Exploration of efficacy and bleeding with combined phosphoinositide 3-kinase ß inhibition and aspirin in man.

BACKGROUND: Based on animal and human data, phosphoinositide 3-kinase (PI3K)ß is a promising antithrombotic target. However, the relation between efficacy and bleeding when combined with current antiplatelet therapies is unclear. OBJECTIVE: To strengthen the PI3Kß target validation using the short-acting inhibitor AZD6482 alone and in different combinations with P2Y12 and cyclooxygenase (COX)-1 inhibition in vitro (human platelets), in vivo (dog), and in healthy subjects. METHODS AND RESULTS: Evaluation of complete target inhibition of PI3Kß (by AZD6482), P2Y12 (by ticagrelor), and COX-1 (by aspirin) alone and in the different combinations vs. concentration responses for a panel of platelet agonists in vitro (adenosine diphosphate, collagen, thrombin receptor activating peptide) indicates that the rank order of antiplatelet efficacy is P2Y12  > PI3Kß > COX-1 as monotherapy and P2Y12 plus PI3Kß > P2Y12 plus COX-1 > PI3Kß plus COX-1 as dual therapy, with little additional effect with triple therapy. Use of a conscious dog model to assess ex vivo antiplatelet effect in parallel with bleeding time prolongation (standard incision in the ear) confirms the wide separation of efficacy vs. bleeding for PI3Kß inhibition and that this separation is reduced when combined with aspirin and more reduced when combined with clopidogrel. In healthy subjects, AZD6482, in combination with aspirin, shows a potential for greater antiplatelet potency but less bleeding potential compared with clopidogrel plus aspirin. CONCLUSIONS: PI3Kß inhibition, in comparison with P2Y12 and COX-1, delivers medium antiplatelet effect but with minimal bleeding. PI3Kß inhibition, in combination with aspirin, in healthy subjects, provides a potential for greater overall antiplatelet effect compared with clopidogrel plus aspirin, but with significantly less bleeding potential.

Does lipoprotein(a) inhibit elastolysis in abdominal aortic aneurysms?

PURPOSE: to test the hypothesis that there is a negative association between serum levels of lipoprotein(a) (Lp(a)) and elastin-derived peptides (EDP) as well as matrix metalloproteinase (MMP)-9 activation in the aneurysm wall in patients with asymptomatic abdominal aortic aneurysms (AAA). MATERIAL AND METHODS: from 30 patients operated for asymptomatic AAAs, preoperative serum samples and AAA biopsies were collected. Lp(a) (mg/L) and EDP (ng/ml) in serum were measured by enzyme linked immunosorbent assays. MMP-9 activity (arbitrary units) in the AAA wall was measured by gelatin zymography and the ratio: active MMP-9/total MMP-9 were calculated. RESULTS: there was a significant negative correlation (Spearman's rho) between serum levels of Lp(a) and EDP (r= -0.707, p<0.001), as well as the share of activated MMP-9 (r= -0.461, p=0.01) in the AAA wall. CONCLUSION: this preliminary study indicate that Lp(a) inhibit elastolysis in asymptomatic AAA.

Serum concentrations of elastin-derived peptides in patients with specific manifestations of atherosclerotic disease.

OBJECTIVE: To measure serum concentrations of elastin-derived peptides (S-EDP) in patients with aneurysmal, occlusive and ulcerative manifestations of atherosclerotic disease. MATERIALS AND METHODS: S-EDP concentrations were measured by a competitive enzyme-linked immunosorbent assay in 10 patients with infrarenal aneurysms 5cm in diameter (AAA), 10 patients with distal aortic occlusive disease (AOD), 10 patients with symptomatic carotid stenosis (>or=70%) and plaque ulceration (SCS) and a control group of 10 patients with no similar specific manifestations of atherosclerotic disease (NAM). RESULTS: S-EDP concentrations (median, range) were significantly higher in patients with AAA (42ng/ml, 35-52, p<0.001) and SCS (49ng/ml, 37-60, p<0.001) but not AOD (28ng/ml, 22-38, p=0.240) compared to NAM (26ng/ml, 19-36) patients. CONCLUSION: Increased concentrations of S-EDP were associated with aneurysmal and ulcerative, but not occlusive, manifestations of atherosclerosis.

