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Candesartan attenuates diabetic retinal vascular pathology by restoring glyoxalase-I function.

Miller AG, Tan G, Binger KJ, Pickering RJ, Thomas MC, Nagaraj RH, Cooper ME, Wilkinson-Berka JL - Diabetes (2010)

Bottom Line: In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO(•)), the latter being a known negative regulator of GLO-I in BRP.In BREC and BRP, candesartan restored GLO-I and reduced NO(•).Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress Laboratory, Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia. antonia.miller@monash.edu

ABSTRACT

Objective: Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy.

Research design and methods: Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats.

Results: In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO(•)), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO(•). Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO(•), and restored GLO-I.

Conclusions: We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.

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BREC and BRP apoptosis as detected by TUNEL staining and flow cytometry, respectively, after treatment with Ang II. In BREC, TUNEL staining is increased after treatment with 100 nmol/l Ang II for 24 h (A) compared with control (C). DAPI nuclear staining of Ang II-treated (B) and control (D) BREC. Arrows denote TUNEL-positive BREC, and arrowhead denotes cellular blebbing, a common feature of apoptosis. Magnification ×200. Representative example of Annexin V-FITC (x-axis) and propidium iodide (PI) staining (y-axis) to detect apoptotic cells after treatment of BRP with 100 nmol/l Ang II (E) or control (F) for 24 h. Increases were observed in the Annexin V positive, or early apoptotic phase (bottom right-hand quadrant), and Annexin V positive/PI positive (top right-hand quadrant), or late apoptotic phase (E). Bottom left quadrant, viable cells; Top left quadrant, necrotic cells (PI staining only). G: Graphical representation of BREC apoptosis detected by TUNEL staining; *P < 0.01 versus control. N = 3 samples and is a representative dataset of three independent experiments. H: Graphical representation of BRP apoptosis detected by Annexin/PI staining; *P < 0.03 versus control. All data were analyzed by unpaired t tests. N = 3 independent experiments. Values are mean ± SEM. (A high-quality color representation of this figure is available in the online issue.)
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Figure 1: BREC and BRP apoptosis as detected by TUNEL staining and flow cytometry, respectively, after treatment with Ang II. In BREC, TUNEL staining is increased after treatment with 100 nmol/l Ang II for 24 h (A) compared with control (C). DAPI nuclear staining of Ang II-treated (B) and control (D) BREC. Arrows denote TUNEL-positive BREC, and arrowhead denotes cellular blebbing, a common feature of apoptosis. Magnification ×200. Representative example of Annexin V-FITC (x-axis) and propidium iodide (PI) staining (y-axis) to detect apoptotic cells after treatment of BRP with 100 nmol/l Ang II (E) or control (F) for 24 h. Increases were observed in the Annexin V positive, or early apoptotic phase (bottom right-hand quadrant), and Annexin V positive/PI positive (top right-hand quadrant), or late apoptotic phase (E). Bottom left quadrant, viable cells; Top left quadrant, necrotic cells (PI staining only). G: Graphical representation of BREC apoptosis detected by TUNEL staining; *P < 0.01 versus control. N = 3 samples and is a representative dataset of three independent experiments. H: Graphical representation of BRP apoptosis detected by Annexin/PI staining; *P < 0.03 versus control. All data were analyzed by unpaired t tests. N = 3 independent experiments. Values are mean ± SEM. (A high-quality color representation of this figure is available in the online issue.)

Mentions: To determine whether Ang II induces apoptosis in retinal vascular cells, BREC and BRP were incubated with 100 nmol/l Ang II for 24 h. In BREC and BRP, Ang II treatment resulted in a 500 and 30% increase in either TUNEL or Annexin-positive cells, respectively, compared with control (Fig. 1). Given this finding, we next evaluated GLO-I in BREC and BRP and found that, in both cell types, Ang II decreased GLO-I activity and mRNA by 20% compared with controls (Fig. 2). To confirm the involvement of Ang II in the downregulation of GLO-I, we next measured GLO-I after treatment with the AT1-RB, candesartan. In both BREC and BRP, candesartan restored both GLO-I activity and mRNA to control levels in Ang II-treated cells (Fig. 2), and in the case of BREC, GLO-I mRNA was elevated above control levels (Fig. 2). Overall, BREC was more responsive to the actions of candesartan in terms of restoration of GLO-I function. Candesartan in the absence of Ang II, in almost all instances, did not influence GLO-I levels (see supplementary Fig. 1, available in the online appendix).


