Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor.

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and its tissue inhibitor (TIMP-2) are mainly known for their roles in the (patho)physiological remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. A mechanism of action of MMP-2 is the proteolytic breakdown of specific extracellular matrix proteins. The amino acid sequences in interstitial collagen (Gly-Leu/Ile) and laminin-5 (Ala-Leu) that are cleaved by MMP-2 are homologous to a region (Gly(32)-Leu(33)) within human big endothelin-1[1 to 38] (big ET-1). Big ET-1 requires cleavage to an active form to produce vasoconstriction. We tested the hypothesis that vascular MMP-2 can cleave big ET-1, thus generating a vasoconstrictor peptide. In perfused rat mesenteric arteries with an intact endothelium, inhibition of vascular MMP-2 with TIMP-2 reduced (by 16.2+/-4.2%) the vasoconstrictor effects of big ET-1 (50 pmol). However, when the endothelium was mechanically removed, TIMP-2 abolished (>90%) the vasoconstriction of big ET-1, and this effect was mimicked by an anti-MMP-2 antibody. Incubation of big ET-1 with recombinant human MMP-2 resulted in the specific cleavage of the Gly(32)-Leu(33) bond of big ET-1. Moreover, the resultant peptide ET-1[1 to 32] exerted greater vasoconstrictor effects than big ET-1. We conclude that vascular MMP-2 contributes to the vasoconstrictor effects of big ET-1 by cleaving big ET-1 to yield a novel and potent vasoconstrictor, ET-1[1 to 32]. These data implicate, for the first time, the endogenous MMP-2/TIMP-2 system in the regulation of vascular reactivity.

Vascular matrix metalloproteinase-2-dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction.

Matrix metalloproteinase (MMP)-2 has been historically associated with the process of vascular remodeling through the cleavage of extracellular matrix proteins. However, we recently found that MMP-2 also cleaves the endothelium-derived peptide big endothelin-1, ET-1[1-38] and yields the novel vasoconstrictor ET-1[1-32]. We therefore investigated the effects of MMP-2 inhibitors as potential vasodilators. MMP inhibition with ortho-phenanthroline (0.3 to 30 micromol/L) induced vasorelaxation of isolated rat mesenteric arteries (maximum of relaxation=74.5+/-27.6% at 30 micromol/L). However, phosphoramidon (0.3 to 30 micromol/L), which inhibits some metalloenzymes, but not MMP-2, did not dilate the arteries. Selective inhibition of endogenous MMP-2 with the novel tissue-permeable cyclic peptide CTTHWGFTLC (CTT, 10 micromol/L) also caused vasorelaxation (by 85+/-6%), whereas STTHWGFTLS (10 micromol/L), an inactive CTT analogue, did not dilate the arteries. Interestingly, the vasorelaxation that results from MMP-2 inhibition was endothelium-independent. Thus, we examined whether MMP-2 acted on peptides derived from the smooth muscle or the perivascular nerves. Recombinant human MMP-2 cleaved calcitonin gene-related peptide (CGRP) specifically at the Gly(14)-Leu(15) peptide bond and reduced the vasodilatory potency of CGRP by 20-fold. Inhibition of MMP-2 increased the amount of intact CGRP in arteries and enhanced vasorelaxation induced by anandamide, which stimulates CGRP release. Vasorelaxation in response to MMP-2 inhibition was abolished by CGRP[8-37], a selective CGRP receptor antagonist, and by capsaicin, which depletes arterial perivascular nerves of CGRP. We conclude that vascular MMP-2 cleaves endogenous CGRP and promotes vasoconstriction. These data suggest a novel mechanism of regulating the vasoactive and, possibly, the neurohormonal actions of CGRP and establish MMP-2 as a modulator of vascular function.

Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1-32].

We recently reported that matrix metalloproteinase 2 (MMP-2, gelatinase A) cleaves big endothelin 1 (ET-1), yielding the vasoactive peptide ET-1[1-32]. We tested whether ET-1[1-32] could affect the adhesion of human neutrophils to coronary artery endothelial cells (HCAEC). ET-1[1-32] rapidly down-regulated the expression of L-selectin and up-regulated expression of CD11b/CD18 on the neutrophil surface, with EC50 values of 1-3 nM. These actions of ET-1[1-32] were mediated via ETA receptors and did not require conversion of ET-1[1-32] into ET-1 by neutrophil proteases, as revealed by liquid chromatography and mass spectroscopy. Moreover, ET-1[1-32] evoked release of neutrophil gelatinase B, which cleaved big ET-1 to yield ET-1[1-32], thus revealing a positive feedback loop for ET-1[1-32] generation. Up-regulation of CD11b/CD18 expression and gelatinase release was tightly associated with activation of extracellular signal-regulated kinase (Erk). Stimulation of Erk activity was due to activation of Ras, Raf-1, and MEK (MAPK kinase). ET-1[1-32] also produced slight increases in the expression of ICAM-1 and E-selectin on HCAEC, and markedly enhanced beta2 integrin-dependent adhesion of neutrophils to activated HCAEC. These results are the first indication that gelatinolytic MMPs via cleavage of big ET-1 to yield ET-1[1-32] activate neutrophils and promote leukocyte-endothelial cell adhesion and, consequently, neutrophil trafficking into inflamed tissues.

Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.

Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins.

We report here a modification to copper and zinc chloride staining methods. The introduction of a preincubation of the gels, prior to metal staining, with 0.2 M imidazole allows the formation of a homogeneous background for the subsequent precipitation of the metal chelate. The reported imidazole-zinc staining takes minutes, resulting in reproducible staining patterns with only slightly lower sensitivity than silver staining. The method allows efficient recovery of proteins from previously stained gels and is compatible with immunoidentification on Western blots and also with amino acid analysis and NH2-terminal sequence analysis of transferred proteins. A mechanism is proposed to explain the observed improvement in reproducibility and sensitivity of imidazole preincubation to zinc staining.

Rapid release of matrix metalloproteinase (MMP)-2 by thrombin in the rat aorta: modulation by protein tyrosine kinase/phosphatase.

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and thrombin contribute to many long-term (patho)physiological processes requiring the proteolytic breakdown of the vascular extracellular matrix (e.g., normal tissue repair, remodeling, tumor invasion, atherosclerosis plaque rupture). Thrombin (10 to 1000 nM, 0.5 to 50 U/ml) induced a rapid secretion of MMP-2 from freshly isolated rat aortic tissue (detectable after 1 min of thrombin exposure). This secretion was mediated by an unidentified thrombin receptor, distinct from the proteinase activated receptors (PAR)-1 and -2. Protein tyrosine kinase/phosphatase activity differentially modulated the basal and the thrombin-induced release of MMP-2. The inhibitors of protein tyrosine kinase, herbymicin A, genistein, and tyrphostin 1288 (1 to 100 microM), enhanced the basal release of MMP-2 but did not affect the thrombin-induced secretion of MMP-2. The inhibitor of phosphotyrosine phosphatases, vanadate (100 microM), selectively inhibited the thrombin-induced, but not the basal, release of MMP-2. Rapid release of vascular MMP-2 by thrombin could contribute to short-term processes where thrombin is involved such as the regulation of platelet aggregation and vascular reactivity. Vascular tyrosine kinase/phosphatase likely modulates this action of thrombin to prevent exaggerated platelet aggregation, thrombosis, and vasospasm.

Differential regulation of platelet aggregation by matrix metalloproteinases-9 and -2.

We have recently found matrix metalloproteinase-2 (MMP-2) in human platelets and reported that the release of this enzyme during platelet activation stimulates aggregation. We have now identified matrix metalloproteinase-9 (MMP-9) in human platelets and resistance-sized (approximately 200 microm) arteries. Resting platelets released small quantities of pro-MMP-9. Maximal release of MMP-9 was detected during partial (appr. 30% maximum) aggregation with thrombin. However, maximal release of MMP-2 was associated with maximal aggregation. MMP-9 antibodies induced aggregation of resting platelets and potentiated aggregation of platelets induced by thrombin and collagen. Moreover, MMP-9 microisolated from arteries as well as recombinant human MMP-9 (0.1-30 ng/ml) inhibited thrombin and collagen-induced aggregation. We conclude that MMP-9 is an inhibitor of aggregation and in this action opposes the effects of MMP-2. The MMP-2/MMP-9 system may play an important role in the regulation of platelet-platelet and platelet-vessel wall interactions.

Matrix metalloproteinase-2 is elevated in the plasma of women with preeclampsia.

