Reduced expression of angiotensin I-converting enzyme in caveolin-1 knockout mouse lungs

Abstract

Reduced lung capillary expression of angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, and of caveolin-1, an important regulator of endothelial cell signalling, has been demonstrated in various models of pulmonary arterial hypertension (PAH). We addressed the relationship between PAH and ACE expression in caveolin-1 knockout mice (Cav1(-/-)), which have moderate PAH. Tissue ACE activity was reduced by 50% in lungs from 3-month-old Cav1(-/-) mice compared to wild type (WT). A similar reduction in lung endothelial ACE expression was observed by measuring the lung uptake of (125)I-labeled monoclonal anti-ACE antibody and by quantitative immunohistochemistry. These alterations in ACE are limited to capillary segments of the pulmonary circulation. Functionally, the increase in pulmonary artery pressure (PAP) in response to ACE conversion of angiotensin I to angiotensin II in isolated, perfused mouse lungs was reduced significantly in Cav1(-/-) mice compared to WT. Thus, these complementary approaches demonstrate the dependence of lung microvascular endothelial cell ACE protein expression on caveolin-1 expression and underscore the vital role of caveolin-1-regulated pulmonary vascular homeostasis on endothelial ACE expression and activity. In summary, we have revealed a novel role of caveolin-1 in the regulation of ACE expression in pulmonary capillary endothelial cells. Further understanding of the mechanism by which reduced caveolin-1 expression leads altered pulmonary vascular development, PAH, and reduced ACE expression may have important clinical implications in patients with these severe lung diseases.

Figure 1. Tissue ACE activity

Mice were sacrificed, blood and tissues weighed, and homogenates (1:10 w/v ratio) prepared as described in Methods. ACE activity was measured fluorimetrically with Hip-Hs-Leu as substrate. A. ACE activity of blood (mU/ml) and tissues (mU/g of tissue) from WT mice. (B) ACE activity in Cav1^−/− mice expressed as percentage of wild-type mice. Data are mean ± SD, N =7; * p< 0.05.

Figure 2. Endothelial ACE expression

Accumulation of intravenously injected ¹²⁵I-labeled mAb 4B10.5 was used to assess intravascular endothelial cell surface ACE expression. Radiolabeled mAb injected intravenously into WT and Cav1^−/− mice was measured in blood and tissues after circulating for 1 hr. A, B. ¹²⁵I-mAb 4B10.5 accumulation is expressed as cpm/ml of plasma or cpm/gram of tissue (A) and as the organ/blood ratio (B). C. Accumulation of mAb 4B10.5 in organs of Cav1^−/− mice is expressed as a % of WT animals. Data are mean ±SD, n=7; * p< 0.05.

Figure 3. Tissue histology and immunohistochemistry of lung ACE

Representative paraffin sections from Cav1^−/− (A, C, and E) and WT mice (B, D, and F). Hematoxylin-eosin staining of lung tissue sections shows a substantial thickening of the alveolar wall and collapsed alveolae apparent in Cav1^−/− mice (A) compared with the WT in B. Original magnification, x400, H&E. ACE immunostaining shows uniform ACE labelling in the endothelium of the entire alveolar microcirculation of Cav1^−/− mice (C) and WT (D). Quantitative immunostaining for ACE revealed a 40% decrease in ACE expression in Cav1^−/− lungs compared to WT, as quantified by computer-assisted pixel analysis, based on at least 10 lung sections from 3 animals from each strain (WT and Cav1^−/−) and at least 5 views from each section. C/D mouse lung: mAb 4G6, original magnification × 200, rat APAAP In all organs the percentage of ACE-positive EC in Cav1^−/− mice (E) and WT (F) is equal, but the intensity of staining per EC is significantly reduced in Cav1^−/− mice. E and F show heart muscle with a small muscular artery. Visual quantification revealed weak expression in EC of Cav1^−/− mice (arrow in E) and strong expression in EC of WT (arrow in F). E/F mouse heart: mAb 4G6, original magnification x400, rat APAAP.

Figure 4. Effect of AI Infusion on Pulmonary Artery Pressure in the Isolated Perfused Mouse lung

Pulmonary artery pressure (PAP) was measured in isolated buffer-perfused and ventilated mouse lung preparations (flow rate = 2.0 ml/min) at baseline and following infusion of angiotensin I (ATI) and angiotensin II (ATII). Data presented in panel A are absolute PAP values in cmH2O and in panel B are as percent change from baseline following infusion of ATI and ATII. PAP at baseline was significantly increased (by 15%) in Cav1^−/− lungs compared to WT (A). Infusion of 1 μM and 10 μM ATI increased PAP in both groups (A, B), but the response was attenuated in Cav1^−/− lungs (A, B). Infusion of 10μM ATII also raised PAP to a greater extent in WT lungs compared to Cav1^−/− lungs (A, B) (*p<0.05, n=3–4).