Angiotensin-converting enzyme 2 (ACE2) expression and activity in human carotid atherosclerotic lesions.

Angiotensin-converting enzyme (ACE)2 is a recently identified homologue of ACE. As ACE2 inactivates the pro-atherogenic angiotensin II, we hypothesize that ACE2 may play a protective role in atherogenesis. The spatiotemporal localization of ACE2 mRNA and protein in human vasculature and a possible association with atherogenesis were investigated using molecular histology (in situ hybridization, immunohistochemistry). Also, the ACE : ACE2 balance was investigated using enzymatic assays. ACE2 mRNA was expressed in early and advanced human carotid atherosclerotic lesions. In addition, ACE2 protein was present in human veins, non-diseased mammary arteries and atherosclerotic carotid arteries and expressed in endothelial cells, smooth muscle cells and macrophages. Quantitative analysis of immunoreactivity showed that total vessel wall expression of ACE and ACE2 was similar during all stages of atherosclerosis. The observed ACE2 protein was enzymatically active and activity was lower in the stable advanced atherosclerotic lesions, compared to early and ruptured atherosclerotic lesions. These results suggest a differential regulation of ACE2 activity during the progression of atherosclerosis and suggest that this novel molecule of the renin-angiotensin system may play a role in the pathogenesis of atherosclerosis.

Genome-wide expression studies of atherosclerosis: critical issues in methodology, analysis, interpretation of transcriptomics data.

During the past 6 years, gene expression profiling of atherosclerosis has been used to identify genes and pathways relevant in vascular (patho)physiology. This review discusses some critical issues in the methodology, analysis, and interpretation of the data of gene expression studies that have made use of vascular specimens from animal models and humans. Analysis of gene expression studies has evolved toward the genome-wide expression profiling of large series of individual samples of well-characterized donors. Despite the advances in statistical and bioinformatical analysis of expression data sets, studies have not yet fully exploited the potential of gene expression data sets to obtain novel insights into the molecular mechanisms underlying atherosclerosis. To assess the potential of published expression data, we compared the data of a CC chemokine gene cluster between 18 murine and human gene expression profiling articles. Our analysis revealed that an adequate comparison is mainly hindered by the incompleteness of available data sets. The challenge for future vascular genomic profiling studies will be to further improve the experimental design, statistical, and bioinformatical analysis and to make data sets freely accessible.

The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity.

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues His(185), Arg(186), and Arg(187) formed the major sites of interaction for MA-44E4. In contrast, the epitopes of MA-42A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350), Asp(355), and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.

Elucidation of the binding regions of PAI-1 neutralizing antibodies using chimeric variants of human and rat PAI-1.

Increased levels of plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, are a known risk factor for thromboembolic and cardiovascular diseases. The elucidation of the binding site of inhibitory monoclonal antibodies may contribute to the rational design of PAI-1 modulating therapeutics. In this study, homolog-scanning mutagenesis was used to identify the binding region of a variety of human PAI-1 inhibitory antibodies, lacking cross-reactivity with rat PAI-1. Therefore. eight chimeric human/rat PAI-1 variants, containing rat PAI-1 substitutions at the N-terminal or C-terminal end with splicing sites at positions 26, 81, 187, 277 or 327, were generated and purified. Biochemical characterization revealed that all chimeras were folded properly. Subsequently, surface plasmon resonance was used to determine the affinity of various monoclonal antibodies for these chimera. Comparative analysis of the affinity and ELISA data allowed the identification of the major binding region of the inhibitory antibodies MA-8H9D4, MA-33B8F7, MA-44E4, MA-42A2F6 and MA-56A7C10. Thus, three segments in human PAI-1 containing each at least one site involved in the neutralization of PAI-1 activity could be identified, i.e. (1) the segment from residue 81 to residue 187 (comprising alpha-helices hD, hE and hF, beta-strands s4C, s3A, s2A and s1A and the loops connecting these elements). (2) the segment between residues 277 and 327 (hI, thIs5A, s5A and s6A) and (3) the region C-terminal from amino acid 327, including the reactive site loop. The current data. together with previous data, indicate that PAI-1 contains at least four different regions that could be considered as putative targets to modulate its activity.

Importance of the hinge region between alpha-helix F and the main part of serpins, based upon identification of the epitope of plasminogen activator inhibitor type 1 neutralizing antibodies.

