On-treatment function testing of platelets and long-term outcome of patients with peripheral arterial disease undergoing transluminal angioplasty.

OBJECTIVE: To assess the clinical importance of on-treatment function testing of platelets in patients on aspirin after catheter-based vascular interventions. MATERIALS AND METHODS: In 109 patients with symptomatic peripheral arterial disease (PAD) of the lower limbs, platelet function testing (adenosine diphosphate-, collagen- and epinephrine-induced aggregation using light transmission aggregometry) was performed before and at multiple time points up to 1 year after a percutaneous angioplasty. Using univariate mixture models and Box-Cox transformation to ensure normally distributed individual variances, we investigated if an intraindividual variability exists and if it has a consequence for clinical outcome. RESULTS: Response to aspirin as measured by platelet aggregometry varies considerably over time in most patients. However, the intraindividual variance over time was not significantly correlated either with restenosis/reocclusion after 1 year or with adverse long-term outcome (occurrence of death for cardiovascular cause, stroke or myocardial infarction in up to 8 years follow-up). CONCLUSIONS: Response to aspirin does not seem to have a role in determining long-term outcome in patients with symptomatic PAD. The fact that testing of platelet function at only one time point has reduced significance may have implications for all clinical settings in which aspirin is used for the prevention of thrombo-embolic events.

Wall shear stress in the superficial femoral artery of healthy adults and its response to postural changes and exercise.

OBJECTIVES: Alterations of wall shear stress (WSS) are considered to precede atherosclerosis. Local variations of WSS might contribute to the typical distribution of atherosclerotic lesions along the superficial femoral artery (SFA). We investigated the course of WSS and its response to postural changes and exercise along the SFA of healthy adults. METHODS: In forty-six healthy subjects, we determined flow velocities and internal vessel diameters in five predefined segments of the SFA using duplex ultrasound; measurements were done at rest, following exercise (30 toe raises) and after postural changes (supine and sitting). Peak and mean WSS were calculated from peak systolic and mean velocities, vessel diameter and whole blood viscosity. RESULTS: At rest, peak and mean WSS did not vary along the femoro-popliteal axis (p > 0.05); peak and mean WSS were lower in the sitting than in the supine position (p < 0.0001). After exercise, peak and mean WSS increased in all segments (p < 0.0001), showing the lowest increase in the distal Hunter's canal. CONCLUSION: Healthy adults do not exhibit local variations of WSS in the SFA at rest, but segmental differences in WSS occur after exercise. Whether these findings are related to the typical distribution of atherosclerotic lesions later in life requires further investigation.

Lack of major gender-related differences in patients undergoing elective endovascular or surgical repair of abdominal aortic aneurysm.

AIM: The aim of this single center retrospective study was to determine gender-related differences in patients undergoing repair of aortic aneurysm. METHODS: A total of 296 consecutive patients with an abdominal aneurysm undergoing elective endovascular or surgical repair was included in the study. Among these, 24 (8.1%) were females and 272 (91.9%) were males. Demographic and clinical characteristics as well as laboratory values in terms of any potential gender-specific differences were compared. RESULTS: Females were significantly older at the time of repair than males. No gender-related differences were found analysing major clinical and laboratory parameters. CONCLUSIONS: The clinical and laboratory profile with which males and females present at the time of elective endovascular or surgical repair for aortic aneurysm is very similar. It does not reveal any potential gender-specific risk constellation. It eventually remains unclear why the prevalence of aortic aneurysm is higher in male than in females.

Isolated ventricular non-compaction of the myocardium in adults.

Isolated ventricular non-compaction (IVNC) in adults is a genetic cardiac disease of emerging importance with a distinct clinical and pathophysiological presentation. The body of evidence for the underlying genetic basis of the disease has also grown. Prognosis remains poor for patients with impaired systolic left ventricular function, as treatment options are very limited. The diagnosis of IVNC, however, is often missed, most often as a consequence of ignorance of the condition. The relevant clinical issues and the emerging concepts of the aetiology of IVNC are summarised.

