Left atrial pump strain predicts long-term survival after transcatheter aortic valve implantation.

BACKGROUND: This study aims at investigating left atrial (LA) deformation by left atrial reservoir (LARS) and pump strain (LAPS) and its implications for long-term survival in patients with severe aortic stenosis (AS) undergoing transcatheter aortic valve implantation (TAVI). METHODS: Speckle tracking echocardiography was performed in 198 patients with severe AS undergoing TAVI. Association of strain parameters with cardiovascular mortality was determined. RESULTS: Over a follow-up time of 5 years, 49 patients (24.7%) died. LAPS was more impaired in non-survivors than survivors (P = 0.010), whereas no difference was found for LARS (P = 0.114), LA ejection fraction (P = 0.241), and LA volume index (P = 0.292). Kaplan-Meier analyses yielded a reduced survival probability according to the optimal threshold for LAPS (P = 0.002). A more impaired LAPS was associated with increased mortality risk (HR 1.12 [95% CI 1.02-1.22]; P = 0.014) independent of LVEF, LAVI, age, and sex. Addition of LAPS improved multivariable echocardiographic (LVEF, LAVI) and clinical (age, sex) models with potential incremental value for mortality prediction (P = 0.013 and P = 0.031, respectively). In contrast, LARS and LAVI were not associated with mortality. CONCLUSIONS: In patients undergoing aortic valve replacement for severe AS, LAPS was impaired in patients dying during long-term follow-up after TAVI, differentiated survivors from non-survivors, was independently associated with long-term mortality, and yielded potential incremental value for survival prediction after TAVI. LAPS seems useful for risk stratification in severe AS and timely valve replacement.

Mitral annular disjunction in patients with severe aortic stenosis: Extent and reproducibility of measurements with computed tomography.

OBJECTIVES: To determine with CT the prevalence and extent of mitral annular disjunction (MAD) in patients undergoing transcatheter aortic valve replacement (TAVR) and its association with mitral valve disease and arrhythmia. METHODS: We retrospectively evaluated 408 patients (median age, 82 years; 186 females) with severe aortic stenosis undergoing ECG-gated cardiac CT with end-systolic data acquisition. Baseline and follow-up data were collected in the context of a national registry. Two blinded, independent observers evaluated the presence of MAD on multi-planar reformations. Maximum MAD distance (left atrial wall-mitral leaflet junction to left ventricular myocardium) and circumferential extent of MAD were assessed on CT using dedicated post-processing software. Associated mitral valve disease was determined with echocardiography. RESULTS: 7.8 % (32/408) of patients with severe aortic stenosis had MAD. The maximum MAD was 3.5 mm (interquartile range: 3.0-4.0 mm). The circumferential extent of MAD comprised 34 ±â€¯15 % of the posterior and 26 ±â€¯12 % of the entire mitral annulus. Intra- and interobserver agreement for the detection of MAD on CT were excellent (kappa: 0.90 ±â€¯0.02 and 0.92 ±â€¯0.02). Mitral regurgitation (p = 1.00) and severe mitral annular calcification (p = 0.29) were similarly prevalent in MAD and non-MAD patients. Significantly more patients with MAD (6/32; 19 %) had mitral valve prolapse compared to those without (6/376; 2 %; p < 0.001). MAD was not associated with arrhythmia before and after TAVR (p > 0.05). CONCLUSIONS: Using CT, MAD was found in 7.8 % of patients with severe aortic stenosis, with a higher prevalence in patients with mitral valve prolapse. We found no association of MAD with arrhythmia before or after TAVR.

A Prospective Pilot Study to Identify a Myocarditis Cohort who may Safely Resume Sports Activities 3 Months after Diagnosis.

International cardiovascular society recommendations to return to sports activities following acute myocarditis are based on expert consensus in the absence of prospective studies. We prospectively enrolled 30 patients with newly diagnosed myocarditis based on clinical parameters, laboratory measurements and cardiac magnetic resonance imaging with mildly reduced or preserved left ventricular ejection fraction (LVEF) with a follow-up of 12 months. Cessation of physical activity was recommended for 3 months. The average age was 35 (19-80) years with 73% male patients. One case of non-sustained ventricular tachycardia was recorded during 48-h-Holter electrocardiogram. Except for this case, all patients were allowed to resume physical exercise after 3 months. At 6- (n = 26) and 12-month (n = 19) follow-up neither cardiac events nor worsening LVEF were recorded. The risk of cardiac events at 1 year after diagnosis of myocarditis appears to be low after resumption of exercise after 3 months among patients who recover from acute myocarditis.

