Novel mass spectrometric methods for evaluation of plasma angiotensin converting enzyme 1 and renin activity

This article demonstrates the applicability of quantitative proteomics to assays of proteolytic enzyme activity. A novel assay was developed for measurement of renin and angiotensin-converting enzyme (ACE) activity in plasma. The method was validated in animal models associated with alterations of the renin angiotensin system (RAS). Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with a ProteinChip Array technology, plasma renin and ACE1 could be measured in <0.5 microL of plasma. Plasma is incubated with peptide substrates for renin and ACE, tetradecapeptide (TDP), and angiotensin I (Ang I), respectively. The reactions mixtures are spotted onto the ProteinChip WCX2 and detected using SELDI-TOF-MS. Peak height or area under curve for TDP, Ang I, and angiotensin II (Ang II) peaks are measured. There was a linear relationship between disappearance of substrate and appearance of products for both renin and ACE (R2=0.95 to 0.98). ACE1 activity was blocked with chelating agents (EDTA and 1,10 phenanthrolene), indicating action of a metalloprotease. The ACE1 inhibitor, captopril, selectively blocked ACE1. Renin activity was specifically blocked with renin inhibitor and was not affected by phenanthrolene or captopril. Animal models tested were Ang AT1a receptor-deficient and streptozotocin (STZ) diabetic mice. Plasma renin activity was increased >2-fold in AT1a(-/-) as compared with AT1a(+/+). In STZ diabetic mice, ACE1 was increased 2-fold as compared with controls. The advantage of the method is that it is tagless, does not require additional purification steps, and is extremely sensitive. The approach can be multiplexed and used for identification of novel substrates/inhibitors of the RAS.     

Tryptic Generation of an Angiotensin Binding Substance

Following trypsin treatment of rat or human plasma, the level of angiotensin I, generated by renin, can be significantly underestimated by radioimmunoassay due to tryptic generation of an angiotensin binding substance. The precursor of the binding substance (void volume-AcA 44 gel) was converted by trypsin to 45K. Analogous to PRC methodology, known concentrations of angiotensin I were added to control and trypsin treated human plasma after the renin incubation step to determine the influence of the binding substance on the measured levels of generated angiotensin I. Using this technique, renin levels in trypsin exposed plasma were approximately two fold higher than when measured by single point conventional assay. If plasma levels of the binding precursor change in response to renin stimulation or suppression, its activation during the trypsin treatment step of the renin assay may explain the relative lack of change of inactive renin observed following numerous in vivo maneuvers.     

Assessment of the renin-angiotensin system in cirrhotic patients. Comparison between plasma renin activity and direct measurement of immunoreactive renin.

The renin-angiotensin system plays an important physiological role and has prognostic significance in cirrhotics with ascites. The degree of stimulation of this system is usually estimated by measuring plasma renin activity after incubation periods of 2-3 h. Recent investigations showed that the direct measurement of immunoreactive renin also estimates the degree of activity of the system. In this study, immunoreactive renin and plasma renin activity (measured at incubation periods of 10, 20, 50 and 180 min) were determined in ten healthy subjects, five hyperreninemic non-hepatic patients and 47 cirrhotics with ascites. Cirrhotic patients showed significantly higher plasma renin activity (5.1 +/- 0.9 ng/ml per h, p less than 0.05) and immunoreactive renin (145.4 +/- 24.4 pg/ml, p less than 0.01) than healthy subjects (1.2 +/- 0.15 ng/ml per h and 25.1 +/- 1.1 pg/ml, respectively). The angiotensin I generation rate was constant during the 3-h incubation in 22 cirrhotics and a close relationship (r = 0.956, p less than 0.001) between plasma renin activity (3.5 +/- 1.6 ng/ml per h) and immunoreactive renin (71 +/- 25 pg/ml) was observed in these patients. In the remaining 25 cirrhotics the generation rate of angiotensin I declined with time and the calculated plasma renin activity at 180 min was lower than the activity calculated at 10 min by 50.7%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin II-producing enzyme III from acidified serum of nephrectomized dogs

