Critical role of TXNIP in oxidative stress, DNA damage and retinal pericyte apoptosis under high glucose: implications for diabetic retinopathy - PubMed (original) (raw)

Critical role of TXNIP in oxidative stress, DNA damage and retinal pericyte apoptosis under high glucose: implications for diabetic retinopathy

Takhellambam S Devi et al. Exp Cell Res. 2013.

Abstract

Diabetic retinopathy (DR) is characterized by early loss of retinal capillary pericytes and microvascular dysfunction. We recently showed that pro-oxidative stress and pro-apoptotic thioredoxin interacting protein (TXNIP) is significantly up-regulated in rat retinas in experimental diabetes and mediates inflammation and apoptosis. Therefore, we hypothesize here that TXNIP up-regulation in pericyte plays a causative role in oxidative stress and apoptosis under sustained high glucose exposure in culture. We maintained a rat retinal capillary pericyte cell line (TR-rPCT1) for 5 days under low glucose (LG, 5.5mM) or high glucose (HG, 25 mM) with or without anti-oxidant N-acetylcysteine (5mM, NAC), Azaseine (2 μM, AzaS), an inhibitor of TXNIP, and TXNIP siRNA (siTXNIP3, 20 nM). The results show that HG increases TXNIP expression in TR-rPCT1, which correlates positively with ROS generation, protein S-nitrosylation, and pro-apoptotic caspase-3 activation. Furthermore, pericyte apoptosis is demonstrated by DNA fragmentation (alkaline comet assay) and a reduction in MTT survival assay. Treatment of TR-rPCT1 with NAC or an inhibition of TXNIP by AzaS or siTXNIP3 each reduces HG-induced ROS, caspase-3 activation and DNA damage demonstrating that TXNIP up-regulation under chronic hyperglycemia is critically involved in cellular oxidative stress, DNA damage and retinal pericyte apoptosis. Thus, TXNIP represents a novel gene and drug target to prevent pericyte loss and progression of DR.

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Figures

Fig. 1

HG induces TXNIP expression and ROS/RNS generation in retinal pericytes. (a) Quantitative RT-PCR for TXNIP. HG increases TXNIP mRNA level significantly in retinal pericytes at day 5 (1.45+/−0.14; _n_=4; _p_=0.01) when compared with LG. (b) IHC of TXNIP in pericytes cultured under HG for 5 days shows enhanced TXNIP staining than in LG (arrows). A representative of _n_=3 is shown. (c) WB of TXNIP in TR-rPCT1. There is also a significant increase (p<0.05) in TXNIP protein in HG at day 5 (2.58+/−0.77; _n_=4) when compared with LG.

Fig. 2

HG induces pericyte ROS/RNS levels and apoptosis in retinal pericytes. (a) Reactive oxygen species (ROS) detected by CM-H2DCFDA. ROS level is significantly increased by HG at day 5 (p<0.05) compared to LG. On the other hand, a 20 mM mannitol plus 5.5 mM LG, an equimolar osmotic effect of HG, does not increase ROS suggesting a specific effect of excess glucose metabolism on ROS production in pericytes. (b) IHC of protein thiol (S) nitrosylation using an anti-_S_-nitroso-cysteine (SNO-Cys) antibody shows enhanced _S_-nitrosylation of proteins in pericytes under HG (arrows). A representative of _n_=3 is shown. (c) Cell viability assay by MTT. HG significantly decreases MTT activity (p<0.0002) when compared with LG. Mannitol has no significant effect on MTT activity in pericytes. (d) IHC of active caspase-3: Cells were cultured in 4 well slide chambers with HG or LG for 5 days. Red SR FLIVO, which is cell membrane permeable and binds to active caspase-3, was added for 30 min, washed and fixed in 4% paraformaldehyde. DAPI was used to stain nuclei. HG activates caspase-3 in retinal pericytes as revealed by enhanced staining of SR FLIVO (arrows). A representative of _n_=3 is shown.

Fig. 3

HG induces DNA breakage and chromatin condensation in retinal pericytes. (a) DAPI staining (IHC) of DNA break and chromatin condensation in pericytes by HG maintained for 5 days (arrows). Note that TXNIP staining (red) colocalizes with broken chromatin. (b) A SINGLE CELL ALKALINE COMET Assay for DNA break also shows DNA tailing in HG suggesting chromatin breakage. A central chromatin nucleoid is also present in HG condition indicating chromatin condensation. The tail portion and chromatin nucleoid are absent in LG indicating intact chromatin. A representative of _n_=3 is shown. (c) Western Blot for histone H3 lysine 9 trimethylation (H3K9Me3), a heterochromatin mark for chromatin condensation, and total H3. HG treatment of TR-rPCT1 for 5 days leads to increases in H3K9Me3 but not at day 3. A representative of _n_=3 is shown.