OBJECTIVE: We have recently demonstrated that matrix metalloproteinase-2 (MMP-2) alters vascular function through cleavage of vasoactive peptides, resulting in increased vasoconstriction and reduced vasodilation. We, therefore, hypothesized that MMP levels are increased in women with preeclampsia. In addition, because vascular endothelial growth factor (VEGF) has been implicated in the pathophysiology of preeclampsia and is involved in angiogenesis that requires the release of proteases to allow for migration of endothelial cells, we hypothesized that VEGF increases release of MMPs from endothelial cells. METHODS: We used zymographic analysis to evaluate MMP-2/MMP-9 levels in plasma of women with preeclampsia (n=12) compared to women with uncomplicated pregnancies (n=12). In addition, we evaluated the changes in the levels of MMP-2 and MMP-9 as well as tissue inhibitors of MMPs (TIMP-1 and TIMP-2) released by cultured human umbilical vein endothelial cells in response to VEGF (0.1-10 ng/mL). RESULTS: Our data showed that plasma MMP-2 levels were significantly higher in women with preeclampsia compared to women with uncomplicated pregnancies (arbitrary intensity units: 690 +/-111 and 252 +/-56, respectively, p<0.05). MMP-9 levels were below the level of detection. In addition, VEGF stimulated endothelial MMP-2 and MMP-9 release in a concentration- and time-dependent (6-24 h) manner. Moreover, VEGF stimulation of MMP release occurs without significantly affecting the release of TIMP-1 and TIMP-2. CONCLUSIONS: These data suggest that VEGF promotes secretion of MMPs from endothelial cells that, in turn, could alter vascular function in women with preeclampsia.

Role of matrix metalloproteinase-2 in thrombin-induced vasorelaxation of rat mesenteric arteries.

The vasodilator effects of thrombin depend on activation of proteinase-activated receptor (PAR)-1 and the subsequent release of endothelin (ET)-1, which stimulates the generation of nitric oxide and PGs. We recently showed that thrombin released matrix metalloproteinase-2 (MMP-2) from rat arteries. We have now studied the significance of this release for the vasodilator effects of thrombin. Thrombin (>/=100 pmol), but not a PAR-1-activating peptide (TFLLR-NH(2)), produced a long-lasting (>10 min) vasorelaxation of rat mesenteric arteries, as detected by a microperfusion bioassay. Thrombin induced a simultaneous release of vascular MMP-2 into arterial perfusates, as revealed by zymography. Interestingly, the vasodilator effects of thrombin were inhibited by a tissue inhibitor of MMP-2 (TIMP-2, 10 pmol). Moreover, infusion of exogenous MMP-2 (5 pmol) resulted in vasorelaxation. These vasodilatory effects of thrombin and MMP-2 were significantly (P < 0.05) inhibited by endothelium denudation and by PD-142893 (2 nmol), an antagonist of ET receptors. Furthermore, both thrombin and MMP-2 constricted endothelium-denuded arteries. These results show that the vasodilator effects of thrombin may depend, in part, on a release of vascular MMP-2 and downstream activation of ETs. Thus MMP-2-dependent signaling may complement the PAR-1-dependent pathway of vasodilator action of thrombin.

Sensitive reverse staining of bacterial lipopolysaccharides on polyacrylamide gels by using zinc and imidazole salts.

We present a new method for visualizing bacterial lipopolysaccharides (LPS)/lipooligosaccharides (LOS) electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are washed in boiling water to appreciably remove remaining electrophoresis reagents, then incubated in 10 mM zinc sulfate for 15 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate all over the gel surface except in the zones occupied by LPS/LOS, which appear as transparent, colorless bands. Gels can be stored in distilled water for weeks without loss of the negative image. The sensitivity of this stain is similar to that of silver. We believe that zinc-imidazole may be a suitable nontoxic alternative to silver in the rapid analysis of LPS/LOS by polyacrylamide gel electrophoresis.

Zinc-imidazole positive: a new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity.

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological activity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new nontoxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.

A procedure for protein elution from reverse-stained polyarcylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis.

We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 x 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance liquid chromatography (HPLC) and automatic sequencing. A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E. coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein staining and of sample reduction prior to electrophoresis on elution yields. For N-terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean-up were monitored at 206 nm. By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N-terminal sequenced.

Single-step electrotransfer of reverse-stained proteins from sodium dodecyl sulfate-polyacrylamide gel onto reversed-phase minicartridge and subsequent desalting and elution with a conventional high-performance liquid chromatography gradient system for analysis.