A highly active angiotensin-producing enzyme (enzyme III) was obtained from the serum of bilaterally nephrectomized dogs by acid treatment and ammonium sulfate fractionation. An inactive precursor (proenzyme III) was converted to enzyme III during prolonged storage (or by treatment with acid or with cathepsin G or by incubation at 38 degrees C as described in the following paper). Enzyme III reacted maximally at pH 7.7 and it produced up to 400 ng of angiotensin II/mL serum/h (i.e., amounts 4000 times higher than that generated by the endogenous renin present in serum after bilateral nephrectomy). Enzyme III produced angiotensin II at identical rates when either dog angiotensinogen or angiotensin I was used as substrate, but the rate was 710 times higher with synthetic tetradecapeptide renin substrate. Enzyme III is not identical to renin, cathepsin G, tonin, enzyme I, enzyme II, the calcium-dependent angiotensin I-converting enzyme, or the calcium-independent carboxy peptidase, which acts by sequential cleavage of angiotensin I. Enzyme III was inhibited by alpha-1-antitrypsin, diisopropyl fluorophosphate, and lima bean trypsin inhibitor (hence it is a serine proteinase). It was not inhibited by Captopril, Teprotide, or Enalapril. It had been reported previously that cathepsin G released from neutrophil granulocytes, by producing high local concentrations of angiotensin II, may provide a mobile means for modulating blood flow in tissue microvasculature during the inflammatory response. The present study offers a new, additional pathway, by enzyme III, for a similar rapid formation of angiotensin II from serum protein substrate or angiotensin I.     

Studies on the human ovarian renin-angiotensin system: optimization of assay methodology and effects of follicular stimulants

The renal and genital tracts share a common embryological origin; it is thus not surprising that tissues from both can synthesize renin. Preliminary studies showed extremely high concentrations of renin in follicular fluid (FRC) following ovarian stimulation for in-vitro fertilization. This necessitated complete revalidation of the renin assays and showed that data obtained using commercial kits were invalid. An assay protocol was developed using a 1:2 dilution of follicular fluid taken into EDTA (0.3 mol/l) and o-phenanthroline (0.05 mol/l). The assay was performed at pH 7.5 in the presence of excess exogenous (sheep) renin substrate, with incubation periods of 5, 10 and 15 min at 37 degrees C. This protocol resulted in the linear generation of angiotensin I (AI). Activation of inactive renin was performed using eightfold more trypsin than was required for plasma samples. Follicular renin substrate concentrations (FRS) were measured using the same assay methodology as used for measurement of plasma renin substrate concentrations (PRS). Storage of samples at -18 degrees C for up to 2 months was found not to affect the FRC, although repeated freeze-thaw cycles did. FRC and plasma renin concentrations (PRC) were very similar in 25 unstimulated control women, studied in the follicular phase of the menstrual cycle. Trypsin activation increased follicular total renin concentration (FTRC) more than plasma total renin concentration (PTRC) (P less than 0.0001). FRS was slightly higher than PRS (P less than 0.02). Ovarian stimulation with clomiphene citrate (CC; six women) was without effect on these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

No evidence for the existence of a species-specific renin substrate in plasma from normal and bilaterally nephrectomized rats.

Renin substrate in plasma from normal and biolaterally nephrectomized rats was measured using an excess of rat or rabbit renin which had the same pressor activity when directly assayed in the rat. The amounts of angiotensin I generated with an excess of rat renin were similar to those generated with an excess of rabbit renin in plasma from normal and bilaterally nephrectomized rats. Further addition of an excess of homologous renin to the incubation mixture did not generate more angiotensin I from normal and bilaterally nephrectomized rat plasma which had been incubated before with an excess of rabbit renin. The isoelectric focusing profiles of plasma renin substrate from normal and bilaterally nephrectomized rats were almost identical using an excess of either rat or rabbit renin. It is concluded that there is no species-specific renin substrate for homologous renin in normal or bilaterally nephrectomized rats.     

Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor

OBJECTIVE: Mannose 6-phosphate receptors (M6PR) bind both renin and prorenin, and such binding contributes to renin/prorenin clearance but not to angiotensin generation. Here, we evaluated the kinetics of renin/prorenin binding to the recently discovered human (pro)renin receptor (h(P)RR), and the idea that such binding underlies tissue angiotensin generation. METHODS AND RESULTS: Vascular smooth muscle cells from control rats and transgenic rats with smooth muscle h(P)RR overexpression were incubated at 4 or 37 degrees C with human renin or prorenin. Incubation at 37 degrees C greatly increased binding, suggesting that (pro)renin-binding receptors cycle between the intracellular compartment and the cell surface. Blockade of the M6PR reduced binding by approximately 50%. During M6PR blockade, h(P)RR cells bound twice as much prorenin as control cells, while renin binding was unaltered. Incubation of h(P)RR (but not control) cells with prorenin + angiotensinogen yielded more angiotensin than expected on the basis of the activity of soluble prorenin, whereas angiotensin generation during incubation of both cell types with renin + angiotensinogen was entirely due to soluble renin. The renin + angiotensinogen-induced vasoconstriction of isolated iliac arteries from control and transgenic rats was also due to soluble renin only. The recently proposed (P)RR antagonist 'handle region peptide', which resembles part of the prosegment, blocked neither prorenin binding nor angiotensin generation. CONCLUSIONS: H(P)RRs preferentially bind prorenin, and such binding results in angiotensin generation, most likely because binding results in prorenin activation.     

On the measurement of inactive renin in rat plasma: activation by trypsin generates interfering tetradecapeptide-like material and destroys angiotensinogen

Trypsin cleaved plasma angiotensinogen with apparent first-order kinetics and generated an angiotensin I (Ang I) immunoreactive material. Size exclusion high-performance liquid chromatography (HPLC) of rat plasma proteins demonstrated that the Ang I immunoreactive material was formed in those fractions which contained angiotensinogen. The Ang I immunoreactive material was higher in nephrectomized rat plasma than normal plasma, in accordance with the higher angiotensinogen concentration. These findings indicated that angiotensinogen could be the source of the Ang I immunoreactive material. Purification of the Ang I immunoreactive material by cation-exchange chromatography followed by reverse-phase HPLC demonstrated an elution pattern close to that of human tetradecapeptide. The purified Ang I immunoreactive material was cleaved by pure mouse submandibular renin to Ang I, exclusively. Incubation at 37 degrees C of the Ang I immunoreactive material with plasma partially destroyed the angiotensin immunoreactive material. These findings demonstrated that the angiotensin immunoreactive material was an Ang I containing tetradecapeptide (TDP)-like peptide, unstable during a renin incubation step, leading to erroneous values for plasma inactive renin if not removed. The Ang I immunoreactive material was removed by cation-exchange chromatography of trypsin-activated plasma allowing for a determination of inactive renin. The presence of inactive renin in plasma from normal and nephrectomized rats was confirmed, and identified by neutralization and immunoprecipitation with antirenins. These findings should enable us to develop a routine assay for plasma inactive renin in rat plasma.     

Suppression of adrenal renin in Dahl salt-sensitive rats

We previously showed that adrenal renin is highest in the rat zona glomerulosa (ZG) and that low sodium or high potassium and nephrectomy increase adrenal ZG renin and aldosterone. Dahl salt-sensitive rats (S) have been shown to have lower plasma renin activity and plasma aldosterone and higher plasma 18-hydroxy-11-deoxycorticosterone than Dahl salt-resistant rats (R). In this study we assess the possible role of adrenal ZG renin in the suppression of aldosterone in S rats. Adrenal ZG renin was significantly decreased in S as compared with R rats even at 6 weeks of age, when both S and R rats are still normotensive (S = 7.2 +/- 0.2, R = 18.0 +/- 1.6 ng angiotensin I/mg protein/hr). Adrenal ZG aldosterone was also significantly lower in S than in R rats (S = 21.1 +/- 4.3, R = 39.5 +/- 3.6 ng/mg protein). Furthermore, the rise in adrenal ZG renin and aldosterone after nephrectomy in S rats was significantly less than that in R rats. To determine if the suppressed adrenal ZG renin of S rats is due to volume expansion, we studied the effect of a sodium-deficient diet on adrenal ZG renin in S and R rats. After 2 weeks of a sodium-deficient diet S rats had significantly lower basal adrenal ZG renin than did R rats (S = 7.6 +/- 0.4, R = 21.7 +/- 1.9 ng angiotensin I/mg protein/hr) and a marked blunting of the adrenal ZG renin response to nephrectomy (S = 13.6 +/- 1.1, DR = 167 +/- 16.1 ng angiotensin I/mg protein/hr).(ABSTRACT TRUNCATED AT 250 WORDS)     