Presence of Chlamydia pneumoniae in abdominal aortic aneurysms is not associated with increased activity of matrix metalloproteinases.

OBJECTIVE: to test the hypothesis that the presence of Chlamydia pneumoniae (C. pneumoniae) in the wall of abdominal aortic aneurysms (AAA) is associated with increased activity of matrix metalloproteinase (MMP)-2 and/or MMP-9. DESIGN: case-control study. MATERIAL AND METHODS: in a series of 40 patients with AAA > or =5cm in maximal cross-sectional diameter, C. pneumoniae-DNA was identified in the aneurysm wall by nested PCR in 14 (35%) patients. Another 14 C. pneumoniae-DNA-negative AAA patients from the same series, matched for gender and aneurysm diameter, were used as controls. In each group there were 7 asymptomatic (aAAA) and 7 ruptured (rAAA) aneurysms. MMP-2 and -9 activity was estimated in AAA wall biopsies by gelatin zymography. RESULTS: patients with a C. pneumoniae-DNA-positive aneurysm wall specimen showed an over-all lower activity of MMP-2 and MMP-9 (pro- and active enzyme) compared to the C. pneumoniae-DNA negative patients. However, there were no statistically significant differences in MMP activity between the two groups of patients with aAAA. Among patients with rAAA both pro-MMP-9 (p=0,026) and active-MMP-9 (p=0.007) were significantly lower in C. pneumoniae-DNA-positive patients compared to C. pneumoniae-DNA-negative patients, whereas there were no significant differences in pro-MMP-2 or active-MMP-2. CONCLUSION: this preliminary study does not support the hypothesis that the presence of C. pneumoniae in the AAA wall is associated with increased activity of MMP-2 and MMP-9.

Proteolysis of the abdominal aortic aneurysm wall and the association with rupture.

PURPOSE: to investigate proteolysis of the abdominal aortic aneurysm (AAA) wall and the association with rupture. METHODS: levels of matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) were measured in the walls of medium-sized (5-7 cm in diameter) ruptured AAA (rAAA) (n =30) and large (> or = 7 cm in diameter) asymptomatic AAA (aAAA) (n=30). RESULTS: MMP-2 levels (median, range) were significantly higher in the walls of large aAAA (165 ng/g AAA tissue, 50-840) than from medium-sized rAAA (110 ng/g AAA tissue, 47-547, p=0.007). MMP-9 levels were significantly higher in the walls of medium-sized rAAA (107 ng/g AAA tissue, 19-582) than from large aAAA (55 ng/g AAA tissue, 11-278, p=0.012). TIMP-1 and TIMP-2 levels were equivalent. There was a positive correlation between MMP-2 and the diameter of aAAA (r=0.54, p=0.002), but a negative correlation with MMP-9 (r= -0.44, p=0.017). No significant correlations were found between aAAA diameter and TIMP-1 or TIMP-2. CONCLUSION: AAA rupture is associated with higher levels of MMP-9. There is no association with TIMP-1 or TIMP-2 levels. MMP-2 levels are positively, whereas MMP-9 levels are negatively, correlated with aAAA size. MMP-9 may play a role in the progression towards rupture, whereas MMP-2 may play a role in expansion.

In vitro degradation of aortic elastin by Chlamydia pneumoniae.

