THE MOLECULAR CONTROL OF ANGIOGENESIS

Angiogenesis is a key event in a broad range of pathological conditions including both diseases with an enhanced and insufficient angiogensis. Angiogenesis is often intiated with vasodilation accompanied by an increase in vascular permeability. After destabilization of the vessel wall and degradation of the surrounding extracellular matrix, extravasation of plasma proteins provides a provisional scaffold for the migration of endothelial cells. Endothileal cell proliferation and migration themselves are under tight control by a balance of angionenesis inducers and inhibitors. A large number of angiogenic factors work together in a highly coordinated manner to induce endothelial cell outgrowth and the formation of functional vessels. On the other hand, angiostatic factor may play a critical role in the pathogenesis of ischemic diseases and contribute to the temination of physiological angiogenesis. Angiogenesis ends with the recruitment of pericytes and smooth muscle cells, which stabilize the newly formed vessel. The rapid increase in the knowledge about the molecular mechanisms of angiogenesis has led to first treatment trials in diseases with both enhanced and reduced angiogeneis. Although initial results are promising, much more work has to be done to consdier anti-angiogenic or pro-angiogenic approaches as reliable therapeutic tools.     

Allergy in chronic haemodialysis. 1. Double-blind intravenous challenge with formaldehyde

Patients undergoing chronic haemodialysis are often exposed to formaldehyde, formaldehyde (F) has been reported to cause IgE-mediated anaphylactic shock. Many other patients reported pruritus or anaphylaxis-like symptoms when dialysed with F-sterilized dialysers. Ten patients presenting such symptoms were compared with five control subjects. Intravenous double-blind challenges were performed on six consecutive occasions, with capillary flow dialysers sterilized with or without F. Dialysis was performed by an investigator who was not aware of the sterilization procedure. Among the ten F-sensitive patients, five had symptoms with F-sterilized dialysers and no symptoms with new dialysers, sterilized by ethylene oxide and free of F. Symptoms included pruritus and hypotension. These five patients were subsequently dialysed with new dialysers, not sterilized with F, and symptoms subsided. The five other patients had inconclusive challenges. The five control subjects had no symptoms during challenges. Skin-prick tests with F showed that only one of the five patients who had symptoms with F-dialysers had a strongly positive prick test. RAST to F was titrated with HSA-discs but it was negative in all patients and control subjects. Formaldehyde was shown to cause symptoms in some patients under chronic haemodialysis but an IgE-mediated mechanism was not demonstrated.     

Crystal and molecular structure of two geometrically restricted chemotactic tripeptides,analogues of formyl-methionine-leucine-phenylalanine*

The crystal structures of HCO-Met-Leu-Phe-OC(CH₃)₃, (CH₂₅H₃₉N₃O⁵S), fMLP-OtBu, and HCO-Metψ[CSNH]-Leu-Phe-OCH₃, (C₃₃H₃₃N₃O₄S₂), fM^SLP-OMe, have been determined by single crystal X-ray diffraction, and their conformational properties investigated by molecular mechanics energy calculations. Crystals of fMLP-OtBu are monoclinic, space group P2₁, a = 12.027(4), b = 9.492(3), c = 12.660(4) Å, β= 101.99(3)°, Z = 2; those of fM^SLP-OMe are orthorhombic, space group P2₁2₁2₁, a = 7.130(1), b = 12.097(2), c = 31.060(5) Å, Z = 4. The first compounds fMLP-OtBu is the t-butyl ester of the tripeptide fMLP that represents one of the most potent compounds in inducing the lysozyme release from human neutrophils that reflects the chemotactic activity. From the crystal structure, it is shown that the orientation of the phenylalanine side chain is largely affected by the presence of the bulky group. fM^SLP-OMe was shown to be inactive after thionation of the methionine residue in the original tripeptide. Nevertheless, the crystal structure does not reveal any influence of the presence of the thionated peptidic bond on the backbone conformation. The X-ray results have been used to generate parameters for empirical energy calculations. Subsequently, a strategy based on random generation of conformations followed by energy-minimization was applied to investigate the conformational space of thiopeptides, in comparison with normal peptides. From molecular free energy calculations, it is shown that the main influence of the introduction of a thioamide bond on the molecular structure is to prevent the existence of C⁷_eqconformations involving the thiomethionine residue. Consequently, a larger number of conformers are found to form intramolecular hydrogen bonds involving the formyl group, reducing its availability to interact with the receptor. For the first time, the theoretical prediction of the existence of C⁷_eq conformations for fMLP is made. The resulting conformers are compared to previously active structures of these chemotactic agents.     