Mitral annular calcification in the elderly - Quantitative assessment.

PURPOSE: To determine the reliability of subjective and objective quantification of mitral annular calcification (MAC) in elderly patients with severe aortic stenosis, to define quantitative sex- and age-related reference values of MAC, and to correlate quantitative MAC with mitral valve disease. METHODS: In this retrospective, IRB-approved study, we included 559 patients (268 females, median age 81 years, inter-quartile range 77-85 years) with severe aortic stenosis undergoing CT. Four independent readers performed subjective MAC categorization as follows: no, mild, moderate, and severe MAC. Two independent readers performed quantitative evaluation of MAC using the Agatston score method (AgatstonMAC). Mitral valve disease was determined by echocardiography. RESULTS: Subjective MAC categorization showed high inter-reader agreement for no (k â€‹= â€‹0.88) and severe MAC (k â€‹= â€‹0.75), whereas agreement for moderate (k â€‹= â€‹0.59) and mild (k â€‹= â€‹0.45) MAC was moderate. Intra-reader agreement for subjective MAC categorization was substantial (k â€‹= â€‹0.69 and 0.62). Inter- and intra-reader agreement for AgatstonMAC were excellent (ICC â€‹= â€‹0.998 and 0.999, respectively), with minor inconsistencies in MAC involving the left ventricular outflow tract/aortic valve. There were significantly more women than men with MAC (n â€‹= â€‹227, 85% versus n â€‹= â€‹209, 72%; p â€‹< â€‹0.001), with a significantly higher AgatstonMAC (median 597, range 81-2055 versus median 244; range 0-1565; p â€‹< â€‹0.001), particularly in patients ≥85 years of age. AgatstonMAC showed an area-under-the-curve of 0.84 to diagnose mitral stenosis, whereas there was no association of AgatstonMAC with mitral regurgitation (p â€‹> â€‹0.05). CONCLUSIONS: Our study in elderly patients with severe aortic stenosis shows that quantitative MAC scoring is more reliable than subjective MAC assessment. Women show higher AgatstonMAC scores than men, particularly in the elderly population. AgatstonMAC shows high accuracy to diagnose mitral stenosis.

Reproducibility of aortic valve calcification scoring with computed tomography - An interplatform analysis.

BACKGROUND: To investigate whether aortic valve calcification (AVC) scoring performed with different workstation platforms generates comparable and thus software-independent results. METHODS: In this IRB-approved retrospective study, we included 100 consecutive patients with symptomatic aortic stenosis undergoing CT prior to transcatheter aortic valve implantation. Two independent observers performed AVC scoring on non-enhanced images with commercially available software platforms of four vendors (GE, Philips, Siemens, 3mensio). Gender-specific Agatston score cut-off values were applied according to current recommendations to assign patients to different likelihood categories of aortic stenosis (unlikely to very likely). Comparative analysis of Agatston scores between the four platforms were performed by using Kruskal-Wallis analysis, Spearman rank correlation, linear regression analysis, and Bland-Altman analysis. Differences in category assignment were compared using Fisher's exact test and Cohen's kappa. RESULTS: For both observers, each workstation platform produced slightly different numeric AVC Agatston scores, however, without statistical significance (p = 0.96 and p = 0.98). Excellent correlation was found between platforms, with r = 0.991-0.996 (Spearman) and r2 = 0.981-0.992 (regression analysis) for both observers. Bland-Altman analyses revealed small mean differences with narrow limits of agreement between platforms (mean differences: 6 ±â€¯128 to 100 ±â€¯179), for inter-observer (mean differences: 1 ±â€¯43 to 12 ±â€¯70), and intra-observer variability (mean differences: 9 ±â€¯42 to 20 ±â€¯96). Observer 1 assigned 11 (kappa: 0.85-0.97) and observer 2 assigned 10 patients (kappa: 0.88-0.95) to different likelihood groups of severe aortic stenosis with at least one platform. Overall, there was no significant difference of likelihood assignment between platforms (p = 0.98 and p = 1.0, respectively). CONCLUSION: While absolute values differ slightly, common commercially available software platforms produce comparable results for AVC scoring, which indicates software-independence of the method.

