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High-glucose-induced endothelial cell injury is inhibited by a Peptide derived from human apolipoprotein E.

Bhattacharjee PS, Huq TS, Potter V, Young A, Davenport IR, Graves R, Mandal TK, Clement C, McFerrin HE, Muniruzzaman S, Ireland SK, Hill JM - PLoS ONE (2012)

Bottom Line: We found that elevated expression of heparanase by high glucose was associated with increased shedding of heparan sulfate (ΔHS) and the tight junction protein occludin.Treatment of hRECs with 100 µM apoEdp in the presence of high glucose significantly reduced the expression of heparanase, shedding of ΔHS, and loss of occludin as detected by Western blot analysis.Thus, strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treating diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Xavier University of Louisiana, New Orleans, Louisiana, USA. pbhattac@xula.edu

ABSTRACT
Although the importance of human apolipoprotein E (apoE) in vascular diseases has clearly been established, most of the research on apoE has focused on its role in cholesterol metabolism. In view of the observation that apoE and its functional domains impact extracellular matrix (ECM) remodeling, we hypothesized that apoE could also confer protection against ECM degradation by mechanisms independent of its role in cholesterol and lipoprotein transport. The ECM degrading enzyme, heparanase, is secreted by cells as pro-heparanase that is internalized through low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1) to become enzymatically active. Both apoE and pro-heparanase bind the LRP-1. We further hypothesized that an apoE mimetic peptide (apoEdp) would inhibit the production of active heparanase by blocking LRP-1-mediated uptake of pro-heparanase and thereby decrease degradation of the ECM. To test this hypothesis, we induced the expression of heparanase by incubating human retinal endothelial cells (hRECs) with high glucose (30 mM) for 72 hours. We found that elevated expression of heparanase by high glucose was associated with increased shedding of heparan sulfate (ΔHS) and the tight junction protein occludin. Treatment of hRECs with 100 µM apoEdp in the presence of high glucose significantly reduced the expression of heparanase, shedding of ΔHS, and loss of occludin as detected by Western blot analysis. Either eye drop treatment of 1% apoEdp topically 4 times a day for 14 consecutive days or intraperitoneal injection (40 mg/kg) of apoEdp daily for 14 consecutive days in an in vivo mouse model of streptozotocin-induced diabetes inhibited the loss of tight junction proteins occludin and zona occludin- 1 (ZO-1). These findings imply a functional relationship between apoE and endothelial cell matrix because the deregulation of these molecules can be inhibited by a short peptide derived from the receptor-binding region of apoE. Thus, strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treating diabetic retinopathy.

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ApoEdp treatment prevents retinal tight junction protein loss diabetic mice.(A). RNA samples isolated from different experimental groups of mice retinas were assessed for occludin-specific mRNA using one-step real-time RT-PCR. ApoEdp treatment increased retinal occludin specific mRNA>2-fold in drop-treated eyes and >2.5 fold in animals given intraperitoneal injection. (B). ApoEdp treatment inhibits the loss of retinal occludin-specific protein expression in streptozotocin-induced diabetic mice as determined by Western blot analysis. Significantly upregulated expression of occludin was detected in both routes (topical and intraperitoneal) in the retinas of diabetic mice.
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pone-0052152-g002: ApoEdp treatment prevents retinal tight junction protein loss diabetic mice.(A). RNA samples isolated from different experimental groups of mice retinas were assessed for occludin-specific mRNA using one-step real-time RT-PCR. ApoEdp treatment increased retinal occludin specific mRNA>2-fold in drop-treated eyes and >2.5 fold in animals given intraperitoneal injection. (B). ApoEdp treatment inhibits the loss of retinal occludin-specific protein expression in streptozotocin-induced diabetic mice as determined by Western blot analysis. Significantly upregulated expression of occludin was detected in both routes (topical and intraperitoneal) in the retinas of diabetic mice.

Mentions: To investigate the effect of eye drops (1% apoEdp) given topically for 14 consecutive days or systemic intraperitoneal injection (40 mg/kg) for 14 consecutive days of apoEdp on the retinal tight junction protein occludin, mice were euthanized, the eyes were enucleated, and retinas were dissected. The dissected retinas were processed to harvest total RNA and one-step real-time RT-PCR assays were performed using mouse occludin-specific mRNA primer/probes. As shown in Figure 2A, both topical administration and intraperitoneal injection of apoEdp significantly inhibited the down-regulation of occludin-specific mRNA compared to diabetic non-treated mouse retinas. To correlate the occludin-specific mRNA data at protein level, total proteins extracted from retinas of different experimental groups (Figure 2B) were analyzed using Western-blot to detect occludin protein expression. Both eye drops and intraperitoneal injection of apoEdp significantly attenuated the loss of occludin in diabetic mice (Figure 2B).


