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Release and Degradation of Angiotensin I and Angiotensin II from Angiotensinogen by Neutrophil Serine Proteinases
Archives of Biochemistry and Biophysics Vol. 397, No. 1, January 1, pp. 77– 83, 2002 doi:10.1006/abbi.2001.2687, available online at http://www.idealibrary.com on
Release and Degradation of Angiotensin I and Angiotensin II from Angiotensinogen by Neutrophil Serine Proteinases Ahmed Ramaha* and Philip A. Patston* ,1 *Department of Oral Medicine and Diagnostic Sciences and Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612
Received August 24, 2001; published online December 4, 2001
Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing. © 2001 Elsevier Science Key Words: angiotensinogen; angiotensin; cathepsin G; elastase; proteinase 3; serpin; proteolysis; inflammation; neutrophil.
Angiotensinogen is a 452-amino-acid glycoprotein in the serpin family of proteins (1). In contrast to other serpins, angiotensinogen does not act as a proteinase inhibitor but rather as a precursor for the angiotensin peptides. The renin–angiotensin system involves cleavage of angiotensinogen at the amino-terminal end of the protein by renin to give the decapeptide angioten1 To whom correspondence and reprint requests should be addressed at Department of Oral Medicine and Diagnostic Sciences (MC 838), 801 South Paulina, Chicago, IL 60612. Fax: 312 355-2688. E-mail: [email protected].
0003-9861/01 $35.00 © 2001 Elsevier Science All rights reserved.
sin I (AI) 2 (Fig. 1). This can then be cleaved by angiotensin converting enzyme to the octapeptide angiotensin II (AII) which has numerous biological effects including vasoconstriction, release of aldosterone, activation of neutrophils, and stimulating the growth of smooth muscle cells. Although the renin–angiotensin system is an important regulator of blood pressure, it is clear that AII and other bioactive angiotensin peptides [angiotensin 1–7, angiotensin III (residues 2– 8), and angiotensin IV (residues 3– 8)] also have diverse and tissue specific effects on numerous processes including angiogenesis, fibrinolysis, long term potentiation, natriuresis, and wound healing (2– 6). In addition, there is a growing body of evidence that angiotensins have proinflammatory actions and can contribute to atherosclerosis and cardiovascular disease (7–10). It is from this latter context that we initiated the study reported here. It has long been known that neutrophil cathepsin G can directly cleave both angiotensinogen and AI to release AII (11–16). Cathepsin G is a serine proteinase released from the azurophilic granules of stimulated neutrophils, and therefore is involved in the inflammatory process. Once it is released, much of this proteinase remains bound to the surface of the neutrophil where it is protected from inhibition by plasma proteinase inhibitors (17–19). This surface bound cathepsin G has been shown to release AII from AI (19). The in vivo significance of this reaction is not fully understood but is more likely to be involved with the localized inflammatory process than it is with systemic blood-pressure regulation. Cathepsin G is released from the azurophilic granules along with two other serine proteinases, proteinase 3 (myeloblastin) and elastase, which also bind to the surface of the cell (17, 20, 21). The concentration of each proteinase in the azurophilic 2
Abbreviations used: AI, angiotensin I; AII, angiotensin II. 77
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FIG. 1. Pathways of angiotensin I and angiotensin II production. Angiotensinogen is a 452-amino-acid protein in the serpin family, present in blood at 1 M, and is the precursor for the angiotensin peptides, as follows: (i) The renin–angiotensin pathway. Renin is an aspartic proteinase which cleaves between residues 10 and 11 in the amino-terminus of angiotensinogen to release angiotensin I (AI). Angiotensin I is then cleaved by angiotensin converting enzyme (ACE) between residues 8 and 9 to give angiotensin II (AII). This pathway is important in the systemic regulation of blood pressure, and is implicated in development of hypertension. (ii) Neutrophil proteinase-dependent pathway. It has been reported that cathepsin G can cleave both angiotensinogen and angiotensin I to give angiotensin II. We report here that elastase and, to a lesser extent, proteinase 3 can also release angiotensin I and angiotensin II. Furthermore, we show that elastase and cathepsin G can degrade angiotensin II to inactive fragments, as indicated in the figure. More details of the reactions involving the neutrophil proteinases are given under Results.
granule is extremely high, at about 14 mM. On stimulation of the neutrophil approximately 10% of the total cellular content of each proteinase is released, and these proteinases bind to the surface of the neutrophil in the range of 100 –200 ng/10 6 cells (17, 19, 21). In addition, about 38% of resting neutrophils express cell surface proteinase 3 (21, 22). These cell surface proteinases are proposed to play a role in wound healing (23). Cathepsin G, elastase, and proteinase 3 have somewhat similar protein substrate specificity in vitro (23) although their true substrates in vivo are not clear. Of relevance to the current study is the fact that effects of proteinase 3 and elastase on angiotensinogen have not been studied in much detail (19, 24). In view of the similarities between the proteinases, we considered that elastase and proteinase 3 might also be able to produce AII from angiotensinogen in the same manner as cathepsin G, therefore the actions of the three proteinases on the cleavage of angiotensinogen, AI, and AII were compared. It was found that elastase and proteinase 3 could cleave angiotensinogen to give AI and AII. In addition, it was found that cathepsin G, and to a lesser extent elastase, could rapidly degrade AII. The data support the idea that release of neutrophil proteinases provides a mechanism for the rapid local release of AII at sites of inflammation, as well as providing a mechanism to limit the actions of AII such that it is present only transiently.