Amiodarone inhibits arterial thrombus formation and tissue factor translation.

BACKGROUND: In patients with coronary artery disease and reduced ejection fraction, amiodarone reduces mortality by decreasing sudden death. Because the latter may be triggered by coronary artery thrombosis as much as ventricular arrhythmias, amiodarone might interfere with tissue factor (TF) expression and thrombus formation. METHODS AND RESULTS: Clinically relevant plasma concentrations of amiodarone reduced TF activity and impaired carotid artery thrombus formation in a mouse photochemical injury model in vivo. PTT, aPTT, and tail bleeding time were not affected; platelet number was slightly decreased. In human endothelial and vascular smooth muscle cells, amiodarone inhibited tumor necrosis factor (TNF)-alpha and thrombin-induced TF expression as well as surface activity. Amiodarone lacking iodine and the main metabolite of amiodarone, N-monodesethylamiodarone, inhibited TF expression. Amiodarone did not affect mitogen-activated protein kinase activation, TF mRNA expression, and TF protein degradation. Metabolic labeling confirmed that amiodarone inhibited TF protein translation. CONCLUSIONS: Amiodarone impairs thrombus formation in vivo; in line with this, it inhibits TF protein expression and surface activity in human vascular cells. These pleiotropic actions occur within the range of amiodarone concentrations measured in patients, and thus may account at least in part for its beneficial effects in patients with coronary artery disease.

Prothrombotic gene expression profile in vascular smooth muscle cells of human saphenous vein, but not internal mammary artery.

BACKGROUND: The resistance of internal mammary artery (IMA) toward thrombotic occlusion and accelerated atherosclerosis is not well understood. This study analyzed gene expression profiles of vascular smooth muscle cells (VSMCs) from IMA versus saphenous vein (SV). METHODS AND RESULTS: 54'675 probe sets were examined by Affymetrix microarrays. Thirty-one genes belonged to the coagulation system; 2 were differentially expressed, namely tissue factor (TF) and tissue-type plasminogen activator (tPA). TF was 3.1-fold lower in IMA than SV (P=0.006), whereas tPA was 9.0-fold higher (P<0.001). TF mRNA expression was lower in IMA than SV (P<0.05); tPA was higher (P<0.001). TF protein expression was 4.2+/-0.5-fold lower in IMA than SV (P<0.001); tPA was 2.6+/-0.4-fold higher (P<0.01). In IMA VSMC supernatant, TF protein and activity was lower (P<0.05), TFPI and tPA protein higher (P<0.05 and P<0.005), and clotting time of human plasma prolonged (P<0.05) as compared to SV. Migration to TF/FVIIa (10(-9) mol/L) was 3-fold lower in IMA than SV (P=0.01); PAR-2 protein expression was similar (P=NS), PAR-2 blockade without effect (P=NS). CONCLUSIONS: Among the genes of the coagulation system, TF and tPA are differentially expressed in VSMCs from IMA versus SV. This is consistent with protection of IMA from thrombus formation and vascular remodeling.

Histamine differentially interacts with tumor necrosis factor-alpha and thrombin in endothelial tissue factor induction: the role of c-Jun NH2-terminal kinase.