Carotid intima-media thickness, carotid wall shear stress and restenosis after femoro-popliteal percutaneous transluminal angioplasty (PTA).

OBJECTIVE: To determine the relationship between carotid intima-media thickness (IMT), carotid wall shear stress (WSS) and restenosis after femoro-popliteal percutaneous transluminal angioplasty (PTA). PATIENTS AND METHODS: Thirty-one subjects (18 men, 13 women, median age 69 years) treated with femoro-popliteal PTA for symptomatic peripheral arterial occlusive disease were enrolled. On admission, IMT, internal diameter and blood velocity of the common carotid artery (CCA) were assessed by high-resolution ultrasonography. Blood viscosity was measured and carotid WSS was calculated. Patients were followed up for 6 months for the occurrence of significant restenosis (>50%) as documented by duplex ultrasonography. Two patients were lost to follow-up. RESULTS: Fourteen patients (48%) developed restenosis at 6 months. IMT and WSS were not different in patients without and with restenosis (IMT: 0.90 (0.85-0.97) vs. 0.89 (0.84-0.93) mm, p = 0.51; WSS: 14.1 (11.9-19.2) vs. 15.9 (12.8-21.5) dyne/cm2, p = 0.48). The hazard ratio of incident restenosis as estimated by Cox regression analysis was 0.04 for IMT (p = 0.23; 95% CI 0.0001-8.22) and 1.07 for WSS (p = 0.10; 95% CI 0.98-1.17). CONCLUSIONS: In this pilot study involving a limited number of patients, carotid IMT and carotid WSS are not significantly related to restenosis at 6 months after femoro-popliteal PTA. This might be the result of different underlying pathophysiology for atherosclerosis and restenosis.

[How do blood vessels age? Mechanisms and clinical implications]. / Wie altern Gefässe? Mechanismen und klinische Implikationen.

Ageing is one of the most important cardiovascular risk factors. Vascular ageing is determined by genetic, mechanic and hemodynamic factors. The latter is strengthened by the fact that age-associated changes in capillaries and veins, vessels which are less prone to changes in pulsatility and blood pressure, are less pronounced or even absent. Age-related morphologic changes in large resistance vessels include an intima-media thickening, increased deposition of matrix substances, thus ultimately leading to a reduced compliance. Vascular ageing is mainly characterized by an impaired endothelium-dependent vasorelaxation. The expression of endothelial nitric oxide synthase (eNOS), producing vasodilatatory nitric oxide (NO), is markedly upregulated with increasing age. However, vasorelaxation is impaired, as the production of reactive oxygen species such as superoxide (O2-), concomitantly increases. NO and O2- react to form the powerful oxidant peroxynitrite (ONOO-). Peroxynitrite is known to initiate oxidative modification of proteins, including nitration of aromatic rings, thereby rendering functionally inactive certain regulatory proteins. Deposition of nitrated proteins is mainly found within endothelial mitochondria, suggesting that mitochondrial dysfunction plays a major role in the vascular ageing process. It yet remains to be shown whether oxidative stress, which is, according to the currently accepted "oxidative stress hypothesis", a key event of vascular ageing, can be pharmacologically prevented, e.g. by naturally occurring antioxidant vitamins. However, in a mammalian model of ageing, an unexpected accumulation of vitamin E was found to accumulate within the aortic wall. This may represent a self-regulatory adaptive mechanism to prevent age-associated oxidative stress. In contrast, ascorbic acid was found to decrease with increasing age. Eventually, it remains to be seen if vitamin C or other antioxidative substances may be useful therapies. Statins and ACE inhibitors are known to have effects on mechanisms interfering with the ageing process. Given the strong age-dependency of cardiovascular disease, the developments of therapies to delay vascular ageing might have enormous medical (and economic) consequences in the future.

An unusual consequence of deep vein thrombosis.