MATERIALS AND METHODS Purified human angiotensinogen, cathepsin G, proteinase 3, and elastase were purchased from Athens Research Laboratories (Athens, GA). The concentrations were as designated by the supplier and the proteinases were fully active. Dichloroisocoumarin (DCI) was from Sigma (St. Louis, MO). Enzyme Immunoassay kits for AI and AII were from Bachem-Peninsula (Belmont, CA). Angiotensin II was from Bachem-Peninsula and also synthesized by the University of Illinois at Chicago protein chemistry lab. Experiments investigating the release of AI or AII from angiotensinogen by cathepsin G, proteinase 3, and elastase were conducted as follows. Angiotensinogen (5 g; 4 M) was incubated with either 5 g (8.5 M), 0.5 g (0.85 M), or 0.1 g (0.17 M) of proteinase at 37°C, in 100 mM sodium phosphate buffer, pH 7.5, 0.1% Triton X-100. These are designated as 1:1, 1:10, and 1:50 (w:w) ratio of proteinase:angiotensinogen, respectively. At the times indicated in the results section an aliquot was removed, and the activity of the proteinases inhibited by the addition of DCI (25) to a final concentration of 10 mM, and stored on ice prior to processing for assay of AI or AII by the immunoassay kits according to the manufacturer’s instructions. With these kits there was no need to separate the angiotensin peptides from the reaction mixture as the antibodies have exclusive specificity for the designated peptide. Each data set was repeated in independent experiments from two to five times. Results are presented as the amount of AI or AII released in the incubation mixture in nM. Given that the angiotensinogen concentration used was 4 M, this would also represent the maximum amount of AI or AII which could be released. Note that the scale of the Y axis varies in Figs. 2, 3, and 4. To determine the cleavage site for the degradation pathways, 5 g cathepsin G was incubated for 4 h at 37°C with 5 g AII in 100 mM Tris–HCl, pH 7.5, 50 mM NaCl, and 15 g elastase was incubated for 18 h at 37°C with 30 g AII in 100 mM sodium phosphate, pH 7.5, 0.1% Triton X-100. Automated Edman degradation N-terminal se-
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quencing of the reaction mixtures was carried out at the University of Illinois at Chicago protein sequencing facility using an Applied Biosystems 477A sequencer.
RESULTS AND DISCUSSION
Rationale for the Experimental Conditions Used To evaluate the relative potency of cathepsin G, elastase, and proteinase 3 in releasing AI from angiotensinogen, we considered the following points in order to determine the experimental conditions to use. The concentrations of proteinase to use were decided based on what is known about the relevant biological context. The proteinases would be present at high concentrations because they remain bound to the surface of the neutrophil. This high local concentration of enzyme would remain active for minutes rather than just seconds as would occur if it was free in plasma for two reasons. Firstly, the surface bound proteinases are protected from inhibition by plasma proteinases inhibitors (17–21), such as ␣ 1-proteinase inhibitor which is a serpin important for inhibiting cathepsin G, elastase, and proteinase 3 (28, 29). Secondly, activated neutrophils also secrete MMP-9 (gelatinase B), a proteinase which can inactivate ␣ 1-proteinase inhibitor in vivo (26), but has no effect on AI or AII (27). Therefore, the protection from inhibition of the surface bound proteinases, plus the MMP-9 dependent decrease in ␣ 1-proteinase inhibitor, means that locally very high concentrations of active proteinase will result. Such local high concentrations have been shown to be very important in mediating local proteolytic events (30). Given that the concentrations of the proteinases in the azurophilic granules are similar, it is also likely that the amount of each proteinase released by the neutrophil would be about the same. Therefore, as the molecular weights of the proteinases are very similar (cathepsin G, 27,000 Da; elastase, 30,000 Da; proteinase 3, 29,000 Da), we decided to use them at the same amount of micrograms in our incubations. Together, these considerations caused us to use 5, 0.5, or 0.1 g of proteinase (depending on the experiment) with 5 g of angiotensinogen to give proteinase:angiotensinogen (w:w) ratios of 1:1, 1:10, and 1:50 and molar ratios of 2:1, 1:4.7, and 1:23.5, respectively. Release of Angiotensin I from Angiotensinogen by Elastase and Proteinase 3 Angiotensinogen was incubated with elastase or proteinase 3 and assayed for release of AI. Figure 2 shows the time course for the release of AI using a 1:1 (w:w) ratio of proteinase to angiotensinogen. Elastase (squares) released slightly more AI than proteinase 3 (circles), but in both cases the amount of AII drops at the longer time points. This is attributed to the concurrent conversion of AI to AII (see below). Cathepsin
FIG. 2. Release of angiotensin I from angiotensinogen by elastase and proteinase 3. Angiotensinogen was incubated with elastase (squares) or proteinase 3 (circles) using a 1:1 (w:w) ratio as described under Materials and Methods. At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin I.