BACKGROUND: Histamine plays an important role in vascular disease. Tissue factor (TF) expression is induced in vascular inflammation and acute coronary syndromes. OBJECTIVES: This study examined the effect of histamine on tumor necrosis factor-alpha- (TNF-alpha-) vs. thrombin-induced endothelial TF expression. METHODS AND RESULTS: Histamine (10(-8)-10(-5) mol L-1), TNF-alpha (5 ng mL-1), and thrombin (1 U mL-1) induced TF expression in human endothelial cells. Although TF expression by TNF-alpha and thrombin was identical, histamine augmented TNF-alpha-induced expression 7.0-fold, but thrombin-induced expression only 2.6-fold. Similar responses occurred with TF activity. The H1-receptor antagonist mepyramine abrogated these effects. Differential augmentation by histamine was also observed at the mRNA level. Histamine-induced p38 activation preceded a weak second activation to both TNF-alpha and thrombin. Histamine-induced c-Jun NH2-terminal kinase (JNK) activation was followed by a strong second activation to TNF-alpha, and less to thrombin. Selective inhibition of this second JNK activation by SP600125 reduced TF induction to histamine plus TNF-alpha by 67%, but to histamine plus thrombin by only 32%. Histamine augmented TNF-alpha- and thrombin-induced vascular cell adhesion molecule 1 (VCAM-1) expression to a similar extent. Consistent with this observation, VCAM-1 induction to TNF-alpha and thrombin was mediated by p38, but not by JNK. CONCLUSIONS: Histamine differentially augments TNF-alpha- vs. thrombin-induced TF expression and activity, which is mediated by the H1-receptor, occurs at the mRNA level, and is related to differential JNK activation.

Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation.

BACKGROUND: The cyclin-dependent kinase inhibitors (CKIs) have different patterns of expression in vascular diseases. The Kip/Cip CKIs, p27(Kip1) and p21(Cip1), are upregulated during arterial repair and negatively regulate the growth of vascular smooth muscle cells (VSMCs). In contrast, the Ink CKI, p16(Ink4), is not expressed in vascular lesions. We hypothesized that a variation in the inactivation of cdk2 and cdk4 during the G(1) phase of the cell cycle by p27(Kip1), p21(Cip1), and p16(Ink4) leads to different effects on VSMC growth in vitro and in vivo. METHODS AND RESULTS: The expression of p27(Kip1) and p21(Cip1) in serum-stimulated VSMCs inactivated cdk2 and cdk4, leading to G(1) growth arrest. p16(Ink4) inhibited cdk4, but not cdk2, kinase activity, producing partial inhibition of VSMC growth in vitro. In an in vivo model of vascular injury, overexpression of p27(Kip1) reduced intimal VSMC proliferation by 52% (P<0.01) and the intima/media area ratio by 51% (P<0.005) after vascular injury and gene transfer to pig arteries, when compared with control arteries. p16(Ink4) was a weak inhibitor of intimal VSMC proliferation in injured arteries (P=NS), and it did not significantly reduce intima/media area ratios (P=NS), which is consistent with its minor effects on VSMC growth in vitro. CONCLUSIONS: p27(Kip1) and p21(Cip1) are potent inhibitors of VSMC growth compared with p16(Ink4) because of their different molecular mechanisms of cyclin-dependent kinase inhibition in the G(1) phase of the cell cycle. These findings have important implications for our understanding of the pathophysiology of vascular proliferative diseases and for the development of molecular therapies.

Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation.

BACKGROUND: We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The NO donor diethylenetriamineNONOate (10(-5) to 10(-3) mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BB in a concentration-dependent manner. This effect was not observed with disintegrated diethylenetriamineNONOate or with the parent compound, diethylenetriamine. Adenoviral transfection of endothelial NO synthase (NOS) inhibited proliferation in response to FCS, which was prevented with N(G)-nitro-L-arginine methyl ester. NOS overexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selectively enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G(1) cell cycle arrest. Cytotoxicity was determined with trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas cyclin E remained unchanged. p21 expression was induced, and p27 remained unaltered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% FCS was barely inhibited with 8-bromo-cGMP (10(-3) mol/L) but was blunted with both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects of NO. CONCLUSIONS: NO inhibits VSMC proliferation by specifically changing the expression and activity of cell cycle regulatory proteins, which may occur independent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.

[Gene therapy in heart diseases]. / Gentherapie bei Herzerkrankungen.