The serpin plasminogen activator inhibitor type 1 (PAI-1) is an important protein in the regulation of fibrinolysis and inhibits its target proteinases through formation of a covalent complex. In the present study, we have identified the epitope of two PAI-1 neutralizing monoclonal antibodies (MA-33H1F7 and MA-55F4C12). Based upon differential cross-reactivity data of these monoclonals with PAI-1 from different species and on a sequence alignment between these PAI-1s, combined with the three-dimensional structure, we predicted that the residues Glu(128)-Val(129)-Glu(130)-Arg(131) and Lys(154) (at the hinge region between alpha-helix F and the main part of the PAI-1-molecule) might form the major site of interaction. Therefore a variety of alanine mutants were generated and evaluated for their affinity toward both monoclonal antibodies. The affinity constants of MA-55F4C12 and MA-33H1F7 for PAI-1 were 2.7 +/- 1.6 x 10(9) M(-1) and 5.4 +/- 1.7 x 10(9) M(-1), respectively, but decreased between 13- and 270-fold upon mutation of Lys(154) to Ala(154) or Glu(128)-Val(129)-Glu(130)-Arg(131) to Ala-Ala-Ala-Ala. The combined mutations (PAI-1-EVER/K), however, resulted in an absence of binding to either of the antibodies. Both antibodies bound to PAI-1-wt/t-PA complexes with a similar affinity as to PAI-1-wt (K(A) = 4-5 x 10(9) M(-1)). The epitope localization reveals the molecular basis for the neutralizing properties of both monoclonal antibodies. In addition, it provides new insights into the validity of various models that have been proposed for the serpin/proteinase complex, excluding full insertion of the reactive-site loop.

The importance of helix F in plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is the only functionally labile serpin, as it converts spontaneously into a non-reactive 'latent' conformation. Several studies have suggested an important role for helix F in the functional behavior and stability of the serpins, especially for PAI-1. We constructed a mutant of PAI-1 (PAI-1-delhF) in which residues 127-158 (hF-thFs3A) were deleted. Whereas wild-type PAI-1 (wtPAI-1) exhibits inhibitory properties towards t-PA and u-PA to an extent of 60-80% of the theoretical maximum, PAI-1-delhF did not exert any detectable inhibitory properties, but behaved as a stable substrate. Prolonged incubation at 37 degrees C did not change its functional properties in contrast to wtPAI-1 that under those conditions converts to the latent conformation. In contrast to active wtPAI-1 and other substrate-type PAI-1 mutants, PAI-1-delhF showed a 3000-fold decreased binding to vitronectin. The obtained results clearly show the importance of helix F in the inhibitory activity of PAI-1. The absence of helix F apparently leads to an impaired kinetics of insertion of the reactive site loop upon interaction with its target proteinase resulting in the inability to form a stable covalent complex. Moreover, removal of helix F strongly affects the binding of PAI-1 to vitronectin.

Expression and characterization of recombinant porcine plasminogen activator inhibitor-1.

Porcine models are, among other animal models, very suitable for in vivo investigations in the vascular field especially with respect to the possible relationship between atherosclerosis and thrombosis. In order to use this model to define the in vivo role of PAI-1, the characterization of porcine PAI-1 and its availability for the generation of immunological tools are a prerequisite. Porcine plasminogen activator inhibitor-1 (poPAI-1) cDNA was isolated from a cDNA library prepared from cultured porcine aortic cells and characterized in comparison with PAI-1 cDNA's from other species including human, bovine, rabbit, rat and murine. Subsequently the DNA sequence coding the mature protein was cloned into an appropriate vector for expression in Escherichia coli and recombinant porcine PAI-1 was purified and characterized. On SDS-PAGE the apparent molecular weight was estimated to be 45 kDa, identical to the molecular weight of human PAI-1. The purified recombinant porcine PAI-1 (rpoPAI-1) had a specific activity of 508,800 +/- 800 U/mg (mean +/- SD, n = 3) towards human tissue-type plasminogen activator (ht-PA) and a functional half-life in vitro of 2.1 +/- 0.8 h (n = 3). Incubation with a two fold molar excess of ht-PA (n = 3) or human urokinase-type plasminogen activator (hu-PA, n = 2) followed by analysis by SDS-PAGE revealed reaction products corresponding to active (71 +/- 7% resp. 96 +/- 3.6%), latent (12 +/- 0.4% resp. 2.6 +/- 2.4%) and substrate (16.6 +/- 6.8% resp. 1.5 +/- 1.3) forms. Inactivated samples of porcine PAI-1 could be reactivated with guanidinium chloride up to 52% of its original specific activity towards t-PA and u-PA. The second order rate constant of inhibition of ht-PA was 1.64 +/- 0.37 10(7)M-1 s-1 (n = 9). In gel filtration rpoPAI-1 in buffer eluted at a volume corresponding to 24 kDa, whereas in the presence of porcine plasma, the molecular form containing PAI-1 activity eluted at a volume corresponding to 330 kDa, presumably as a consequence of binding of active PAI-1 to vitronectin. Taken together, these data demonstrate that no obvious functional differences exist between human and porcine PAI-1.

Codon adjustment to maximise heterologous gene expression in Streptomyces lividans can lead to decreased mRNA stability and protein yield.

The impact of the codon bias of the mouse tumour necrosis factor alpha (mTNF) gene cloned in Streptomyces lividans on the efficiency of expression and secretion was analysed. Minor codons occurring in the mTNF gene were therefore adapted to the codon bias of Streptomyces by site-directed mutagenesis. No improvement in mTNF yield could be detected. The stability of the transcript derived from the construct was shown to be more important for determining the final level of mTNF production. A strong correlation was observed between the yield of secreted biologically active mTNF and the amount of mTNF mRNA present in the cells.