G is not shown as no release of AI was detected. Whether this is because cathepsin G only removes AII and not AI, or because any AI released is rapidly converted to AII and cannot be detected is not known. Release of Angiotensin II from Angiotensinogen by Cathepsin G, Elastase, and Proteinase 3 Using the same conditions as above angiotensinogen was incubated with cathepsin G, elastase, and proteinase 3 and assayed for release of AII. Figure 3a shows the time course for the release of AII using a 1:1 ratio (w:w) of proteinase to angiotensinogen. With a 1:1 ratio of cathepsin G to angiotensinogen (triangles), there was a very rapid release of AII, followed by an equally rapid degradation of AII. Elastase (squares) released AII at a slower rate, but this did not keep increasing at longer time points suggesting that it might also be degraded. Proteinase 3 (circles) released AII very slowly. Due to the rapid production and degradation of AII with cathepsin G, the time course was repeated using a 1:10 ratio of proteinase to angiotensinogen (Fig. 3b). With a 1:10 ratio of cathepsin G to angiotensinogen, both the reactions were slower as would be expected, as were those with elastase and proteinase 3. Thus at equivalent concentrations of enzyme the relative AII producing activities are cathepsin G ⬎ elastase ⬎ proteinase 3.
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considered to be of minor importance. In contrast to this conclusion, we suggest that the ability of cathepsin G to degrade AII could be an important regulatory mechanism, such that AII is rapidly produced and can bind to local AII receptors, but would be degraded prior to it escaping into the systemic circulation where it could have unwanted effects on blood pressure. With elastase a decrease in AII concentration was also observed at longer time points. Sequencing of the reaction mixture between elastase and AII revealed the sequences DRV and YIH, indicating that elastase cleaved AII between residues 3 and 4 (V and Y), to give a tripeptide (residues 1–3) and pentapeptide (residues 4 – 8). This cleavage site had been reported to occur as a result of incubation of elastase with angiotensinogen (24) and not considered to be important, but it had not been previously seen with AII and elastase. None of the AII fragments produced by cathepsin G and elastase (residues 1– 4, 5– 8, 1–3, and 4 – 8) are believed to have any bioactivity, and we suggest that degradation of AII by cathepsin G and elastase provides a potential mechanism to limit the action of AII to the local environment of the neutrophil and surrounding tissue, and prevents escape of AII into the systemic circulation where it could have unwanted effects on blood pressure. Release of Angiotensin II from Angiotensinogen by All Proteinases Together
FIG. 3. Release of angiotensin II from angiotensinogen by cathepsin G, elastase, and proteinase 3. (a) Angiotensinogen was incubated with cathepsin G (triangles), elastase (squares), or proteinase 3 (circles) using a 1:1 (w:w) ratio. At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin II. (b) Angiotensinogen was incubated with cathepsin G (triangles), elastase (squares), or proteinase 3 (circles) using a 1:10 (w:w) ratio. At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin II.
Degradation of Angiotensin II by Cathepsin G and Elastase To determine the cleavage site for the degradation pathways, cathepsin G was incubated with AII and the reaction mixture subjected to N-terminal sequencing. Sequencing showed the presence of DRVY and IHPF sequences, indicating that cathepsin G cuts AII between residues 4 and 5 (Y and I), to give two tetrapeptide products, residues 1– 4 and 5– 8, respectively. This cleavage site was observed previously (16), but was
An additional experiment was carried out in which angiotensinogen was incubated with the proteinases separately at 1:50, and with all three together with each individual proteinase being at 1:50 (Fig. 4). Under these conditions the same order of potency for AII release was observed [cathepsin G (triangles) ⬎ elastase (circles) ⬎ proteinase 3 (squares)]. The summation of these three data sets is shown in diamonds. When all three proteinases were incubated together with angiotensinogen the release of AII (crosses) was about twice that of the combined data set (diamonds). Thus, when they are present at the same time, all three proteinases appear to act in a synergistic manner to release AII, suggesting that the neutrophil-dependent pathway to generate AII might be even more potent than previously considered when all proteinases are present, further supporting the idea that AII generation by neutrophils is a biologically significant process in inflammation and wound healing (19, 23). The previous studies with cathepsin G (13–15, 19) have only considered the actions of this proteinase alone, and not in combination with elastase or proteinase 3, and so this synergistic action had not been observed previously.
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FIG. 4. Release of angiotensin II from angiotensinogen by cathepsin G, elastase, and proteinase 3 when combined. Angiotensinogen was incubated with cathepsin G (triangles), elastase (squares), or proteinase 3 (circles) using a 1:50 (w:w) ratio. Angiotensinogen was also incubated with all three proteinases (each at 1:50) at the same time (crosses). At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin II. The sum of the values for cathepsin G, elastase, and proteinase 3 individually (that is, the data shown in triangles plus the data in squares plus the data shown in circles) is shown in diamonds.