Cardiovascular diseases are the most important cause of death and hospitalisation in industrialised countries. Although pharmacological, interventional and surgical therapy has achieved major progress during the past 25 years, most therapeutic measures are only transiently effective or require life-long medication. Molecular cardiology aims at applying molecular biological methods for both diagnosis and treatment of cardiovascular disease. With respect to diagnosis of cardiac diseases such as hypertrophic cardiomyopathy or the long QT syndrome, it has become possible to characterise mutations in the genome responsible for the disease process. It is interesting that different mutations inducing hypertrophic cardiomyopathy are associated with a different prognosis and survival time. This example demonstrates that molecular biological analysis allows a better estimation of the individual risk in patients with a monogenetic disease. Such diseases are an important target for genetic therapies, as transfection of normal copies of the diseased gene would potentially cure the patient. Clinical experience has so far only been obtained in patients with familial hypercholesterolaemia and mutations in the LDL receptor. Molecular biology also permits a better understanding of the pathogenesis of atherosclerosis, which is responsible for most cardiovascular disease. Atherosclerosis is a disease of conduit arteries such as the aorta and the coronary arteries. In recent years it has become possible to characterise better the molecular and cellular changes leading to endothelial dysfunction, coronary vasospasm, adhesion of monocytes and lymphocytes, proliferation and migration of vascular smooth muscle cells, and formation of extracellular matrix. This improved understanding has led to new therapeutic approaches, although a genetic intervention is not probable for the moment due to the complexity of the disease process. Balloon dilatation of coronary arteries has generated a new disease, namely restenosis. Vascular remodelling and proliferation are of major importance for this disease. Many cellular mechanisms have been characterised, and gene therapeutic strategies including signal transduction and cell cycle regulation have already been investigated experimentally. Coronary bypass graft disease represents another target for gene therapy in the vascular system. Many experimental and a few clinical protocols have been performed with the saphenous vein. Yet another strategy for gene therapy is the endogenous formation of new vessels due to the effect of vascular endothelial growth factor. Molecular cardiology is a new and promising approach to a better understanding of cardiovascular disease. Genetic analysis is already established for the diagnosis of single gene disorders and, in addition, allows a more precise prognostic evaluation. Cardiovascular gene therapy has been focussing mainly on angiogenesis; other strategies, however, are under investigation mainly in an experimental setting.

Connective tissue growth factor induces apoptosis in human breast cancer cell line MCF-7.

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor beta (TGF-beta). TGF-beta by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-beta-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF.

Overexpression of connective tissue growth factor gene induces apoptosis in human aortic smooth muscle cells.

BACKGROUND: Connective tissue growth factor (CTGF) is expressed at very high levels particularly in the shoulder of human atherosclerotic lesions but not in normal blood vessels. Thus, CTGF may be important in the regulation of vascular smooth muscle cell function in atherosclerosis, but its precise role remains elusive. METHODS AND RESULTS: Full-length CTGF cDNA driven by a cytomegalovirus promoter was transiently transfected into cultured human aortic smooth muscle cells (HASCs). Northern and Western analysis demonstrated that CTGF was overexpressed in these cells 48 hours after transfection. The effects of CTGF overexpression on cell proliferation were evaluated by [(3)H]thymidine uptake and cell count in quiescent HASCs or those stimulated with platelet-derived growth factor (PDGF). Although mock transfection showed no effect, CTGF overexpression significantly inhibited cell proliferation in cells stimulated by PDGF. Moreover, CTGF overexpression, but not mock transfection, significantly increased apoptosis as assessed by DNA fragmentation associated with histone, TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry. CONCLUSIONS: Our results for the first time demonstrate that CTGF can also act as a growth inhibitor in human aortic smooth muscle cells at least in part by inducing apoptosis. This may be important for the formation and composition of lesions and plaque stability in atherosclerosis.

Expression of cyclin-dependent kinase inhibitors in vascular disease.

