Molecular cytogenetic characterization of the human cerebral microvessel endothelial cell line hCMEC/D3.

The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 has been repeatedly used as a model of human blood-brain barrier (BBB). hCMEC/D3 cells between passage 25 and 35 are most often applied in research, remained phenotypically nontransformed, and cells maintained many characteristics of human brain endothelial cells. Also hCMEC/D3 was thought to have conserved a normal diploid karyotype over all these passages. Here we characterized the cell line using high-resolution multicolor fluorescence in situ hybridization (FISH) approaches and revealed a complex karyotype in the 30th passage. Clonal cryptic unbalanced structural rearrangements and numerical aberrations were discovered and described as follows: 45 approximately 48,XX, -X,del(5)(q11)[2],del(9)(q11)[3],+9[3],del(11)(q13 approximately 14)[2], der(14)t(14;21)(q32.33;q22.3)[28],der(15)t(9;15)(p11;p11)[13], dup(15)(p11q11)[5],der(21)t(17;21)(p12;q22)[9],-22[6][cp28]. In summary, a complex karyotype with clonal unbalanced chromosomal rearrangements is present in hCMEC/D3. Thus, we solicit to include molecular cytogenetics in the testing of all cell lines prior to application of their use in complex studies.

[Hypertrophy and coronary reserve]. / Hypertrophie und Koronarreserve.

Left ventricular hypertrophy represents the structural mechanism of adaptation of the left ventricle as the answer of a chronic pressure overload in arterial hypertension. Initially an increment in left ventricular wall thickness occurs. In this stadium of "concentric hypertrophy" LV systolic wall stress, LV ejection fraction and myocardial oxygen consumption per weight unit myocardium remain unchanged. In the further time course of disease LV dilatation will be present. In this phase of "excentric hypertrophy" LV systolic wall stress and myocardial oxygen consumption per weight unit myocardium rise and LV ejection fraction decreases. Patients with arterial hypertension frequently complain of angina pectoris. Angina pectoris and the positive exercise tolerance test or the positive myocardial scintigraphy are the consequence of the impaired coronary flow reserve. The coronary flow reserve is diminished due to structural and functional changes of the coronary circulation. ACE-inhibitors and AT1-receptor blockers cause a significant improvement of coronary flow reserve and regression of both left ventricular hypertrophy and myocardial fibrosis.

Mineralisation of chitosan scaffolds with nano-apatite formation by double diffusion technique.

The study of inorganic crystal assembly in organic matrices has given rise to increasing interest in various fields of materials science to the natural process of biomineralisation. To mimic the formation of hydroxyapatite as natural bone, a double diffusion technique is utilised in this study to nucleate the hydroxyapatite crystals onto three-dimensional porous polymeric scaffolds. The porous polymer scaffolds were produced from chitosan by a thermally induced lyophilisation technique, which yields highly porous, well-controlled anisotropic open pore architecture. The nucleation of hydroxyapatite crystals was initiated at ambient conditions on the surface of the polymer scaffold, which was in contact with a calcium solution chamber, due to diffusion of phosphate ions through the scaffold. The morphology of the mineralised scaffold as analysed by scanning electron microscopy shows that apatite crystals were not only formed on the surface of the scaffold, but also in the pore channels and attached to the pore walls. The X-ray diffraction and Fourier transformed infrared analyses confirmed the phase purity of the formed apatite crystals. The transmission electron microscopy analysis reveals the microstructure of the entangled nano-apatite in the chitosan polymeric matrix. The in-vitro cytocompatibility tests with osteoblast-like cells (Saos-2) demonstrated that the biomineralised scaffold is a suitable substrate for cell attachment and migration in bone tissue engineering.

[Coronary microangiopathy in hypertensive heart disease: pathogenesis, diagnosis and therapy]. / Koronare Mikroangiopathie bei hypertensiver Herzkrankheit: Pathogenese, Diagnostik und Therapie.

