(954.1) Internalization of Angiotensin-(1-12) in Adult Retinal Pigment Epithelial-19 Cells

Poster Board Number: E576

Sarfaraz Ahmad (Wake Forest University School of Medicine), Kendra Wright (Wake Forest University School of Medicine), Carlos Ferrario (Wake Forest University School of Medicine), Heng-Jie Cheng (Wake Forest University School of Medicine), Mayur Choudhary (Duke University School of Medicine), Goldis Malek (Duke University School of Medicine), Rebecca Sappington (Wake Forest University School of Medicine)

Introduction:   The characterization, by this laboratory, of angiotensin-(1-12) [Ang-(1-12)] as a primary angiotensin II (Ang II)-forming substrate may be an unrecognized source of Ang II-mediated microvascular complication in hypertension-mediated retinopathy. We found that the plasma Ang-(1-12) level was 12-66-fold higher when compared to the angiotensin I in normal human subjects. For the first time, we investigated the endogenous expression and incorporation of Ang-(1-12) in cultured adult retinal pigment epithelial-19 (ARPE-19) cells. We also investigated the internalization of Ang-(1-12) in the presence of a highly specific monoclonal antibody (mAb) that was developed by us against the C-terminus of the human Ang-(1-12) sequence. 

Method: ARPE-19 cells (passage 28-30) were grown to post-confluence in 10% serum supplemented DMEM/F12 medium for 2-3 days. An immunofluorescence (IF) staining was performed to detect the internalized Ang-(1-12) using the human Ang-(1-12) mAb (1:5000 dilution) and the Alexa Flour 488 secondary antibody (1:1000 dilution). Internalization was also confirmed in ARPE-19 using radiolabeled Ang-(1-12) [125I-Ang-(1-12), purity gt;99%] in the presence and absence of the mAb by HPLC. For this, the APRE-19 cells were incubated (4 h) with Ang-(1-12) alone or Ang-(1-12) + mAb. Afterward, cells were washed and harvested in isotonic buffer. The lysed cells were centrifuged at 28,000 g and the membrane pellet and supernatants (cytosolic fractions) were counted on a gamma counter. The cytosolic fractions were further analyzed by HPLC using a C18 column to confirm the internalization of intact Ang-(1-12) sequence.

Results: Our IF staining shows that Ang-(1-12) is endogenously expressed in ARPE-19 cells. Further, an increased intensity of staining was detected in ARPE-19 cells exposed to Ang-(1-12) (100 nM). High Ang-(1-12) radiolabeled counts were detected in the cytosolic fractions (3.1% of the total loaded radiolabeled) and very low counts in the membrane pellet (0.2%). We found that the internalization of 125I-Ang-(1-12) was significantly decreased (~40%, Plt;0.0001) in the presence of the human Ang-(1-12) mAb (Fig. 1). The detection of a large peak (88%) of 125I-Ang-(1-12) in the cytosolic fraction in the HPLC chromatogram further confirms that the intact Ang-(1-12) sequence is internalized by the cells (Fig. 2).

Conclusions: Ang-(1-12) is internalized by ARPE-19 cells from extracellular spaces of the retina and the internalization of Ang-(1-12) decreased following exposure to the human Ang-(1-12) mAb. Overall our data suggests the human Ang-(1-12) mAb may be used as a novel therapeutic agent to prevent circulating Ang-(1-12) internalization into the retinal cells and subsequently reduce the intracellular generation of the Ang II from the Ang-(1-12) substrate.





Fig. 1: Internalization of 125I-Ang-(1-12) by ARPE-19 cells in the presence or absence of human Ang-(1-12) monoclonal antibody (mAb). Cells harvested after 4h treatment, washed in PBS and cells associated radiolabeled 125I-Ang-(1-12) counted on a gamma counter. Results are expressed as counts per mg protein.; Fig. 2: HPLC chromatogram of internalized 125I-Ang-(1-12). Harvested cells were lysed in isotonic buffer and centrifuged at 28,000 g. The supernatant (cytosolic fraction) was further analyzed by HPLC connected to in-line flow-through gamma detector.