Arterial lesions in cardiovascular diseases are characterized by proliferation and migration of smooth muscle cells as well as deposition of connective tissue matrix. Factors that stimulate vascular smooth muscle cell (VSMC) proliferation are well described; however, the role of proteins that limit intimal hyperplasia is not well understood. To examine the function of Kip/Cip and INK cyclin-dependent kinase inhibitors (CKIs) in vascular diseases, the expression of p27Kip1 and p16INK was examined in VSMCs in vitro and in porcine arteries and human atherosclerosis in vivo. Western blot and fluorescence activated cell-sorting analysis demonstrated that levels of p27Kip1, but not p16INK, increased during serum deprivation of primary VSMC cultures and caused G1 arrest. p27Kip1 inhibited Cdk2 activity, suggesting that Kip CKIs promote G1 arrest in VSMCs by binding cyclin E/Cdk2. In porcine arteries, p27Kip1, but not p16INK, was constitutively expressed at low levels. Immediately after balloon injury, cell proliferation increased as p27Kip1 levels declined. Three weeks after injury, p27Kip1 was strongly expressed in intimal VSMCs when VSMC proliferation was < 2%, suggesting that p27Kip1 functions as an inhibitor of cell proliferation in injured arteries. In contrast, p16INK expression was detected only transiently early after injury. CKI expression was examined in 35 human coronary arteries, ranging from normal to advanced atherosclerosis. p27Kip1 expression was abundant in nonproliferating VSMCs and macrophages within normal (7 of 8) and atherosclerotic (25 of 27) arteries. p21Cip1 levels were undetectable in normal arteries but were elevated in atherosclerotic (19 of 27) arteries. p16INK could not be detected in normal or atherosclerotic arteries (0 of 35). Thus, the Kip/Cip and INK CKIs have different temporal patterns of expression in VSMCs in vitro and in injured arteries and atherosclerotic lesions in vivo. In contrast to p16INK, p27Kip1 likely contributes to the remodeling process in vascular diseases by the arrest of VSMCs in the G1 phase of the cell cycle.

Transfection of human endothelial cells.

OBJECTIVE: The introduction of recombinant genes into endothelial cells provides a method to study specific gene products and their effect on cell function. In addition, endothelial cells can be used for implantation into vessels or prosthetic vascular grafts. Because transfection efficiencies in human endothelial cells have been low, it is important to develop improved gene transfer techniques. Therefore, several transfection methods were optimized and transfection efficiencies were determined. METHODS: Transfection by particle-mediated gene transfer (biolistics) or by cationic liposomes were optimized and compared to calcium phosphate and DEAE-dextran. Transfection efficiency was determined using either a beta-galactosidase or placental alkaline phosphatase reporter gene. The effect of promoter strength was analyzed by transfecting plasmids with either the Rous sarcoma virus (RSV) promoter or cytomegalovirus (CMV) promoter regions. RESULTS: Optimal conditions for particle-mediated gene transfer utilized gold particles of 1.6 microns diameter, a target distance of 3 cm, helium pressures of 8.96 MPa (1300 psi) and cell confluence of 75%. Transfection with different cationic liposomes demonstrated that one compound, N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanimi nium bromide/dioleoyl phosphatidylethanolamine (gamma AP-DLRIE/DOPE), was optimal for gene transfer when 5 micrograms of DNA and 10 to 20 micrograms of lipid was used. With both gold particles and gamma AP-DLRIE/DOPE, the alkaline phosphatase reporter was more efficient than beta-galactosidase using comparable promoters and polyadenylation sites. CMV regulatory elements were more efficient than the RSV promoter in optimizing gene expression. Optimal gene transfer efficiency was 20.28% of cells with gamma AP-DLRIE/DOPE, 3.96% with biolistics, 2.09% with calcium phosphate and 0.88% with DEAE-dextran. CONCLUSIONS: Gene expression is detectable in a high percentage of human endothelial cells after liposome-mediated transfection when expression is controlled by a strong promoter. Particle-mediated transfection is less efficient under these conditions, but more effective than liposomes when expression is driven by a relatively weak promoter. Calcium phosphate and DEAE-dextran are less useful.

Lipids and endothelial function: effects of lipid-lowering and other therapeutic interventions.

Coronary arteries are regulated by neuronal mechanisms, hormones and paracrine mediators. The importance of endothelium-dependent mechanisms has recently been recognized. The endothelium responds to mechanical and chemical signals from the blood by releasing mediators that modulate vascular tone and structure, platelet function, coagulation and monocyte adhesion. Important relaxing factors are nitric oxide, prostacyclin and a putative hyperpolarizing factor. Nitric oxide also inhibits smooth muscle proliferation and, together with prostacyclin, platelet function. Bradykinin-induced nitric oxide production is reduced by angiotensin-converting enzyme. Endothelin-1, thromboxane A2 and prostaglandin H2 are contracting factors. Thromboxane A2 and prostaglandin H2 activate platelets, while endothelin has no direct platelet effects, but causes smooth muscle proliferation. In hypercholestermia, endothelium-dependent relaxation is impaired and contraction as well as adhesion of monocytes and platelets enhanced. Pharmacological correction of hyperlipidemia by statins also improves or normalizes endothelial dysfunction in patients. Angiotensin-converting enzyme inhibitors have similar effects.