Radiofrequency Catheter Ablation of Atrial Flutter Induces the Release of Platelet and Leukocyte-Derived Procoagulant Microparticles and a Prothrombotic State

Background: To assess the extent of endothelium, platelet, and leukocyte damage and coagulation activation induced by radiofrequency catheter ablation (RF) of atrial flutter. In the vasculature, procoagulant microparticles (MPs) are reliable markers of vascular damage. They provide an additional phospholipidic surface, enabling the assembly of the enzyme complexes of blood coagulation and consequent thrombin generation.
Methods: MPs were measured in the venous blood of 33 patients with isthmus-dependent atrial flutter undergoing RF before (RF₀), immediately after (RF₁), and at day 1 (RF₂) thereafter. Concentrations of PAI-1, vWF, and D-dimers were simultaneously determined. MPs procoagulant activities were determined using a functional prothrombinase assay. RF induces an early rise of platelet-derived MPs (platelet), vWF Ag, and D-dimers levels, which is concomitant with the decrease of PAI-1 concentrations. Conversely, no significant changes in endothelial-derived MPs could be evidenced. At RF₂, sustained elevation of leukocytes-derived MPs, vWF, and D-dimers testified to an ongoing prothrombotic status.
Conclusion: RF ablation of common flutter induces a prothrombotic state and the release of platelet and leukocyte-derived procoagulant microparticles. Whereas this activation of blood coagulation could be viewed as clinically marginal in right-sided procedures, its relevance in left-sided procedures should be established.     

Conformational behaviour of some tachykinin C-terminal heptapeptides

The conformational behaviour of substance P, physalaemin and eledoisin C-terminal heptapeptides was investigated using empirical energy calculations. Although the conformational distributions of the three heptapeptides are some-what different, they have a few common low energy conformations. Some of them, which satisfy the structure-activity relationships, may fit the well known SP-P receptors.     

Structure-function studies on human growth hormone

The relationship between the conformation of human pituitary growth hormone (hGH), biological activity, and ligand binding activity was studied by comparing conformational details previously published on in vivo and in vitro studies of identical samples of hGH and its known derivatives. In vivo assays included the rat tibia test for somatotropic activity and the pigeon crop-sac assay for lactogenic hormone activity. Relative binding affinities were compared in radioimmuno-assays using ¹²⁵I-hGH as tracer with 1) anti-human chorionic somatomammotropin (hCS) serum (low discriminatory hybrid assay), 2) anti-hGH sera (in conventional assays), 3) monospecific anti-hGH serum (absence of cross-reaction with hCS) and 4) human anti-hGH sera obtained from GH-deficient patients on replacement therapy. In addition, binding affinities were examined in two receptor-binding assays, one specific for somatotropic activity (rabbit liver membranes, ¹²⁵I-hGH), and the other, for lactogenic hormones (rabbit mammary membranes, ¹²⁵I-oPRL). The conformational properties of native hGH and various chemically and enzymatically modified derivatives of the hormone were evaluated primarily from circular dichroism spectra, while conformational stabilities were estimated from the relative rates of tryptic digestion. Unfragmented, but chemically modified derivatives, exhibited good parallelism between retention or loss of native conformation and the in vivo potencies and in vitro binding affinities. None of the fragments of hGH showed activity in any of the radioreceptor assays or radioimmunoassays. Two derivatives of hGH, which contain gaps of 6 or 12 residues in the polypeptide backbone produced by partial enzymatic digestion, had full or increased in vivo potencies, full activities in the radioimmunoassays, and were the most active derivatives in both radio-receptor assays. One of these, missing the hexapeptide corresponding to residues 135–140, was also found to retain nearly all the conformational properties of native hGH. These studies proved further evidence that 1) retention by modified forms of hGH of a high degree of in vivo biological potency or in vitro binding affinity is causally related to the retention of most of the conformation and conformational stability of the molecule, and 2) the biologically acitve, receptor-binding and immunoreactive sites on the hGH molecule are 3-dimensional in nature.     

Atrial Refractory Periods after Atrial Premature Beats in Patients with Paroxysmal Atrial Fibrillation

To study the effects of an atrial premature beat on atrial refractory periods, we investigated 11 patients (group A) who were the control group, 12 patients suffering from paroxysmal atrial fibrillation (group B), and 10 patients (group C) without arrhythmias but with cardiopathy or cardiomyopathy. At every eighth complex of a constant atrial electrostimulated rhythm a fixed premature extrastimulus was introduced, and effective and functional refractory periods (ERP and FRP) were measured in three different sites of the right atrium, before and after introduction of this extrastimulus. Average ERP and FRP shortened respectively in group A, from 220.28 ± 25.68 msec and 281.17 ± 28.15 msec before extrastimulation, to 190.58 ± 22.74 msec and 245.88 ± 19.86 msec after; in group B, from 219.44 ± 27.38 msec and 284 ± 30.06 msec to 191.66 ± 28.72 msec and 253.23 ± 34.01 msec; and in group C from 229.03 ± 29.65 msec and 289.67 ± 51.62 msec to 194.19 ± 24.6 msec and 237.74 ± 39.59 msec. The average dispersions of ERP and FRP in group A were, respectively: 41.81 ± 21.36 msec and 36.36 ± 18.04 msec before extrastimulation, 28.18 ± 18.14 msec and 35.45 ± 15.72 msec after. In group B: 26.66 ± 19.46 msec and 41.66 ± 16.96 msec versus 45.83 ± 23.91 msec and 45 ± 34.77 msec and in group C: 27 ±11.59 msec and 45 ± 29.15 msec versus 29 ± 18.52 and 27 ± 18.88. It is concluded that an atrial premature beat tends to shorten the dispersion of atrial refractory periods when patients are free of arrhythmias, and to lengthen them when paroxysmal atrial fibrillation are documented.