The Existence of Inactive (Trypsin-Activated) Renin in Dog Plasma and Renin Granules from the Kidney

Trypsin-activated renin (inactive renin) was detected in the break-through fraction when dog plasma or renin extracted from renin granules (stored renin) was applied to a pepstatin column, respectively. The appearance of the renin activity by trypsin treatment was not due to acid protease. Production of angiotensin I from homologous renin substrate by the trypsin-activated renin was proportional to the time of incubation. The trypsin-activated renin had an affinity for the pepstatin column. The maximum amount of trypsin-activated renin was obtained with incubation for 15 min at 37°C at 1000 µg/ml in plasma or at 100 µg/ml in case of stored renin. The ratio of inactive to active renin was calculated to be 1.6 or 0.002 in plasma or stored renin, respectively, under conditions of a standard sodium diet.     

Effects of trypsin on the measurement of inactive renin in rat plasma by radio-immunoassay: decrease in inactive renin following nephrectomy

We have examined the effect of trypsin treatment of rat plasma on the rate of angiotensin (Ang) I generation and measurement of this peptide by radio-immunoassay. Trypsin increased the renin incubation blank but did not alter the kinetics of the renin reaction with exogenous renin. The quantity of immunoreactive material detected in trypsin-treated plasma was not proportional to the volume of plasma assayed. Consequently, the level of inactive renin was dependent upon the volume of plasma subjected to the assay. This discrepancy occurred with two independent radio-immunoassay systems. The rate of Ang I generation was linear and significantly elevated following the addition of renin substrate to trypsin-treated plasma. However, if trypsin degradation of endogenous renin substrate was extensive and additional renin substrate was not provided, non-linear rates of Ang I generation occurred. Multiple additions of trypsin were necessary to activate maximally inactive rat plasma renin. Inactive renin accounted for 79 +/- 2% of the total enzyme activity in normal rats. Although active renin declined following bilateral nephrectomy, the ratio of active to inactive renin did not change. The data suggest that the kidney is the primary source of inactive renin in the normal rat.     

Quantitation of Inactive Renin in Human and Dog Plasma: Techniques for Activation

For human samples quantitation of inactive renin can be carried out by incubation with trypsin under defined conditions, followed by RIA of the activated renin. for dog samples we were unable to obtain evidence for the presence of inactive renin in the plasma by using trypsin, acid or cold to activate. Increases in angiotensin generation did occur with trypsin and acid but they both changed renin substrate such that the rate of angiotensin generation by exogenous renin was increased at pH 7.4, but not at pH 5.7; also following trypsin or acid treatment angiotensin I was cleaved from renin substrate by a plasma acid protease that normally does not cleave renin substrate in plasma. Therefore, for dog samples, it is important to demonstrate that an increase in the rate of angiotensin generation is indeed due to activation of inactive renin and not to changes in pH optimum of renin with angiotensinogen or to the effect of another enzyme.     

Characterization of angiotensin peptides in plasma of anephric man.

Recent evidence suggests that a considerable proportion of plasma angiotensin is generated not in blood but in peripheral tissues. Through the measurement of angiotensin peptides and renin in the plasma of 11 anephric subjects, we have investigated whether kidney-derived renin, or some other tissue mechanism for angiotensin generation, is the major determinant of plasma angiotensin. Particular care was taken to prevent inadvertent activation of inactive renin and possible generation, conversion and metabolism of angiotensin peptides during processing of blood samples. Initial experiments revealed that plasma from anephric subjects contains high amounts of material which interferes in radioimmunoassays for angiotensin, even after high-performance liquid chromatography (HPLC). Therefore, in order to obtain an unambiguous identification of angiotensin peptides, a dual HPLC method was developed in which angiotensin peptides were first separated by HPLC, then acetylated and run again on HPLC before radioimmunoassay for angiotensin I and II (detection limits, 0.25 and 0.2 fmol/ml, respectively). The levels of angiotensin I and II were 1.2 +/- 1.6 and 0.7 +/- 0.5 fmol/ml (mean +/- s.d., n = 9-10), respectively, being 6% of levels in normal subjects, and were consistent with the active renin levels (1.8 +/- 1.7 muIU/ml, n = 11) which were 7% of levels in normal subjects. Artefactual activation of prorenin and angiotensin generation during sample processing were excluded as significant causes of the low levels of active renin and angiotensin I and II in anephric plasma. These data indicate that kidney-derived renin is the major determinant of angiotensin levels in normal human plasma. However, the present demonstration of low levels of active renin and angiotensin I and II in plasma of anephric subjects provides unequivocal evidence for a functional extrarenal renin-angiotensin system in man.     

