the p38 inhibitor or JNK inhibitor for 2 h, followed (more ...)
p38 inhibition decreased CML-collagen-induced apoptosis 34%, and the JNK inhibitor decreased apoptosis by 41%, both of which were statistically significant (p<0.05; ). However, when both inhibitors were used simultaneously, CML-collagen-stimulated apoptosis was reduced by 58%, which was greater than either inhibitor alone (p<0.05; ). Thus, both p38 and JNK arms of this signaling pathway are involved in CML-collagen-induced apoptosis. Doubling the concentration of p38 and JNK inhibitors did not significantly increase their inhibitory effect on TNF-α- or CML-collagen-stimulated apoptosis (data not shown).
Akt, also known as protein kinase B, inhibits apoptosis through multiple mechanisms, one of which is the phosphorylation of FOXO1, which prevents FOXO1 translocation to the nucleus [19
]. Similarly, NF-κB in many cell types is directly anti-apoptotic [34
]. We further investigated apoptosis by determining whether Akt or NF-κB were affecting TNF-α-induced apoptosis. When Akt was inhibited, the capacity of TNF-α to stimulate apoptosis increased by 34% (), which was statistically significant (p<0.05). To investigate the role of NF-κB in apoptosis of pericytes, apoptosis of pericytes in the presence of TNF-α and NF-κB inhibitor was measured. Inhibition of NF-κB enhanced TNF-α-induced apoptosis by 41% (p<0.05; ).
Figure 5 Inhibition of AKT or nuclear factor kappa B (NF-κB) enhances tumor necrosis factor-alpha (TNF-α)-induced apoptosis. Primary bovine retinal pericytes were preincubated with or without the Akt inhibitor, the specific NF-κB inhibitor (more ...)