Endothelial regulation of vascular tone and growth.

The endothelium regulates vascular tone by releasing factors involved in relaxation and contraction, in coagulation and thrombus formation, and in growth inhibition and stimulation. Endothelium-dependent relaxations are elicited by transmitters, hormones, platelet substances, and the coagulation system, and by physical stimuli such as the shear stress from circulating blood. They are mediated by the endothelium-derived relaxing factor, recently identified as nitric oxide, which causes vasodilation and platelet deactivation. Other proposed endothelium-derived relaxing factors include a hyperpolarizing factor, lipooxygenase products, and the cytochrome P450 pathway. Endothelium-derived contracting factors are produced by the cyclooxygenase pathway and by endothelial cells, which produce the peptide endothelin-1, a potent vasoconstrictor that under normal conditions circulates at low levels. The endothelium produces both growth inhibitors--normally dominant--and growth stimuli. Denuded or dysfunctional endothelium leads to a proliferative response and intimal hyperplasia in the vessel wall; moreover, platelets adhere to the site and release potent growth factors. Endothelial dysfunction has numerous causes: Aging is associated with increased formation of contracting factor and decreased relaxing factor; denudation, such as by coronary angioplasty, impairs the capacities of regenerated endothelial cells; oxidized low-density lipoproteins and hypercholesterolemia interfere with nitric oxide production; hypertension morphologically and functionally alters the endothelium; and atherosclerosis markedly attenuates some endothelium-dependent relaxations. For patients with coronary bypass grafts, differences in endothelium-derived vasoactive factors between the internal mammary artery and the saphenous vein may be important determinants of graft function, with the mammary artery having more pronounced relaxations than the saphenous vein and thus a higher patency rate.

Endothelium-derived nitric oxide, endothelin, and platelet vessel wall interaction: alterations in hypercholesterolemia and atherosclerosis.

The endothelium modulates vascular tone by releasing NO, which is a potent vasodilator and inhibitor of platelet aggregation. Thus, the endogenous nitrate has an important protective role in preventing vasospasm and thrombus formation. In addition, the endothelium is a source of contracting factors, such as endothelin-1. Due to its strategical anatomic position, the endothelium is a primary target for injurious stimuli and cardiovascular risk factors. Oxidized LDL reduce the endothelial production of NO and enhance that of endothelin-1. The same pattern of endothelial dysfunction occurs in hypercholesterolemia and in part in atherosclerosis. These alterations of endothelial function may contribute to vasospasm and thrombus formation, which are common events in patients with atherosclerosis.

Endothelial dysfunction in coronary artery disease.

The endothelium is a physical barrier between the blood and vascular smooth muscle, a source of enzymes activating and deactivating cardiovascular hormones and a site of production of relaxing and contracting factors. In addition, the endothelium is a source of growth inhibitors and promoters of vascular smooth muscle cells. Monoaminooxidase deactivates catecholamines and serotonin. Angiotensin converting enzyme transforms angiotensin I into angiotensin II and breaks down bradykinin into inactive products. Nitric oxide is a potent vasodilator and inhibitor of platelet function that under most circumstances is released together with prostacyclin, which exerts similar effects. Both substances play an important protective role in the coronary circulation in that they cause continuous vasodilation and inhibition of platelet function. In addition, the endothelium is a source of contracting factors such as endothelin-1, thromboxane A2, and endoperoxides. Endothelium-derived growth inhibitors include heparin (sulfates) and transforming growth factor beta 1, while basic fibroblast growth factors and platelet-derived growth factor and possibly endothelin promote proliferation. Because of its strategic anatomic position, the endothelium is a primary target for injuries and cardiovascular risk factors. In particular, aging, low density lipoproteins, hypertension, diabetes, and ischemia alter endothelium function. In arterial coronary bypass grafts, the release of nitric oxide is more pronounced than in vein grafts. Alterations of endothelial function may contribute to vasospasm, thrombus formation, and vascular proliferation and in turn myocardial ischemia, all common events in patients with coronary artery disease.