Direct radioimmunoassay of human renin: comparison with renin activity in plasma and amniotic fluid

Human plasma and amniotic liquid were activated by dialysis at pH 3.3. Then, renin before and after acidification was determined by two methods: enzymatic activity measurement, and direct radioimmunoassay. The identity between nonactivated and activated renin in plasma and amniotic fluid on the one hand, and pure renin on the other, was demonstrated by the dilution curves in radioimmunoassay. After acidification, mean plasma renin activity in 17 patients with high renin activity rose from 26.8 +/- 11.7 pmoles A I ml-1 h-1 to 67.9 +/- 29.3 pmoles A I ml-1 h-1, whereas the mean renin concentration tested by direct radioimmunoassay remained constant at 13.8 +/- 10.5 and 14.8 +/- 11.2 fmol/ml before and after acidification respectively. In amniotic fluid, renin activity increased from 9.7 to 227 pmoles angiotensin I/ml/h, but the renin concentration did not change. Direct radioimmunoassay of renin may therefore be considered as measuring total renin, regardless of its enzymatic activity. In 12 hypertensive patients undergoing bilateral renal-vein catheterization, the direct measurement of renin was very significantly correlated to the non-activated (r = 0.883) and activated renin values (r = 0.963).     

Changes in active and inactive renin and in angiotensin II across the kidney in essential hypertension and renal artery stenosis

Investigations were performed in 26 patients with essential hypertension and 24 with unilateral renal artery stenosis. In each patient blood was drawn simultaneously and in triplicate, from both renal veins and aorta, for measurement of plasma concentrations of active and inactive renin and of angiotensin II. In 19 patients estimates of individual renal plasma flow were obtained in order to calculate secretion rates for active and inactive renin, and to assess the contribution of renin secretion rate and of renal plasma flow to the renal vein renin ratio. In patients with essential hypertension there was evidence that the kidney secreted active renin (18% mean increase in renal vein concentration above that of arterial plasma; P less than 0.001), but no evidence of secretion of inactive renin (4% mean increase; NS). There was a tendency for the kidney to extract angiotensin II (8% mean decrease in renal vein concentration below that of arterial plasma; P = 0.07). The affected kidney in patients with renal artery stenosis showed marked secretion of active renin (364% mean increase; P less than 0.001) and also secreted inactive renin (80% mean increase; P less than 0.05) with net generation of angiotensin II across the renal circulation (100% mean increase; P less than 0.05). The contralateral kidney exhibited suppressed secretion of active renin (3% mean increase; NS) with no evidence of secretion of inactive renin (2% mean increase; NS), and marked extraction of angiotensin II (50% mean decrease; P less than 0.001). The correlation between combined secretion rate of active renin by both kidneys and the arterial concentration of active renin in patients with essential and renovascular hypertension taken together was strongly positive (r = 0.82; P less than 0.01). The same correlation for inactive renin was weak (r = 0.32; NS). The correlation between the combined secretion rates of active renin by both kidneys and the circulating plasma concentration of angiotensin II (r = +0.60; P less than 0.05) was both significant and positive. By contrast, the total 'secretion' rate of angiotensin II by both kidneys was inversely related to arterial plasma angiotensin II (r = -0.92; P less than 0.001). This latter relationship suggests an important role for the kidney in clearing angiotensin II from the circulation, this being more marked the higher the arterial angiotensin II concentration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methodologic problems in plasma renin activity measurements.