Isolation of proteins from polyacrylamide electrophoresis gels by a novel combination of techniques is described. A given protein band from a reverse stained (imidazol-sodium dodecyl sulfate--zinc salts) gel can be directly electrotransferred onto a reversed-phase chromatographic support, packed in a self-made minicartridge (2 mm in thickness, 8 mm in internal diameter, made of inert polymeric materials). The minicartridge is then connected to a high-performance liquid chromatography system and the electrotransferred protein eluted by applying an acetonitrile gradient. Proteins elute in a small volume ( < 700 microL) of high-purity volatile solvents (water, trifluoroacetic acid, acetonitrile) and are free of contaminants (gel contaminants, salts, etc). Electrotransferred proteins were efficiently retained, e.g., up to 90% for radioiodinated alpha-lactalbumin, by the octadecyl matrix, and their recovery on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73% for alpha-lactalbumin. The technique was successfully applied to a variety of proteins in the molecular mass range 6-68 kDa, and with amounts between 50 and 2000 pmol. The good mechanical and chemical stability of the developed minicartridges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single-step separation of two proteins unresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to their different elution from the reversed-phase support. The isolated proteins were amenable to analysis by N-terminal sequencing, enzymic digestion and mass spectrometry of their proteolytic fragments. Chromatographic elution of proteins from the reversed-phase mini-cartridge was apparently independent of the specific loading mode employed, i.e., loading by conventional loop injection or by electrotransfer.

High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level. Application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins.

A simple, reliable procedure for practically quantitative (90-98%) and fast (< 30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-micron gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 microliters for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.

Double staining of coomassie blue-stained polyacrylamide gels by imidazole-sodium dodecyl sulfate-zinc reverse staining: sensitive detection of coomassie blue-undetected proteins.

The sensitivity, simplicity, and relative rapidity of Coomassie blue staining have made this technique the method of choice for routine detection and quantitative analysis of gel electrophoresis-separated protein bands in many applications. To extend the usefulness of this technique, we have developed a new double-staining method for visualizing SDS-PAGE-separated protein bands that were undetected by Coomassie blue staining of the gel. Coomassie blue-stained gels are washed in distilled water (15 min, two times) and then subjected to imidazole-zinc reverse staining. As a result of the method, a homogeneous white-stained background is generated and two types of protein bands can be observed: (a) typical Coomassie blue-stained bands, which appear superposed on larger transparent bands; and (b) reverse-stained (transparent) bands, which were previously undetected by the Coomassie blue staining. The method is rapid, simple, and reproducible and double-staining gels can be kept in distilled water for months without loss of the protein pattern. The overall sensitivity is high (e.g., 1.6 ng for recombinant streptokinase, 47 kDa) over a wide range of protein molecular weights (10 to 100 kDa) and independent of the degree of Coomassie blue destaining of the gel. Furthermore, a mechanism offering a consistent explanation for the role of imidazole, SDS, and zinc in the reverse staining of gels, particularly after Coomassie blue staining is proposed.

Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: generalization of the reverse-staining technique.

We have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels can be detected by reverse-staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gel-isolated nucleic acids and bacterial glycolipids. Because there is no recourse to chemical modifiers, the reverse-staining technique has been valuable when micropreparing these biomacromolecules for later use or characterization. The mechanism underlying the reverse-staining effect, however, remains incompletely understood and this has prevented a further generalization of the technique. Here, we have conducted physicochemical experiments and identified zinc imidazolate (ZnIm2) as the main component of the precipitate that forms along the surface of zinc-imidazole reverse-stained gels. Many staining effects observed when gels containing electrophoretically separated biopolymers are subjected to zinc-imidazole stains have been rationalized. The reverse-staining method has been vastly generalized, now allowing the detection of proteins and glycolipids as well as complexes of these macromolecules in native gels. We demonstrate the application of the reverse-staining technique in situations where Coomassie blue or silver staining was inappropriate or failed to produce detection of the species of interest. The present generalization of the reverse-staining method facilitated the characterization of biomacromolecular interaction partners in mixtures of bacterial glycolipids and human tears.

Protein reverse staining: high-efficiency microanalysis of unmodified proteins detected on electrophoresis gels.

A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water (even during years); and (c) protein bands result immobilized, i.e., they do not diffuse upon gel storage. To recover reverse-stained proteins or fragments thereof from the gel, the immobilization of bands must first be abrogated by chelating the zinc ions from stain (protein mobilization). We had originally described mobilization at low pH by using citric acid. Here, we improve this procedure regarding the protein electrotransfer. We demonstrate that mobilization is efficiently done at neutral to alkaline pH by short-term (5 to 10 min) incubation of the gel in a buffer containing glycine or dithiothreitol prior to transfer. Moreover, mobilization was most simply performed by just adding the zinc chelating agent to the transfer buffer. Reverse staining and the new mobilization procedure made electrotransferring single protein bands from gel onto small-sized (13 x 5 mm2) PVDF membrane pieces in mini sandwich-like assemblies practical. Equipment is described for the protein electroblotting in such minisandwiches. Microsequence analysis of the electroblotted proteins showed initial yields in the range of those achieved when the transfer was done from unstained control gels. Protein bands kept in the reverse-stained gel for prolonged time periods (even for as long as 2 years) could be similarly analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)