High-glucose-induced endothelial cell injury is inhibited by a Peptide derived from human apolipoprotein E.

Bhattacharjee PS, Huq TS, Potter V, Young A, Davenport IR, Graves R, Mandal TK, Clement C, McFerrin HE, Muniruzzaman S, Ireland SK, Hill JM - PLoS ONE (2012)

ApoEdp treatment prevents retinal tight junction protein loss diabetic mice.(A). RNA samples isolated from different experimental groups of mice retinas were assessed for occludin-specific mRNA using one-step real-time RT-PCR. ApoEdp treatment increased retinal occludin specific mRNA>2-fold in drop-treated eyes and >2.5 fold in animals given intraperitoneal injection. (B). ApoEdp treatment inhibits the loss of retinal occludin-specific protein expression in streptozotocin-induced diabetic mice as determined by Western blot analysis. Significantly upregulated expression of occludin was detected in both routes (topical and intraperitoneal) in the retinas of diabetic mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526597&req=5

pone-0052152-g002: ApoEdp treatment prevents retinal tight junction protein loss diabetic mice.(A). RNA samples isolated from different experimental groups of mice retinas were assessed for occludin-specific mRNA using one-step real-time RT-PCR. ApoEdp treatment increased retinal occludin specific mRNA>2-fold in drop-treated eyes and >2.5 fold in animals given intraperitoneal injection. (B). ApoEdp treatment inhibits the loss of retinal occludin-specific protein expression in streptozotocin-induced diabetic mice as determined by Western blot analysis. Significantly upregulated expression of occludin was detected in both routes (topical and intraperitoneal) in the retinas of diabetic mice.
Mentions: To investigate the effect of eye drops (1% apoEdp) given topically for 14 consecutive days or systemic intraperitoneal injection (40 mg/kg) for 14 consecutive days of apoEdp on the retinal tight junction protein occludin, mice were euthanized, the eyes were enucleated, and retinas were dissected. The dissected retinas were processed to harvest total RNA and one-step real-time RT-PCR assays were performed using mouse occludin-specific mRNA primer/probes. As shown in Figure 2A, both topical administration and intraperitoneal injection of apoEdp significantly inhibited the down-regulation of occludin-specific mRNA compared to diabetic non-treated mouse retinas. To correlate the occludin-specific mRNA data at protein level, total proteins extracted from retinas of different experimental groups (Figure 2B) were analyzed using Western-blot to detect occludin protein expression. Both eye drops and intraperitoneal injection of apoEdp significantly attenuated the loss of occludin in diabetic mice (Figure 2B).

Bottom Line: We found that elevated expression of heparanase by high glucose was associated with increased shedding of heparan sulfate (ΔHS) and the tight junction protein occludin.Treatment of hRECs with 100 µM apoEdp in the presence of high glucose significantly reduced the expression of heparanase, shedding of ΔHS, and loss of occludin as detected by Western blot analysis.Thus, strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treating diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Xavier University of Louisiana, New Orleans, Louisiana, USA. pbhattac@xula.edu

ABSTRACT
Although the importance of human apolipoprotein E (apoE) in vascular diseases has clearly been established, most of the research on apoE has focused on its role in cholesterol metabolism. In view of the observation that apoE and its functional domains impact extracellular matrix (ECM) remodeling, we hypothesized that apoE could also confer protection against ECM degradation by mechanisms independent of its role in cholesterol and lipoprotein transport. The ECM degrading enzyme, heparanase, is secreted by cells as pro-heparanase that is internalized through low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1) to become enzymatically active. Both apoE and pro-heparanase bind the LRP-1. We further hypothesized that an apoE mimetic peptide (apoEdp) would inhibit the production of active heparanase by blocking LRP-1-mediated uptake of pro-heparanase and thereby decrease degradation of the ECM. To test this hypothesis, we induced the expression of heparanase by incubating human retinal endothelial cells (hRECs) with high glucose (30 mM) for 72 hours. We found that elevated expression of heparanase by high glucose was associated with increased shedding of heparan sulfate (ΔHS) and the tight junction protein occludin. Treatment of hRECs with 100 µM apoEdp in the presence of high glucose significantly reduced the expression of heparanase, shedding of ΔHS, and loss of occludin as detected by Western blot analysis. Either eye drop treatment of 1% apoEdp topically 4 times a day for 14 consecutive days or intraperitoneal injection (40 mg/kg) of apoEdp daily for 14 consecutive days in an in vivo mouse model of streptozotocin-induced diabetes inhibited the loss of tight junction proteins occludin and zona occludin- 1 (ZO-1). These findings imply a functional relationship between apoE and endothelial cell matrix because the deregulation of these molecules can be inhibited by a short peptide derived from the receptor-binding region of apoE. Thus, strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treating diabetic retinopathy.

Show MeSH
Related in: MedlinePlus