The influence of pH and angiotensinase inhibitors on the in vitro generation of angiotensin I during PRA measurements has been investigated. PRA values obtained at pH 5.7 are higher than those obtained at pH 7.4. At pH 5.7, values obtained using diisopropylfluorophosphate (DRP 9 mM) as an angiotensinase inhibitor are higher than values obtained with a mixture of dimercaprol (BAL, 1.6 mM) and hydroxyquinoline (8-OHQ, 3 to 4 mM). Since the two methods for inhibiting angiotensinase are completely and equally efficient, it is suggested that these inhibitors might interfere with the renin angiotensinogen reaction. Significant correlations are observed between the PRA values obtained by the different methods which have been studied. Using an incubation pH of 5.7, and BAL and 8-OH quinoline as angiotensinase inhibitors, the distribution of PRA values in a population of 124 hospitalized hypertensive patients ingesting a normal sodium diet had been studied, and it has been demonstrated that the sensitivity of this method of measurement can detect small changes in PRA in patients with low renin activity.     

Searching out low renin patients: limitations of some commonly used methods

This study was designed to determine optimum conditions for measuring plasma renin activity in low renin samples. Optimum conditions were found to be an 18 hour incubation at pH 5.7 and 37 °C in the presence of ethylenediaminetetracetic acid (EDTA), diisopropyl fluorophosphate (DFP) and neomycin. Alkaline pH was disadvantageous because of lower rates of generation of angiotensin I, inability to maintain constant pH without addition of buffer and because the incubation time cannot be prolonged beyond 3 hours. An 18 hour incubation increases sensitivity and eliminates the need for blank subtractions. Dilution prior to incubation is detrimental since the reaction rate is slowed due to dilution of both enzyme and substrate with an inability to correct for the effect of substrate dilution. It was also found, in both acid and alkaline incubations, that dimercaprol (BAL) and 8-hydroxyquinoline are considerably less effective in protecting against angiotensinase than DFP.Commercial kits provide conveniently packaged reagents for renin radioimmunoassay. However, incubation steps currently recommended are inadequate for physiologic studies. Renin measurements were consistently lower using the kits and the amounts of angiotensin generated in low renin samples were only 1 to 4 per cent of those generated using our method. Thus, most low renin samples are undetectable by kit radioimmunoassays and therefore often cannot be discriminated with confidence from many normal renin samples. Moreover, incomplete angiotensinase inhibition and the necessity of subtracting a blank from each value lead to considerable loss of accuracy in both the low and normal renin samples when measured with commercial kits.     

The prolonged pressor response to renin in the nephrectomized rat.

Angiotension I dose-response curves and renin clearances were studied in nephrectomized and paired sham-nephrectomized control rats under pentobarbital anesthesia. Both threshold and slope of the angiotensin dose-response curves were decreased 22 hours after nephrectomy. In addition, the ratio of renin clearance (determined during renin infusions) in the 22-hour-nephrectomized rat to that in paired 22-hour sham-nephrectomized controls was 0.50 +/- 0.03 (mean +/- SEM, P less than 0.001, n = 12 pairs). The finding of reduced renin clearance was confirmed by an indirect assessment of "effective renin clearance" based on a comparison of the blood pressure decline after renin injections with angiotensin I dose-response curves in the same rat. Overall, approximately half of the 50% fall in renin clearance could be accounted for by an immediate effect of removal of the kidney on renin clearance. This role of the kidney in renin clearance was confirmed by the finding of a renal venous-arterial renin ratio of 0.9 +/- 0.03 (P less than 0.005) during renin infusion in normal rats. It is concluded that both changes in the angiotensin I dose-response curve and decrease in plasma renin clearance contribute to the postnephrectomy prolongation of the renin pressor response in the rat.     

Molecular biology of the vascular renin-angiotensin system.

Considerable evidence has been accumulated for a renin-angiotensin system in the blood vessel wall with local generation of both angiotensin I and angiotensin II that plays an important role in blood pressure regulation. A major source for vascular renin is renal renin taken up by the arterial wall from the circulation. However, recent studies suggest that, in addition, local synthesis of components of the renin-angiotensin cascade also takes place in the vessel wall. The contribution that these locally derived components make to the functions of the vascular renin-angiotensin system remains to be elucidated. Studies, particularly in vitro, suggest that vascular pathways for angiotensin generation not involving renin or angiotensin-converting enzyme may also exist. As in the case of the locally derived components of the renin-angiotensin cascade, the role of these alternate pathways in the physiology of the vascular wall remain to be defined.