Coronary reserve plays an important role in myocardial oxygen supply. During rest, oxygen consumption is near to maximal. An increase in myocardial oxygen demand can only be covered by an increase in coronary flow by dilation of coronary vessels. The maximal achievable rise in coronary blood flow is called coronary reserve. Coronary reserve is not only enhanced in patients with coronary artery disease but also in patients with disorders of coronary microcirculation for example in arterial hypertension. The following review will deal especially with disorders of the microcirculation in arterial hypertension. The impairment of coronary reserve is a result of structural and functional alterations. Structural alterations include an increase in media wall thickness of the small coronary arteries and a reduction of coronary capillaries. Extravascular myocardial forces which determine coronary resistance include myocardial hypertrophy and qualitative changes of myocardium like interstitial and perivascular fibrosis. The role of functional alterations like endothelial related vasomotion is discussed. The renin-angiotensin system modulates the growth of the small muscle cells of the vessels and induces protooncogenes and other growth factors. Therefore the renin-angiotensin system may also play an important role in hypertensive remodeling. Hypertensive coronary microangiopathy is diagnosed by exercise stress test and ST-segment-monitoring over 24 hours to show myocardial ischemia. Also nuclear medicine technics can be used if conventional methods of showing ischemia don't work. The diagnosis is definite if the determination of coronary reserve shows that the maximal coronary blood flow is not achieved. Coronary flow can be measured by the argon-gas-method, the thermodulation-technic or by the doppler-method. Also by nuclear medicine technics (PET) the coronary flow reserve can be determined. The advantages of these methods are discussed. In experimental studies calcium-channel-blockers, ACE-inhibitors and moxonidine showed an increase in density of capillaries and also a reduction of myocardial hypertrophy, which both result in an improvement of coronary reserve. Clinical studies of our group demonstrate that coronary microangiopathy in hypertensives can be improved by calcium-channel-blockers and ACE-inhibitors after one year treatment. Beta-receptor-blockers show no clear improvement of coronary reserve. It has to be shown by further studies whether the improvement of coronary reserve is more important for prognosis than the regression of myocardial hypertrophy.

Evaluation of cardiac damage in hypertension.

Functional and structural alterations of the coronary microcirculation and left ventricular hypertrophy represent the two key cardiac manifestations of arterial hypertension. In addition, qualitative changes in myocardial tissue composition and structure, such as reactive interstitial fibrosis, are frequently present. The extent of the impairment of coronary flow reserve can be measured using Doppler, argon gas chromatography or thermodilution techniques. Impaired flow reserve can be detected non-invasively using positron emission tomography. The combination of both a normal coronary angiogram and a positive exercise tolerance test (electrocardiogram or thallium) strongly indicates the presence of a hypertension-related disorder of the coronary microcirculation. Left ventricular hypertrophy can easily be quantified using echocardiography (Penn formula). Diastolic function can be assessed by measuring mitral inflow using pulsed-wave Doppler echocardiography (E/A ratio). Non-invasive assessment of myocardial structural alternations may be possible in the future using backscatter echocardiography.

Mechanism of angina pectoris in patients with systemic hypertension and normal epicardial coronary arteries by arteriogram.

Patients with arterial hypertension frequently have angina pectoris despite a normal coronary angiogram. This angina pectoris syndrome often goes along with an impaired coronary vasodilator reserve. The aim of the study was to find out whether an impaired coronary flow reserve is associated with electrographic signs of transient myocardial ischemia. Forty-three hypertensive patients not taking cardiovascular medication were studied with 24-hour Holter monitoring. Coronary blood flow and resistance were measured before and after intravenous administration of dipyridamole (0.5 mg/kg body weight). Coronary reserve was determined as the relation of coronary resistance before and after dipyridamole. For control purposes 9 normotensive subjects were studied with the same protocol. Hypertensive patients with ST-segment depressions (n = 31) had a significantly impaired coronary reserve (2.3 +/- 0.5) compared with normotensive subjects (4.9 +/- 1.0, p < 0.01). Coronary reserve in hypertensive patients without ST-segment depressions was only slightly impaired (4.0 +/- 1.8). Arterial pressure and left ventricular mass did not differ between hypertensive patients with and without ST-segment depressions. Left ventricular mass had no effect on coronary reserve. It is concluded that neither left ventricular hypertrophy nor arterial pressure were determinants for ST-segment depressions. Consequently primary functional and structural alterations on the level of the microcirculation appear to be responsible for the occurrence of transient ischemic episodes in the Holter electrocardiogram.

[Improvement of coronary reserve following regression of hypertrophy resulting from blood pressure lowering therapy with a beta-receptor blocker]. / Verbesserung der Koronarreserve nach Hypertrophieregression durch blutdrucksenkende Therapie mit einem beta-Rezeptorenblocker.

The coronary microcirculation was investigated by the argon gas method in ten patients (eight men, two women; mean age 56 [44-63] years) before and after an average of 12.9 (9-18) months of treatment with the cardioselective beta-receptor blocker bisoprolol. Left ventricular muscle mass was measured echocardiographically, before and after the treatment. All these patients were known to have hypertension, with exercise-related angina and ischaemic signs in the resting ECG, but normal coronary angiograms. After the treatment period both maximal coronary perfusion and coronary reserve had increased by 22%. At the same time, left ventricular muscle mass had decreased from 161 +/- 18 to 146 +/- 21 g/m2. These results demonstrate that blood pressure reducing treatment can bring about not only a regression of left ventricular hypertrophy, but also an improvement in coronary reserve.

Pharmacotherapeutic effects of antihypertensive agents on myocardium and coronary arteries in hypertension.