Proteolysis of Angiotensinogen by Cathepsin G, Elastase, and Proteinase 3: Analysis by SDS–PAGE Renin is a very specific proteinase with only one known substrate, namely angiotensinogen which it cleaves between residues 10 and 11 to release AI. Similarly, angiotensin converting enzyme is specific only for cleavage between residues 8 and 9 in AI to release AII (Fig. 1). In contrast, cathepsin G, elastase, and proteinase 3 are much less specific, as judged by their numerous potential substrates (23). Therefore, it was considered possible that they could cause much more significant digestion of angiotensinogen than just release of AI and AII. To test this idea, angiotensinogen was digested with the proteinases and the reaction mixture run on SDS gels. In the experiment shown in Fig. 5, angiotensinogen (5 g) was incubated with 0.5 or 5 g of proteinase 3, elastase, or cathepsin G for 60 min. Native angiotensinogen runs as a doublet of 66,000 and 60,000 Da (lane 10). The reason for the doublet might be due to glycosylation heterogeneity, although this is not fully understood (31). At the 1:10 ratio angiotensinogen was converted to discrete lower molecular weight species. With proteinase 3 these had molecular weights of 63,000 and 57,000 Da. With elas-
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tase and cathepsin G they had a molecular weight of 55,000 and 50,000 Da. It has been shown that removal of AI by renin does not result in an increase in the mobility of the des-AI-angiotensinogen (32), thus we can conclude that these lower molecular weight bands are also cleaved elsewhere in the N or C terminal regions. At the 1:1 ratios extensive degradation of angiotensinogen occurred, such that discrete protein bands no longer existed. These data show that the proteinases cause more degradation of angiotensinogen than just removal of AI and AII. Whether this degradation is of any biological significance is not clear as the only known function of angiotensinogen is as a precursor of AI and AII. Animal studies have shown that angiotensinogen cleaved by renin is removed from the circulation by the kidney (33–35). Renin only cleaves between residues 10 and 11 and so the resulting des-AI-angiotensinogen is uncleaved elsewhere within the molecule, in contrast to the angiotensinogen resulting from cathepsin G, elastase, and proteinase 3 digestion. Angiotensinogen is a member of the serpin family, but unlike many other serpins it does not inhibit proteinases because it cannot undergo the conformational changes required by the serpin inhibitory mechanism (24). Nevertheless, it is useful to consider some of the properties of other serpins. One feature is that new chemotactic activity has been detected on serpins which have been cleaved by proteinases. In the case of ␣ 1-proteinase inhibitor (36) and ␣ 1-antichymotrypsin (37) this activity resides in the carboxy-terminal region of the molecule. With heparin cofactor II, the chemotactic activity resides in the amino-terminal half of the molecule (38, 39), and is generated as a result of degradation by elastase and cathepsin G (40, 41). Whether any peptide fragments resulting from the more extensive degradation of an-
FIG. 5. Degradation of angiotensinogen by cathepsin G, elastase, and proteinase 3. Angiotensinogen (5 g, lane 10) was incubated at 37°C with proteinase 3 (0.5 g, lane 2; or 5 g, lane 3), elastase (0.5 g, lane 5; or 5 g, lane 6), or cathepsin G (0.5 g, lane 8; or 5 g, lane 9). After incubation for 60 min the reactions were stopped by addition of DCI, sample buffer, and placing on ice, prior to running on a 10% acrylamide SDS gel. Lane 1 shows molecular weight markers of 204,000, 124,000, 80,000, and 49,000 Da. Lanes 4 and 7 are empty. The gel was stained with Coomassie blue.
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giotensinogen by neutrophil proteinases have novel biological activities is currently not known. CONCLUDING REMARKS
As our results are from in vitro studies using purified proteins, the data does not prove a physiological role for the reactions described here. However, cathepsin G knockout mice have a normal phenotype and their neutrophils have normal function (42), suggesting that elastase and proteinase 3 might substitute for cathepsin G in vivo. These mice were, however, deficient in wound healing (43), and there is evidence that AII is involved in the wound healing process (23). An additional consideration is the link between inflammation, AII, and atherosclerosis. Endothelial injury and inflammation are risk factors for development of atherosclerosis (44), as is AII (10, 45). AII can stimulate the synthesis of plasminogen activator inhibitor-1 (46), itself a risk factor for atherosclerosis (47, 48). Thus, neutrophil-dependent AII generation might contribute to the development of atherosclerosis in part through increased expression of plasminogen activator inhibitor-1. Our results are in keeping with the hypothesis that AII generation and inflammation are key processes in the development of cardiovascular disease (9). We suggest that the combination of cathepsin G, elastase, and proteinase 3 released from neutrophils is likely to cause rapid production of AII at the site of injury and inflammation. AII could then cause localized vasoconstriction to reduce blood loss, and have effects on gene expression related to the inflammatory and wound healing processes. This localized release of AII is probably not relevant to the control of blood pressure, as the ability of cathepsin G and elastase to subsequently degrade AII suggests a mechanism by which AII activity will be limited to the environment around the neutrophils. ACKNOWLEDGMENT This work was supported by National Institutes of Health Grant HL-64013.