OBJECTIVES: to investigate whether Chlamydia pneumoniae (C. pneumoniae) may increase elastin degradation in the aortic wall. MATERIALS AND METHODS: eighteen full thickness aortic wall samples from non-aneurysmal infrarenal abdominal aortas were collected from autopsies. Two adjacent and equally large pieces were cut out of each aortic sample. From each sample, one piece was incubated in a HEp-2 cell culture infected with C. pneumoniae and the other piece was incubated in an uninfected HEp-2 cell culture. The incubation time was one week at 35 degrees C. The concentration of elastin-derived peptides (EDP) (ng/ml) in the medium of each cell culture was measured in duplicate. For each paired sample, delta-EDP (EDP in HEp-2 cell culture infected with C. pneumoniae- EDP in uninfected HEp-2 cell culture) was calculated. RESULT: there was a significantly increased degradation of aortic elastin, estimated by EDP concentrations in cell culture conditioned medium, when aortic wall samples were incubated in C. pneumoniae cultures compared with uninfected cultures (p=0.025, Wilcoxon signed ranks test). CONCLUSION: these results indicate that there is a relationship between the presence of C. pneumoniae and increased elastin degradation in the aortic wall in vitro. This suggests C. pneumoniae in the aortic wall directly or indirectly leads to the degradation of aortic elastin.

Serum levels of elastin-derived peptides in patients with ruptured and asymptomatic abdominal aortic aneurysms.

OBJECTIVE: to determine whether serum elastin-derived peptides (S-EDP), are lower in patients with ruptured abdominal aortic aneurysms (rAAA) than asymptomatic (aAAA). MATERIALS AND METHODS: serum samples were collected preoperatively from 45 consecutive patients with aAAA and 15 haemodynamically stable patients with rAAA. S-EDP (ng/ml) was measured by a competitive enzyme-linked immunosorbent assay (ELISA). RESULTS: S-EDP (mean +/- s.d.) was significantly lower in patients with rAAA (31.6 ng/ml +/- 6.8) than in patients with aAAA (39.4 ng/ml +/- 8.0 p=0.001). CONCLUSION: patients with rAAA had significantly lower levels of S-EDP than patients with aAAA. The possibility that S-EDP can be used to identify patients at increased risk of rupture requires further investigation.

Activity of matrix metalloproteinase-2 and -9 in abdominal aortic aneurysms. Relation to size and rupture.

OBJECTIVES: to investigate the activity of matrix metalloproteinase (MMP)-2 and -9 in asymptomatic abdominal aortic aneurysms (aAAAs) and ruptured abdominal aortic aneurysms (rAAAs). DESIGN: cross-sectional study. MATERIALS AND METHODS: MMP-2 and MMP-9 activity was estimated in biopsies from the anterior wall of 60 AAAs using gelatin zymography. There were 20 medium-sized (diameter 5<7 cm) aAAAs, 20 large (>57 cm) aAAAs and 20 rAAAs. MMP activity was quantified using a laser densitometer and expressed as arbitrary units (au). RESULTS: mean (SEM) MMP-9 activity was significantly lower in large aAAAs (1190 au +/-247) than in rAAAs (2647 au +/-498, p<0.05). There was no difference in MMP-2 activity. CONCLUSION: High MMP-9 activity in the AAA wall is associated with rupture.

Two glutamate decarboxylase forms corresponding to the mammalian GAD65 and GAD67 are expressed during development of the chick telencephalon.

The gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamate decarboxylase (GAD) was studied during development of the chick telencephalon. By means of reverse-phase HPLC analysis, we showed that GABA indeed accumulates during embryogenesis, whereas the levels of glutamate, the substrate for GAD, are more or less unchanged up to later developmental stages. The enzyme activity increased approximately 25-fold from embryonic day 3 to embryonic day 17. Immunoblotting data revealed that two GAD proteins, of approximately 65 and 67 kDa, were present during the period investigated. Furthermore, Northern blot analysis with probes obtained from rat cDNA sequences, as well as a chicken-specific probe for GAD65 generated by means of reverse transcriptase-polymerase chain reaction (RT-PCR), strengthened the interpretation that the chick embryo expresses genes corresponding to GAD65 and GAD67. The rat probes recognized transcript sizes of 3.9 kb (GAD65) and 5.6 kb (GAD67), sizes which are different from those of the rat brain (Erlander et al., Neuron, 7, 91-100, 1991). Sequencing of the RT-PCR products revealed a high level of homology (82% at the nucleotide level) between the mammalian and chick GAD65 genes. Taken together, these findings suggest that the chick embryo expresses two GAD genes during embryogenesis. The functional properties of each gene product remain to be investigated.