Limits...
Plasma Kallikrein-Kinin System as a VEGF-Independent Mediator of Diabetic Macular Edema

View Article: PubMed Central - PubMed

ABSTRACT

This study characterizes the kallikrein-kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased twofold and 11.0-fold (both P < 0.0001), respectively, in vitreous from subjects with DME compared with those with a macular hole (MH). While the vascular endothelial growth factor (VEGF) level was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r = 0.266, P = 0.112). Using mass spectrometry–based proteomics, we identified 167 vitreous proteins, including 30 that were increased in DME (fourfold or more, P < 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin-induced retinal thickening in mice was not affected by blockade of VEGF receptor 2. Diabetes-induced RVP was decreased by up to 78% (P < 0.001) in Klkb1 (PPK)-deficient mice compared with wild-type controls. B2- and B1 receptor–induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (NOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF-independent mediator of DME.

No MeSH data available.


Role of NOS on RVP induced by BK peptides. A: Effect of l-NAME on DABK- and BK-induced RVP measured using vitreous fluorophotometry. B: Phosphorylation state of eNOS by BRECs in the presence of BK (100 nmol/L) or DABK (100 nmol/L). C: Effects of BK on RVP in WT and eNOS-deficient mice. D: Western blot analysis of iNOS normalized to GAPDH in diabetic and NDM mice injected with DABK. E: Effect DABK on iNOS levels in astrocytes. F: Effects of DABK on RVP in WT and iNOS-deficient mice. **P < 0.01. a.u., arbitrary units; p, phosphorylated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4587649&req=5

Figure 7: Role of NOS on RVP induced by BK peptides. A: Effect of l-NAME on DABK- and BK-induced RVP measured using vitreous fluorophotometry. B: Phosphorylation state of eNOS by BRECs in the presence of BK (100 nmol/L) or DABK (100 nmol/L). C: Effects of BK on RVP in WT and eNOS-deficient mice. D: Western blot analysis of iNOS normalized to GAPDH in diabetic and NDM mice injected with DABK. E: Effect DABK on iNOS levels in astrocytes. F: Effects of DABK on RVP in WT and iNOS-deficient mice. **P < 0.01. a.u., arbitrary units; p, phosphorylated.

Mentions: Increased nitric oxide level contributes to the action and vascular permeability of BK in DR (17,34,35); however, the role of eNOS and iNOS in the effects of the KKS on the retina are not yet available. We examined the role of nitric oxide in BK- and DABK-induced RVP using l-NAME, which is hydrolyzed to NG-nitro-l-arginine, a competitive inhibitor of eNOS, iNOS, and neuronal nitric oxide synthase. Systemic administration of l-NAME in DM rats blocked both the short-term effect of BK and the delayed effect of DABK on RVP (Fig. 7A). The effects of BK and DABK on eNOS phosphorylation were examined using BRECs. The addition of BK to the culture media increased eNOS phosphorylation at Ser1177 within 5 min, and maximum phosphorylation was observed after 30 min. In contrast, the incubation of BRECs with DABK did not alter eNOS phosphorylation at this site (Fig. 7B). The potential role of eNOS on BK-induced RVP was examined in WT and eNOS knockout mice in the absence and presence of 8 weeks of DM. In WT mice, BK increased RVP by 2.1-fold and 1.7-fold at 40 min after intravitreal injection compared with vehicle-injected eyes (P < 0.01) in NDM and DM mice, respectively, whereas intravitreal injection of BK did not alter RVP in eNOS-deficient mice (with/without DM) (Fig. 7C).


Plasma Kallikrein-Kinin System as a VEGF-Independent Mediator of Diabetic Macular Edema
Role of NOS on RVP induced by BK peptides. A: Effect of l-NAME on DABK- and BK-induced RVP measured using vitreous fluorophotometry. B: Phosphorylation state of eNOS by BRECs in the presence of BK (100 nmol/L) or DABK (100 nmol/L). C: Effects of BK on RVP in WT and eNOS-deficient mice. D: Western blot analysis of iNOS normalized to GAPDH in diabetic and NDM mice injected with DABK. E: Effect DABK on iNOS levels in astrocytes. F: Effects of DABK on RVP in WT and iNOS-deficient mice. **P < 0.01. a.u., arbitrary units; p, phosphorylated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4587649&req=5

Figure 7: Role of NOS on RVP induced by BK peptides. A: Effect of l-NAME on DABK- and BK-induced RVP measured using vitreous fluorophotometry. B: Phosphorylation state of eNOS by BRECs in the presence of BK (100 nmol/L) or DABK (100 nmol/L). C: Effects of BK on RVP in WT and eNOS-deficient mice. D: Western blot analysis of iNOS normalized to GAPDH in diabetic and NDM mice injected with DABK. E: Effect DABK on iNOS levels in astrocytes. F: Effects of DABK on RVP in WT and iNOS-deficient mice. **P < 0.01. a.u., arbitrary units; p, phosphorylated.
Mentions: Increased nitric oxide level contributes to the action and vascular permeability of BK in DR (17,34,35); however, the role of eNOS and iNOS in the effects of the KKS on the retina are not yet available. We examined the role of nitric oxide in BK- and DABK-induced RVP using l-NAME, which is hydrolyzed to NG-nitro-l-arginine, a competitive inhibitor of eNOS, iNOS, and neuronal nitric oxide synthase. Systemic administration of l-NAME in DM rats blocked both the short-term effect of BK and the delayed effect of DABK on RVP (Fig. 7A). The effects of BK and DABK on eNOS phosphorylation were examined using BRECs. The addition of BK to the culture media increased eNOS phosphorylation at Ser1177 within 5 min, and maximum phosphorylation was observed after 30 min. In contrast, the incubation of BRECs with DABK did not alter eNOS phosphorylation at this site (Fig. 7B). The potential role of eNOS on BK-induced RVP was examined in WT and eNOS knockout mice in the absence and presence of 8 weeks of DM. In WT mice, BK increased RVP by 2.1-fold and 1.7-fold at 40 min after intravitreal injection compared with vehicle-injected eyes (P < 0.01) in NDM and DM mice, respectively, whereas intravitreal injection of BK did not alter RVP in eNOS-deficient mice (with/without DM) (Fig. 7C).

View Article: PubMed Central - PubMed

ABSTRACT

This study characterizes the kallikrein-kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased twofold and 11.0-fold (both P &lt; 0.0001), respectively, in vitreous from subjects with DME compared with those with a macular hole (MH). While the vascular endothelial growth factor (VEGF) level was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r = 0.266, P = 0.112). Using mass spectrometry&ndash;based proteomics, we identified 167 vitreous proteins, including 30 that were increased in DME (fourfold or more, P &lt; 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin-induced retinal thickening in mice was not affected by blockade of VEGF receptor 2. Diabetes-induced RVP was decreased by up to 78% (P &lt; 0.001) in Klkb1 (PPK)-deficient mice compared with wild-type controls. B2- and B1 receptor&ndash;induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (NOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF-independent mediator of DME.

No MeSH data available.