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Release and Degradation of Angiotensin I and Angiotensin II from Angiotensinogen by Neutrophil Serine Proteinases Ahmed Ramaha* and Philip A. Patston* ,1 *Department of Oral Medicine and Diagnostic Sciences and Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612
Received August 24, 2001; published online December 4, 2001
Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing. © 2001 Elsevier Science Key Words: angiotensinogen; angiotensin; cathepsin G; elastase; proteinase 3; serpin; proteolysis; inflammation; neutrophil.
Angiotensinogen is a 452-amino-acid glycoprotein in the serpin family of proteins (1). In contrast to other serpins, angiotensinogen does not act as a proteinase inhibitor but rather as a precursor for the angiotensin peptides. The renin–angiotensin system involves cleavage of angiotensinogen at the amino-terminal end of the protein by renin to give the decapeptide angioten1 To whom correspondence and reprint requests should be addressed at Department of Oral Medicine and Diagnostic Sciences (MC 838), 801 South Paulina, Chicago, IL 60612. Fax: 312 355-2688. E-mail: [email protected].
0003-9861/01 $35.00 © 2001 Elsevier Science All rights reserved.
sin I (AI) 2 (Fig. 1). This can then be cleaved by angiotensin converting enzyme to the octapeptide angiotensin II (AII) which has numerous biological effects including vasoconstriction, release of aldosterone, activation of neutrophils, and stimulating the growth of smooth muscle cells. Although the renin–angiotensin system is an important regulator of blood pressure, it is clear that AII and other bioactive angiotensin peptides [angiotensin 1–7, angiotensin III (residues 2– 8), and angiotensin IV (residues 3– 8)] also have diverse and tissue specific effects on numerous processes including angiogenesis, fibrinolysis, long term potentiation, natriuresis, and wound healing (2– 6). In addition, there is a growing body of evidence that angiotensins have proinflammatory actions and can contribute to atherosclerosis and cardiovascular disease (7–10). It is from this latter context that we initiated the study reported here. It has long been known that neutrophil cathepsin G can directly cleave both angiotensinogen and AI to release AII (11–16). Cathepsin G is a serine proteinase released from the azurophilic granules of stimulated neutrophils, and therefore is involved in the inflammatory process. Once it is released, much of this proteinase remains bound to the surface of the neutrophil where it is protected from inhibition by plasma proteinase inhibitors (17–19). This surface bound cathepsin G has been shown to release AII from AI (19). The in vivo significance of this reaction is not fully understood but is more likely to be involved with the localized inflammatory process than it is with systemic blood-pressure regulation. Cathepsin G is released from the azurophilic granules along with two other serine proteinases, proteinase 3 (myeloblastin) and elastase, which also bind to the surface of the cell (17, 20, 21). The concentration of each proteinase in the azurophilic 2
Abbreviations used: AI, angiotensin I; AII, angiotensin II. 77
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FIG. 1. Pathways of angiotensin I and angiotensin II production. Angiotensinogen is a 452-amino-acid protein in the serpin family, present in blood at 1 M, and is the precursor for the angiotensin peptides, as follows: (i) The renin–angiotensin pathway. Renin is an aspartic proteinase which cleaves between residues 10 and 11 in the amino-terminus of angiotensinogen to release angiotensin I (AI). Angiotensin I is then cleaved by angiotensin converting enzyme (ACE) between residues 8 and 9 to give angiotensin II (AII). This pathway is important in the systemic regulation of blood pressure, and is implicated in development of hypertension. (ii) Neutrophil proteinase-dependent pathway. It has been reported that cathepsin G can cleave both angiotensinogen and angiotensin I to give angiotensin II. We report here that elastase and, to a lesser extent, proteinase 3 can also release angiotensin I and angiotensin II. Furthermore, we show that elastase and cathepsin G can degrade angiotensin II to inactive fragments, as indicated in the figure. More details of the reactions involving the neutrophil proteinases are given under Results.
granule is extremely high, at about 14 mM. On stimulation of the neutrophil approximately 10% of the total cellular content of each proteinase is released, and these proteinases bind to the surface of the neutrophil in the range of 100 –200 ng/10 6 cells (17, 19, 21). In addition, about 38% of resting neutrophils express cell surface proteinase 3 (21, 22). These cell surface proteinases are proposed to play a role in wound healing (23). Cathepsin G, elastase, and proteinase 3 have somewhat similar protein substrate specificity in vitro (23) although their true substrates in vivo are not clear. Of relevance to the current study is the fact that effects of proteinase 3 and elastase on angiotensinogen have not been studied in much detail (19, 24). In view of the similarities between the proteinases, we considered that elastase and proteinase 3 might also be able to produce AII from angiotensinogen in the same manner as cathepsin G, therefore the actions of the three proteinases on the cleavage of angiotensinogen, AI, and AII were compared. It was found that elastase and proteinase 3 could cleave angiotensinogen to give AI and AII. In addition, it was found that cathepsin G, and to a lesser extent elastase, could rapidly degrade AII. The data support the idea that release of neutrophil proteinases provides a mechanism for the rapid local release of AII at sites of inflammation, as well as providing a mechanism to limit the actions of AII such that it is present only transiently.