Fig. 4

_N_-acetylcysteine prevents HG-induced ROS generation and DNA damage in retinal pericytes. (a) ROS generation. _N_-acetylcysteine (NAC, 5 mM) was added during the last 2 days of treatment while HG was present for 5 days. NAC significantly reduces HG-induced ROS generation in TR-rPCT1 (p<0.001; HG vs. HG+NAC). (b) IHC of DNA break and chromatin condensation (DAPI staining). HG induces TXNIP and chromatin breakage (middle panel, arrows). TXNIP up regulation and chromatin breakage by HG are reduced by NAC (right panel), which is more of less comparable to LG (left panel). A representative of _n_=3 is shown. (c) NAC also reduces COMET tailing by DNA break in HG treated pericytes suggesting a role for ROS in chromatin instability and breakage. A representative of _n_=3 is shown.

Fig. 5

Azaserine prevents HG-induced ROS generation, maintains MTT activity, and reduces DNA break in retinal pericytes. (a) ROS generation. Azaserine (AzaS, 2 μM) was added during the last 2 days of treatment while HG was present for 5 days. ROS generation was determined by CM-H2DCFDA. AzaS has not effect on ROS level in LG but prevents the HG-induced ROS generation in TR-rPCT1. (b) MTT activity. In the presence of AzaS, a reduction in the MTT activity by HG is reversed in retinal pericytes. In fact there is a significant increase (p<0.001) in MTT activity in HG+AzaS. (c) IHC of DNA break and chromatin condensation. HG induces chromatin breakage (middle panel, arrows), which is reduced by AzaS (right panel), which is more of less comparable to LG (left panel). A representative of _n_=3 is shown.

Fig. 6

TXNIP siRNA prevents HG-induced ROS generation and DNA damage in retinal pericytes. Scramble (scr)RNA or siTXNIP3 were transiently transfected in pericytes for the last 2 days of 5 days HG treatment. (a) ROS determination by CM-H2DCFDA. HG is able to increase ROS level in scrRNA-treated pericytes (p<0.06 vs. LG+scrRNA). However, after siTXNIP3 transfection, ROS level is significantly reduced (p<0.01; scrRNA+HG vs. siTXNIP3+HG). (b) IHC. SrcRNA transfection has no effect on TXNIP and DNA integrity in LG (left panel). However, HG increases TXNIP staining and DNA damage/breakage (middle panel, arrows) in scrRNA transfected pericytes. On the other hand, siTXNIP3 reduces both TXNIP staining and DNA breakage in pericytes by HG, which is more or less comparable to the level of LG+scrRNA (right panel). (c) Comet assay further shows DNA tailing by HG in scrRNA treated TR-rPCT1 suggesting chromatin breakage. However, with siTXNIP3 transfection, the DNA tailing are absent indicating that TXNIP is involved in chromatin breakage under chronic hyperglycemia (middle panel vs. right panel). A representative of _n_=3 is shown.

Fig. 7

HG does not increase autophagic response in retinal pericytes but induces GADD153 DNA binding activity. (a) IHC of LC3B antibody. The number and size of LC3B punctae (red dots) were marginally increased in IHC. (b) However, on Western blots LC3BII levels, the cleaved and active form, are not significantly different between HG and LG. β-Actin was used for normalization. A representative of _n_=3 is shown for each experiment. ((c)–(d)) EMSA for DNA damage repair protein p53 and pro-apotptotic GADD153. EMSA shows that the DNA binding activity of (c) p53 is unaltered by HG while that of (d) GADD153 is enhanced. The specificity of the reaction is indicated by a competitive inhibition with excess cold probe and a lack of protein–DNA band shift by mannitol. Lane 1. Probe alone; Lane 2. LG; Lane 3. HG; Lane 4. Mannitol; and Lane 5. HG+cold probe (_n_=3–4 for each experiment).

Fig. 8

Summary: A potential role of TXNIP in hyperglycemia-induced ROS/RNS stress, DNA damage and pericyte demise (Pericytopathy) in DR. Chronic hyperglycemia-induced TXNIP up-regulation leads to cellular ROS/RNS stress, mitochondrial membrane depolarization, bioenergetic imbalance (low ATP), chromatin (DNA) fragmentation and pericyte apoptosis. Retinal pericytes appear to have a weaker anti-oxidant and mitophagic response to cellular stress to scavenge ROS and remove depolarized mitochondria, which leak ROS and are inefficient in ATP production. In addition, the DNA damage repair mechanism (p53 activation) is not evoked that may result in chromatin breakage and early demise of pericytes in DR. Anti-oxidant treatment such as NAC and a blockade of TXNIP via an inhibition of the HBP may represent potential therapeutic approaches to ameliorate DR pathogenesis.

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Critical role of TXNIP in oxidative stress, DNA damage and retinal pericyte apoptosis under high glucose: implications for diabetic retinopathy - PubMed (original) (raw)