Vascular immunotargeting is a mean for a site-selective delivery of drugs and genes to endothelium. In this study, we compared recognition of pulmonary and systemic vessels in rats by candidate carrier monoclonal antibodies (MAbs) to endothelial antigens platelet endothelial cell adhesion molecule (PECAM)-1 (CD31), intercellular adhesion molecule (ICAM)-1 (CD54), Thy-1.1 (CD90.1), angiotensin-converting enzyme (ACE; CD143), and OX-43. Tissue immunostaining showed that endothelial cells were Thy-1.1 positive in capillaries but negative in large vessels. In the lung, anti-ACE MAb provided a positive staining in 100% capillaries vs. 5–20% capillaries in other organs. Other MAbs did not discriminate between pulmonary and systemic vessels. We determined tissue uptake after infusion of 1 μg of¹²⁵I-labeled MAbs in isolated perfused lungs (IPL) or intravenously in intact rats. Uptake in IPL attained 46% of the injected dose (ID) of anti-Thy-1.1 and 20–25% ID of anti-ACE, anti-ICAM-1, and anti-OX-43 (vs. 0.5% ID of control IgG). However, after systemic injection at this dose, only anti-ACE MAb 9B9 displayed selective pulmonary uptake (16 vs. 1% ID/g in other organs). Anti-OX-43 displayed low pulmonary (0.5% ID/g) but significant splenic and cardiac uptake (7 and 2% ID/g). Anti-Thy-1.1 and anti-ICAM-1 displayed moderate pulmonary (4 and 6% ID/g, respectively) and high splenic and hepatic uptake (e.g., 18% ID/g of anti-Thy-1.1 in spleen). The lung-to-blood ratio was 5, 10, and 15 for anti-Thy-1.1, anti-ACE, and anti-ICAM-1, respectively. PECAM antibodies displayed low pulmonary uptake in perfusion (2% ID) and in vivo (3–4% ID/g). However, conjugation with streptavidin (SA) markedly augmented pulmonary uptake of anti-PECAM in perfusion (10–54% ID, depending on an antibody clone) and in vivo (up to 15% ID/g). Therefore, ACE-, Thy-1.1-, ICAM-1-, and SA-conjugated PECAM MAbs are candidate carriers for pulmonary targeting. ACE MAb offers a high selectivity of pulmonary targeting in vivo, likely because of a high content of ACE-positive capillaries in the lungs.

Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).

Al injury than lung wet-to-dry weight ratio. The aim of this study was to evaluate the potential of the anesthetic propofol, which has antioxidant properties, to prevent oxidative lung injury by measuring ACE shedding. ACE release from isolated perfused rat lungs increased significantly after ischemia-reperfusion (I/R). Propofol significantly decreased I/R-induced ACE release by 23.4% (P < 0.05). Perfusion with 0.75 mM H2O2 also caused ACE release from the lung microvasculature, which was similarly attenuated by propofol. The protective effect of propofol on H2O2-induced ACE shedding was confirmed in vitro using Chinese Hamster Ovary cells overexpressing human ACE. Thus, propofol can attenuate oxidative injury of the pulmonary endothelium as detected by ACE shedding in I/R and H2O2 models of acute lung injury....

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of 2 homologous domains, each bearing a Zn-dependent active site. The ratio of the rates of hydrolysis of 2 synthetic substrates, Z-Phe-His-Leu (ZPHL) and Hip-His-Leu (HHL), is characteristic for each type of ACE: somatic 2-domain 1, N-domain 4.5, and C-domain 0.7 (Williams et al, 1996). We hypothesized that inactivation or selective inhibition of the C-domain within the somatic ACE should increase the ratio from 1 toward higher values, whereas inactivation or inhibition of the N-domain should decrease the ratio to lower values. Temperatures in the 40-60°C range cause preferential inactivation of the C-domain in somatic ACE and an increase in the ZPHL/HHL ratio. Determination of the ZPHL/HHL ratio in freshly 100-fold concentrated urine ruled out the existence of the N-domain fragment in human urine, which appears only as a result of long storage. Inhibition of ACE by common inhibitors also increases the ZPHL/HHL ratio for 2-domain ACE, thus providing a sensitive method for the detection of ACE inhibitors in biological fluids. Therefore, simultaneous measurements of ACE activity with 2 substrates (ZPHL and HHL) and calculation of their ratio allow us to monitor the status of the ACE molecule and detect ACE inhibitors (endogenous and exogenous) in human blood and other biological fluids. This method should find wide application in monitoring clinical trials with ACE inhibitors and in the development of the approach for personalized medicine by these effective drugs.