MATERIALS AND METHODS Purified human angiotensinogen, cathepsin G, proteinase 3, and elastase were purchased from Athens Research Laboratories (Athens, GA). The concentrations were as designated by the supplier and the proteinases were fully active. Dichloroisocoumarin (DCI) was from Sigma (St. Louis, MO). Enzyme Immunoassay kits for AI and AII were from Bachem-Peninsula (Belmont, CA). Angiotensin II was from Bachem-Peninsula and also synthesized by the University of Illinois at Chicago protein chemistry lab. Experiments investigating the release of AI or AII from angiotensinogen by cathepsin G, proteinase 3, and elastase were conducted as follows. Angiotensinogen (5 g; 4 M) was incubated with either 5 g (8.5 M), 0.5 g (0.85 M), or 0.1 g (0.17 M) of proteinase at 37°C, in 100 mM sodium phosphate buffer, pH 7.5, 0.1% Triton X-100. These are designated as 1:1, 1:10, and 1:50 (w:w) ratio of proteinase:angiotensinogen, respectively. At the times indicated in the results section an aliquot was removed, and the activity of the proteinases inhibited by the addition of DCI (25) to a final concentration of 10 mM, and stored on ice prior to processing for assay of AI or AII by the immunoassay kits according to the manufacturer’s instructions. With these kits there was no need to separate the angiotensin peptides from the reaction mixture as the antibodies have exclusive specificity for the designated peptide. Each data set was repeated in independent experiments from two to five times. Results are presented as the amount of AI or AII released in the incubation mixture in nM. Given that the angiotensinogen concentration used was 4 M, this would also represent the maximum amount of AI or AII which could be released. Note that the scale of the Y axis varies in Figs. 2, 3, and 4. To determine the cleavage site for the degradation pathways, 5 g cathepsin G was incubated for 4 h at 37°C with 5 g AII in 100 mM Tris–HCl, pH 7.5, 50 mM NaCl, and 15 g elastase was incubated for 18 h at 37°C with 30 g AII in 100 mM sodium phosphate, pH 7.5, 0.1% Triton X-100. Automated Edman degradation N-terminal se-
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quencing of the reaction mixtures was carried out at the University of Illinois at Chicago protein sequencing facility using an Applied Biosystems 477A sequencer.
RESULTS AND DISCUSSION
Rationale for the Experimental Conditions Used To evaluate the relative potency of cathepsin G, elastase, and proteinase 3 in releasing AI from angiotensinogen, we considered the following points in order to determine the experimental conditions to use. The concentrations of proteinase to use were decided based on what is known about the relevant biological context. The proteinases would be present at high concentrations because they remain bound to the surface of the neutrophil. This high local concentration of enzyme would remain active for minutes rather than just seconds as would occur if it was free in plasma for two reasons. Firstly, the surface bound proteinases are protected from inhibition by plasma proteinases inhibitors (17–21), such as ␣ 1-proteinase inhibitor which is a serpin important for inhibiting cathepsin G, elastase, and proteinase 3 (28, 29). Secondly, activated neutrophils also secrete MMP-9 (gelatinase B), a proteinase which can inactivate ␣ 1-proteinase inhibitor in vivo (26), but has no effect on AI or AII (27). Therefore, the protection from inhibition of the surface bound proteinases, plus the MMP-9 dependent decrease in ␣ 1-proteinase inhibitor, means that locally very high concentrations of active proteinase will result. Such local high concentrations have been shown to be very important in mediating local proteolytic events (30). Given that the concentrations of the proteinases in the azurophilic granules are similar, it is also likely that the amount of each proteinase released by the neutrophil would be about the same. Therefore, as the molecular weights of the proteinases are very similar (cathepsin G, 27,000 Da; elastase, 30,000 Da; proteinase 3, 29,000 Da), we decided to use them at the same amount of micrograms in our incubations. Together, these considerations caused us to use 5, 0.5, or 0.1 g of proteinase (depending on the experiment) with 5 g of angiotensinogen to give proteinase:angiotensinogen (w:w) ratios of 1:1, 1:10, and 1:50 and molar ratios of 2:1, 1:4.7, and 1:23.5, respectively. Release of Angiotensin I from Angiotensinogen by Elastase and Proteinase 3 Angiotensinogen was incubated with elastase or proteinase 3 and assayed for release of AI. Figure 2 shows the time course for the release of AI using a 1:1 (w:w) ratio of proteinase to angiotensinogen. Elastase (squares) released slightly more AI than proteinase 3 (circles), but in both cases the amount of AII drops at the longer time points. This is attributed to the concurrent conversion of AI to AII (see below). Cathepsin
FIG. 2. Release of angiotensin I from angiotensinogen by elastase and proteinase 3. Angiotensinogen was incubated with elastase (squares) or proteinase 3 (circles) using a 1:1 (w:w) ratio as described under Materials and Methods. At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin I.