We report here an unusual case in which deep vein thrombosis, limited to the infrapopliteal region, led to an anterior tibial compartment syndrome as a major complication in a patient who had undergone heart surgery shortly before.

Enhanced peroxynitrite formation is associated with vascular aging.

Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (.O(2)(-)) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented.O(2)(-) release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

HMG-CoA reductase inhibition improves endothelial cell function and inhibits smooth muscle cell proliferation in human saphenous veins.

OBJECTIVES: This study examined effects of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor cerivastatin on human saphenous vein (SV), endothelial cells (EC) and smooth muscle cells (SMC). BACKGROUND: Venous bypass graft failure involves EC dysfunction and SMC proliferation. Substances that improve EC function and inhibit SMC proliferation would be of clinical relevance. METHODS: Both EC and SMC were isolated from SV. Endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production were analyzed by immunoblotting and porphyrinic microsensor. The SMC proliferation was assayed by 3H-thymidine incorporation. Protein kinases and cell cycle regulators were analyzed by immunoblotting. RESULTS: Cerivastatin (10(-9) to 10(-6) mol/liter) enhanced eNOS protein expression and NO release (about two-fold) in EC in response to Ca2+ ionophore (10(-6) mol/liter). This was fully abrogated by the HMG-CoA product mevanolate (2 x 10(-4) mol/liter). In SMC, platelet-derived growth factor (5 ng/ml) enhanced 3H-thymidine incorporation (298 +/- 23%, n = 4), activated cyclin-dependent kinase (Cdk2), phosphorylated Rb and down-regulated p27Kip1 (but not p21CiP1). Cerivastatin reduced the 3H-thymidine incorporation (164 +/- 11%, p < 0.01), inhibited Cdk2 activation and Rb phosphorylation, but did not prevent p27Kip1 down-regulation, nor p42mapk and p70S6K activation. Mevalonate abrogated the effects of cerivastatin on Cdk2 and Rb but only partially rescued the 3H-thymidine incorporation (from 164 +/- 11% to 211 +/- 13%, n = 4, p < 0.01). CONCLUSIONS: In humans, SVEC inhibition of HMG-CoA/mevalonate pathway contributes to the enhanced eNOS expression and NO release by cerivastatin, whereas in SMC, inhibition of this pathway only partially explains cerivastatin-induced cell growth arrest. Inhibition of mechanisms other than p42mapk and p70S6K or Cdk2 are also involved. These effects of cerivastatin could be important in treating venous bypass graft disease.

A role for changes in platelet production in the cause of acute coronary syndromes.

Platelets are heterogeneous with respect to their size, density, and reactivity. Large platelets are more active hemostatically, and platelet volume has been found to be increased both in patients with unstable angina and with myocardial infarction. Furthermore, platelet volume is a predictor of a further ischemic event and death when measured after myocardial infarction. Platelets which are anucleate cells with no DNA are derived from their precursor, the megakaryocyte. Therefore, it is suggested that changes in platelet size are determined at thrombopoiesis in the megakaryocyte and that those changes might precede acute cardiac events. Understanding of the signaling system that controls platelet production may also further elucidate the cascade of events leading to acute vascular occlusion in some patients.

Cytochemical detection of a senescence-associated beta-galactosidase in endothelial and smooth muscle cells from human and rabbit blood vessels.

