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

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) [125 I-Ang-(1-12), purity >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 125 I-Ang-(1-12) was significantly decreased (~40%, P<0.0001) in the presence of the human Ang-(1-12) mAb (Fig. 1). The detection of a large peak (88%) of 125 I-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. © FASEB.