G is not shown as no release of AI was detected. Whether this is because cathepsin G only removes AII and not AI, or because any AI released is rapidly converted to AII and cannot be detected is not known. Release of Angiotensin II from Angiotensinogen by Cathepsin G, Elastase, and Proteinase 3 Using the same conditions as above angiotensinogen was incubated with cathepsin G, elastase, and proteinase 3 and assayed for release of AII. Figure 3a shows the time course for the release of AII using a 1:1 ratio (w:w) of proteinase to angiotensinogen. With a 1:1 ratio of cathepsin G to angiotensinogen (triangles), there was a very rapid release of AII, followed by an equally rapid degradation of AII. Elastase (squares) released AII at a slower rate, but this did not keep increasing at longer time points suggesting that it might also be degraded. Proteinase 3 (circles) released AII very slowly. Due to the rapid production and degradation of AII with cathepsin G, the time course was repeated using a 1:10 ratio of proteinase to angiotensinogen (Fig. 3b). With a 1:10 ratio of cathepsin G to angiotensinogen, both the reactions were slower as would be expected, as were those with elastase and proteinase 3. Thus at equivalent concentrations of enzyme the relative AII producing activities are cathepsin G ⬎ elastase ⬎ proteinase 3.
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considered to be of minor importance. In contrast to this conclusion, we suggest that the ability of cathepsin G to degrade AII could be an important regulatory mechanism, such that AII is rapidly produced and can bind to local AII receptors, but would be degraded prior to it escaping into the systemic circulation where it could have unwanted effects on blood pressure. With elastase a decrease in AII concentration was also observed at longer time points. Sequencing of the reaction mixture between elastase and AII revealed the sequences DRV and YIH, indicating that elastase cleaved AII between residues 3 and 4 (V and Y), to give a tripeptide (residues 1–3) and pentapeptide (residues 4 – 8). This cleavage site had been reported to occur as a result of incubation of elastase with angiotensinogen (24) and not considered to be important, but it had not been previously seen with AII and elastase. None of the AII fragments produced by cathepsin G and elastase (residues 1– 4, 5– 8, 1–3, and 4 – 8) are believed to have any bioactivity, and we suggest that degradation of AII by cathepsin G and elastase provides a potential mechanism to limit the action of AII to the local environment of the neutrophil and surrounding tissue, and prevents escape of AII into the systemic circulation where it could have unwanted effects on blood pressure. Release of Angiotensin II from Angiotensinogen by All Proteinases Together
FIG. 3. Release of angiotensin II from angiotensinogen by cathepsin G, elastase, and proteinase 3. (a) Angiotensinogen was incubated with cathepsin G (triangles), elastase (squares), or proteinase 3 (circles) using a 1:1 (w:w) ratio. At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin II. (b) Angiotensinogen was incubated with cathepsin G (triangles), elastase (squares), or proteinase 3 (circles) using a 1:10 (w:w) ratio. At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin II.
Degradation of Angiotensin II by Cathepsin G and Elastase To determine the cleavage site for the degradation pathways, cathepsin G was incubated with AII and the reaction mixture subjected to N-terminal sequencing. Sequencing showed the presence of DRVY and IHPF sequences, indicating that cathepsin G cuts AII between residues 4 and 5 (Y and I), to give two tetrapeptide products, residues 1– 4 and 5– 8, respectively. This cleavage site was observed previously (16), but was
An additional experiment was carried out in which angiotensinogen was incubated with the proteinases separately at 1:50, and with all three together with each individual proteinase being at 1:50 (Fig. 4). Under these conditions the same order of potency for AII release was observed [cathepsin G (triangles) ⬎ elastase (circles) ⬎ proteinase 3 (squares)]. The summation of these three data sets is shown in diamonds. When all three proteinases were incubated together with angiotensinogen the release of AII (crosses) was about twice that of the combined data set (diamonds). Thus, when they are present at the same time, all three proteinases appear to act in a synergistic manner to release AII, suggesting that the neutrophil-dependent pathway to generate AII might be even more potent than previously considered when all proteinases are present, further supporting the idea that AII generation by neutrophils is a biologically significant process in inflammation and wound healing (19, 23). The previous studies with cathepsin G (13–15, 19) have only considered the actions of this proteinase alone, and not in combination with elastase or proteinase 3, and so this synergistic action had not been observed previously.
CLEAVAGE OF ANGIOTENSINOGEN AND ANGIOTENSIN
FIG. 4. Release of angiotensin II from angiotensinogen by cathepsin G, elastase, and proteinase 3 when combined. Angiotensinogen was incubated with cathepsin G (triangles), elastase (squares), or proteinase 3 (circles) using a 1:50 (w:w) ratio. Angiotensinogen was also incubated with all three proteinases (each at 1:50) at the same time (crosses). At the indicated times an aliquot of each reaction was removed for immunoassay of angiotensin II. The sum of the values for cathepsin G, elastase, and proteinase 3 individually (that is, the data shown in triangles plus the data in squares plus the data shown in circles) is shown in diamonds.
