Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator

Abstract

MMPs (matrix metalloproteinases) play a major role in the pathogenesis of hypertension by altering the extracellular matrix during cardiovascular remodelling. In the present study we show that MMP-2, but not MMP-9, cleaves the vasodilator peptide AM (adrenomedullin). Addition of the AM-binding protein, complement factor H, prevents this cleavage, providing a hitherto unknown mechanism of action for this binding protein. We identified the signature cleavage fragments and found some of them in human urine, suggesting that MMP-2 processing of AM may occur in vivo. Synthetic AM fragments regulated blood pressure in rats. The larger peptides are vasodilators, as is intact AM, whereas intermediate fragments did not affect blood pressure. In contrast, AM(11-22) elicited vasoconstriction. Studies of AM receptor activation in Rat2 cells confirm that the larger AM cleavage peptides activated this receptor, whereas AM(11-22) did not. The present study defines a new mechanism through which MMP-2 may regulate blood pressure by simultaneously eliminating a vasodilator and generating a vasoconstrictor.

Figure 1. MMP-2, but not MMP-9, degraded AM in the absence of factor H

Synthetic AM was exposed to MMP-2 (lanes 1–8) or MMP-9 (lanes 9–12) in the presence (lanes 5–8) or absence (lanes 1–4 and 9–12) of factor H. Individual reactions were stopped at the indicated times and the resulting peptides separated by electrophoresis in 16% polyacrylamide gels. Lanes 1–4 show a progressive degradation of the original peptide and a concomitant appearance of digestion products.

Figure 2. MMP-2 digestion of AM generated novel peptide fragments

Digestion reactions were stopped at the indicated times and then analysed by HPLC in a reverse-phase column. The single peak at time=0 corresponds to the intact AM peptide. This peak progressively diminished over time, whereas additional peaks began to appear. The fractions exhibiting new peaks were analysed by MS and some of them are identified in the Figure.

Figure 3. Urine from normal volunteers contained products of MMP-2-dependent AM degradation

After an initial C-18 cartridge extraction, the equivalent of 250 ml of urine was fractionated through a C-18 HPLC preparative column following an acetonitrile gradient (broken line) (a). Select fractions were loaded on to a 12% polyacrylamide gel, transferred on to nitrocellulose, labelled with a polyclonal antibody against AM and developed by chemiluminescence (b). Synthetic AM (3 ng) was added in the first lane (AM) as a positive control.

Figure 4. One of the new fragments elevated blood pressure in rats

Typical recordings of the blood pressure modifications elicited by intact AM (a) and its fragments (b–d) in anaesthetized rats. The peptides AM(8–52) and AM(11–52) induced hypotension, and only the effect of the latter is shown (b). The fragments AM(23–52), AM(29–52) and AM(11–28) did not have any effect, and only the effect of the AM(11–28) is shown (c). The small peptide AM(11–22) induced vasoconstriction several minutes after injection (d). The arrow indicates the time when the peptides were injected. The horizontal bar represents 1 min. The vertical bar represents 50 mmHg. Schematic drawings of the structure of the AM peptides are provided underneath their name. The solid circle represents the amide group at the carboxy end and the rectangle indicates the intramolecular disulphide bond.

Figure 5. Some digestion products were no longer able to activate the AM receptor

Intracellular levels of cAMP were quantified by radioimmunoassay as an indirect measurement of AM receptor activation in Rat2 cells. (a) Intact AM and the two larger fragments induced a significant elevation of cAMP when compared with the addition of PBS (Control), whereas the rest of the test peptides did not have any effect on the levels of cAMP (*P<0.001). (b) Addition of different concentrations of AM(11–22) did not affect the response elicited by the intact peptide AM(1–52). The control value is significantly different from all the treatments (*P<0.001), but these are not statistically significant among themselves. The results are expressed as the means±S.D. for 8 independent measurements.

Figure 6. Diagram of the sequential degradation of AM into smaller peptides and the physiological implications of the process

The larger peptides maintain the vasodilator capability characteristic of intact AM, whereas intermediate peptides lack vasomotor activity, and the small peptide AM(11–22) is a vasoconstrictor. Schematic diagrams are as in Figure 4.