Candesartan attenuates diabetic retinal vascular pathology by restoring glyoxalase-I function.

Miller AG, Tan G, Binger KJ, Pickering RJ, Thomas MC, Nagaraj RH, Cooper ME, Wilkinson-Berka JL - Diabetes (2010)

BREC and BRP apoptosis as detected by TUNEL staining and flow cytometry, respectively, after treatment with Ang II. In BREC, TUNEL staining is increased after treatment with 100 nmol/l Ang II for 24 h (A) compared with control (C). DAPI nuclear staining of Ang II-treated (B) and control (D) BREC. Arrows denote TUNEL-positive BREC, and arrowhead denotes cellular blebbing, a common feature of apoptosis. Magnification ×200. Representative example of Annexin V-FITC (x-axis) and propidium iodide (PI) staining (y-axis) to detect apoptotic cells after treatment of BRP with 100 nmol/l Ang II (E) or control (F) for 24 h. Increases were observed in the Annexin V positive, or early apoptotic phase (bottom right-hand quadrant), and Annexin V positive/PI positive (top right-hand quadrant), or late apoptotic phase (E). Bottom left quadrant, viable cells; Top left quadrant, necrotic cells (PI staining only). G: Graphical representation of BREC apoptosis detected by TUNEL staining; *P < 0.01 versus control. N = 3 samples and is a representative dataset of three independent experiments. H: Graphical representation of BRP apoptosis detected by Annexin/PI staining; *P < 0.03 versus control. All data were analyzed by unpaired t tests. N = 3 independent experiments. Values are mean ± SEM. (A high-quality color representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 1: BREC and BRP apoptosis as detected by TUNEL staining and flow cytometry, respectively, after treatment with Ang II. In BREC, TUNEL staining is increased after treatment with 100 nmol/l Ang II for 24 h (A) compared with control (C). DAPI nuclear staining of Ang II-treated (B) and control (D) BREC. Arrows denote TUNEL-positive BREC, and arrowhead denotes cellular blebbing, a common feature of apoptosis. Magnification ×200. Representative example of Annexin V-FITC (x-axis) and propidium iodide (PI) staining (y-axis) to detect apoptotic cells after treatment of BRP with 100 nmol/l Ang II (E) or control (F) for 24 h. Increases were observed in the Annexin V positive, or early apoptotic phase (bottom right-hand quadrant), and Annexin V positive/PI positive (top right-hand quadrant), or late apoptotic phase (E). Bottom left quadrant, viable cells; Top left quadrant, necrotic cells (PI staining only). G: Graphical representation of BREC apoptosis detected by TUNEL staining; *P < 0.01 versus control. N = 3 samples and is a representative dataset of three independent experiments. H: Graphical representation of BRP apoptosis detected by Annexin/PI staining; *P < 0.03 versus control. All data were analyzed by unpaired t tests. N = 3 independent experiments. Values are mean ± SEM. (A high-quality color representation of this figure is available in the online issue.)
Mentions: To determine whether Ang II induces apoptosis in retinal vascular cells, BREC and BRP were incubated with 100 nmol/l Ang II for 24 h. In BREC and BRP, Ang II treatment resulted in a 500 and 30% increase in either TUNEL or Annexin-positive cells, respectively, compared with control (Fig. 1). Given this finding, we next evaluated GLO-I in BREC and BRP and found that, in both cell types, Ang II decreased GLO-I activity and mRNA by 20% compared with controls (Fig. 2). To confirm the involvement of Ang II in the downregulation of GLO-I, we next measured GLO-I after treatment with the AT1-RB, candesartan. In both BREC and BRP, candesartan restored both GLO-I activity and mRNA to control levels in Ang II-treated cells (Fig. 2), and in the case of BREC, GLO-I mRNA was elevated above control levels (Fig. 2). Overall, BREC was more responsive to the actions of candesartan in terms of restoration of GLO-I function. Candesartan in the absence of Ang II, in almost all instances, did not influence GLO-I levels (see supplementary Fig. 1, available in the online appendix).

Bottom Line: In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO(•)), the latter being a known negative regulator of GLO-I in BRP.In BREC and BRP, candesartan restored GLO-I and reduced NO(•).Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress Laboratory, Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia. antonia.miller@monash.edu

ABSTRACT

Objective: Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy.

Research design and methods: Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats.

Results: In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO(•)), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO(•). Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO(•), and restored GLO-I.

Conclusions: We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.

Show MeSH
Related in: MedlinePlus