A beta-galactosidase activity has recently been used as a histochemical marker of replicative senescence in human fibroblasts and keratinocytes. To establish whether this marker could be used to detect senescence of vascular cells, we have investigated its presence in cultures of serially passaged human umbilical vein endothelial cells and rabbit aortic smooth muscle cells. beta-Galactosidase activity was detected by light microscopy using the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside. In endothelial cell cultures, lysosomal beta-galactosidase activity, which is detected at pH 4.0, was present in all cells regardless of their replicative age. In contrast, senescence-associated beta-galactosidase activity, which is detected at pH 6.0, was absent in the majority of cells in early passage cultures (<15 cumulative population doublings), but was present in a large proportion of cells (up to 62%) in late passage cultures (>30 cumulative population doublings); in intermediate passage cultures (15-30 cumulative population doublings) it was found in fewer than 15% of the cells. The increase in the percentage of senescence-associated beta-galactosidase-positive cells correlated with a decrease in the cell density at confluence and with a marked increase in cell size. Counterstaining with an antibody directed against the endothelial cell marker CD31 showed that senescent cells retained the expression of this antigen. Senescence-associated beta-galactosidase was also detected in serially passaged, but not in primary explant cultures of rabbit aortic vascular smooth muscle cells. The presence of senescence-associated beta-galactosidase in cultured vascular smooth muscle cells and endothelial cells suggests that this marker could be used to study the role of cellular senescence in vascular disease.

Protein kinase C mediates the mitogenic action of thrombopoietin in c-Mpl-expressing UT-7 cells.

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms alpha and beta found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms alpha and beta to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

The adventitia, endothelium and atherosclerosis.

Despite many theories, the initiating circumstances for the development of atherosclerosis remain obscure. The development of animal models of atherosclerosis was based upon the different theories of the origins of atherosclerosis which suggested that it originates at the intimal surface of the vessel. A more recent model of atherosclerosis involves perivascular manipulation of the vessel by positioning of a hollow silastic collar around the artery. In this model, several of the features seen in early human atherosclerosis are generated within a period of 7 days. It is hypothesized that arterial wall hypoxia following occlusion of the vasa vasorum may be the initial lesion of atherosclerosis, and hence that in some cases atherosclerosis is a disease of the outer layers of the arterial wall.

Megakaryocytes and platelets in vascular disease.

Platelets are anucleate cells with no DNA. They are derived from their precursor, the megakaryocyte (MK), whose differentiation is characterized by nuclear polyploidization through a process called endomitosis. Changes in the MK-platelet-haemostasis axis may precede acute thrombotic events. Changes in MK ploidy distribution may be associated with the production of large platelets. Mean platelet volume (MPV) is an important biological variable as it is a determinant of platelet reactivity. Large platelets are denser and more active haemostatically. MPV is increased in patients after myocardial infarction (MI) and is a predictor of a further ischaemic event and death when measured after MI. It has been suggested that changes not only in platelets but also in the parental MK are associated with chronic and acute vascular events. The regulation of megakaryocytopoiesis depends on several haematopoietic factors such as thrombopoietin. An understanding of the signalling system that controls platelet number and size might give insight into a role of platelet production in thrombogenesis and atherogenesis.

Antimicrotubule agents induce polyploidization of human leukaemic cell lines with megakaryocytic features.

In most eukaryotic cells the regular alternation of chromosome reduplication and cell division is controlled by interdependent relationships which prevent progression to the next cell-cycle phase unless the preceding phase has been completed. Megakaryocytes become polyploid by allowing many rounds of DNA replication without completion of intervening mitoses. To assess the role of cell-cycle dependencies in megakaryocytopoiesis we examined human cell lines which express megakaryocytic features for their ability to continue DNA synthesis and undergo polyploidization in the presence of mitotic poisons. Treatment of HEL cells with colcemid blocked cell division but not cellular DNA synthesis. DNA content distributions of cells treated with colcemid for 48 h showed a marked increase in the proportion of polyploid cells (57.6% +/- 9.9%, n = 16), an increase in cellular size and nuclear lobation. Identical effects were observed in HEL cells treated with colchicine, nocodazole or taxol but not with the inactive compound lumicolchicine. Induction of polyploidization by antimicrotubule agents was also observed in the megakaryoblastic cell lines MEG-01, DAMI and UT-7 but not in the T-cell line MOLT-4 or the promyelocytic cell line HL-60. These results suggest that dependency of DNA replication on completion of the previous mitosis is suppressed in the megakaryocytic lineage.