Proteolysis of Angiotensinogen by Cathepsin G, Elastase, and Proteinase 3: Analysis by SDS–PAGE Renin is a very specific proteinase with only one known substrate, namely angiotensinogen which it cleaves between residues 10 and 11 to release AI. Similarly, angiotensin converting enzyme is specific only for cleavage between residues 8 and 9 in AI to release AII (Fig. 1). In contrast, cathepsin G, elastase, and proteinase 3 are much less specific, as judged by their numerous potential substrates (23). Therefore, it was considered possible that they could cause much more significant digestion of angiotensinogen than just release of AI and AII. To test this idea, angiotensinogen was digested with the proteinases and the reaction mixture run on SDS gels. In the experiment shown in Fig. 5, angiotensinogen (5 g) was incubated with 0.5 or 5 g of proteinase 3, elastase, or cathepsin G for 60 min. Native angiotensinogen runs as a doublet of 66,000 and 60,000 Da (lane 10). The reason for the doublet might be due to glycosylation heterogeneity, although this is not fully understood (31). At the 1:10 ratio angiotensinogen was converted to discrete lower molecular weight species. With proteinase 3 these had molecular weights of 63,000 and 57,000 Da. With elas-
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tase and cathepsin G they had a molecular weight of 55,000 and 50,000 Da. It has been shown that removal of AI by renin does not result in an increase in the mobility of the des-AI-angiotensinogen (32), thus we can conclude that these lower molecular weight bands are also cleaved elsewhere in the N or C terminal regions. At the 1:1 ratios extensive degradation of angiotensinogen occurred, such that discrete protein bands no longer existed. These data show that the proteinases cause more degradation of angiotensinogen than just removal of AI and AII. Whether this degradation is of any biological significance is not clear as the only known function of angiotensinogen is as a precursor of AI and AII. Animal studies have shown that angiotensinogen cleaved by renin is removed from the circulation by the kidney (33–35). Renin only cleaves between residues 10 and 11 and so the resulting des-AI-angiotensinogen is uncleaved elsewhere within the molecule, in contrast to the angiotensinogen resulting from cathepsin G, elastase, and proteinase 3 digestion. Angiotensinogen is a member of the serpin family, but unlike many other serpins it does not inhibit proteinases because it cannot undergo the conformational changes required by the serpin inhibitory mechanism (24). Nevertheless, it is useful to consider some of the properties of other serpins. One feature is that new chemotactic activity has been detected on serpins which have been cleaved by proteinases. In the case of ␣ 1-proteinase inhibitor (36) and ␣ 1-antichymotrypsin (37) this activity resides in the carboxy-terminal region of the molecule. With heparin cofactor II, the chemotactic activity resides in the amino-terminal half of the molecule (38, 39), and is generated as a result of degradation by elastase and cathepsin G (40, 41). Whether any peptide fragments resulting from the more extensive degradation of an-
FIG. 5. Degradation of angiotensinogen by cathepsin G, elastase, and proteinase 3. Angiotensinogen (5 g, lane 10) was incubated at 37°C with proteinase 3 (0.5 g, lane 2; or 5 g, lane 3), elastase (0.5 g, lane 5; or 5 g, lane 6), or cathepsin G (0.5 g, lane 8; or 5 g, lane 9). After incubation for 60 min the reactions were stopped by addition of DCI, sample buffer, and placing on ice, prior to running on a 10% acrylamide SDS gel. Lane 1 shows molecular weight markers of 204,000, 124,000, 80,000, and 49,000 Da. Lanes 4 and 7 are empty. The gel was stained with Coomassie blue.
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giotensinogen by neutrophil proteinases have novel biological activities is currently not known. CONCLUDING REMARKS
As our results are from in vitro studies using purified proteins, the data does not prove a physiological role for the reactions described here. However, cathepsin G knockout mice have a normal phenotype and their neutrophils have normal function (42), suggesting that elastase and proteinase 3 might substitute for cathepsin G in vivo. These mice were, however, deficient in wound healing (43), and there is evidence that AII is involved in the wound healing process (23). An additional consideration is the link between inflammation, AII, and atherosclerosis. Endothelial injury and inflammation are risk factors for development of atherosclerosis (44), as is AII (10, 45). AII can stimulate the synthesis of plasminogen activator inhibitor-1 (46), itself a risk factor for atherosclerosis (47, 48). Thus, neutrophil-dependent AII generation might contribute to the development of atherosclerosis in part through increased expression of plasminogen activator inhibitor-1. Our results are in keeping with the hypothesis that AII generation and inflammation are key processes in the development of cardiovascular disease (9). We suggest that the combination of cathepsin G, elastase, and proteinase 3 released from neutrophils is likely to cause rapid production of AII at the site of injury and inflammation. AII could then cause localized vasoconstriction to reduce blood loss, and have effects on gene expression related to the inflammatory and wound healing processes. This localized release of AII is probably not relevant to the control of blood pressure, as the ability of cathepsin G and elastase to subsequently degrade AII suggests a mechanism by which AII activity will be limited to the environment around the neutrophils. ACKNOWLEDGMENT This work was supported by National Institutes of Health Grant HL-64013.
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