Hypertensive left ventricular hypertrophy comprises myocyte hypertrophy, interstitial fibrosis and structural alterations in the coronary microcirculation. This leads to impairment of diastolic function in the left ventricle and coronary flow reserve despite normal epicardial arteries. Consequently, antihypertensive treatment should aim at (1) reversing myocyte hypertrophy, (2) restoring myocardial structure and (3) improving coronary flow reserve without lowering blood pressure. In recent years many clinical studies have shown that regression of hypertensive hypertrophy can be induced by long-term treatment with ACE inhibitors, calcium-channel blockers, beta-receptor blockers and antisympathonic drugs. However, vasodilators and diuretics, which stimulate adrenoceptor activity and increase angiotensin II levels, were found to be less effective in reversing left ventricular hypertrophy. The trophic influence of catecholamines and angiotensin II on the myocardium counteracts the effect of systolic wall stress reduction due to blood pressure lowering. As regards reversal of interstitial fibrosis, ACE inhibitors seem to be effective, because fibroblast growth was found to be stimulated by angiotensin II. Recently, clinical studies have confirmed previous experimental data that improvement in impaired coronary vasodilator reserve can be realized by long-term antihypertensive therapy. In adopting an antihypertensive treatment strategy prime consideration should be given to reversal of cardiac remodelling through restoration of myocardial structure and repair of the coronary microcirculation.

Transient myocardial ischaemia in hypertensives: missing link with left ventricular hypertrophy.

Hypertensive patients often complain of angina pectoris in spite of a normal coronary angiogram. The aim of this study was to establish whether electrographical signs of transient myocardial ischaemia during 24-h ST Holter monitoring are associated with an increased left ventricular muscle mass. Thirty-five hypertensive patients were studied by 24-h Holter monitoring and M-mode and two-dimensional echocardiography. For control purposes nine normotensives were studied by the same protocol. Hypertensives with and without ST-segment depression did not differ in respect of blood pressure or left ventricular muscle mass (162.9 +/- 80 vs. 162.3 +/- 53 g m-2). Since both groups only showed a borderline left ventricular hypertrophy, the myocardial factor does not seem to be important for the occurrence of ST segment depression. Primary functional and structural alterations at the microcirculation level seem to be responsible for the occurrence of transient episodes of ST segment depression in the Holter electrocardiogram, indicating transient myocardial ischaemia.

[Prevention with vasoactive drugs]. / Prophylaxe mit gefässaktiven Substanzen.

Arterial hypertension is the most frequent cause of a disturbance of coronary microcirculation. Inspite of having normal epicardial coronary arteries, patients with arterial hypertension often have symptoms of angina pectoris and a positive exercise tolerance test. The angina pectoris symptoms in patients with arterial hypertension are due to functional and structural alterations of the coronary microcirculation. Consequently, an antihypertensive therapy should not only aim at lowering blood pressure and reversing myocardial hypertrophy, but also to improve coronary microcirculation in order to avoid the consequences of chronic ischemia on the myocardium. Until now, only experimental studies have indicated that antihypertensive therapy can improve coronary flow reserve. To determine (also under clinical conditions) if coronary flow reserve can be improved, in 30 hypertensive patients maximal coronary blood flow, minimal coronary resistance, and coronary reserve (dipyridamol) were studied before and after a long-term antihypertensive treatment (9-12 months) with an ACE-inhibitor (enalapril 10-20 mg/d), a calcium channel blocker (diltiazem 120-180 mg/d) and a beta 1-selective beta-receptor-blocker (bisoprolol 5-10 mg/d). To assess the chronic effects rather than the acute effects of the antihypertensive pharmacon, coronary microcirculation was studied after intermission of medical therapy for a period of 1 week. Along with a comparable decrease in LV muscle mass, coronary reserve was improved after enalapril by 48%, after diltiazem by 48%, and after bisoprolol by 22%. It is possible that the observed increase in coronary reserve is related to the reversal of structural vascular abnormalities on the level of the coronary microcirculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Medical repair of hypertensive left ventricular remodeling.

Hypertensive left ventricular (LV) hypertrophy leads to myocytic hypertrophy, interstitial fibrosis, and structural alterations of the coronary microcirculation. This structural remodeling of the myocardium results in an impairment of diastolic function of the left ventricle and of coronary flow reserve despite normal epicardial arteries. Consequently, an antihypertensive treatment should aim at (a) reversing myocytic hypertrophy, (b) regression of myocardial fibrosis, and (c) improvement of coronary flow reserve apart from blood pressure lowering. In recent years many clinical studies have shown that regression of hypertensive hypertrophy can be induced by long-term treatment with angiotensin-converting enzyme (ACE) inhibitors, calcium-channel blockers, beta-receptor blockers, and antisympathonic drugs. However, vasodilators and diuretics, which stimulate adrenoceptor activity and increase angiotensin II levels, were found to be less effective in reversing LV hypertrophy. The trophic influence of catecholamines and angiotensin II on the myocardium counteracts the effect of systolic wall stress reduction due to blood pressure lowering. In respect of reversal of interstitial fibrosis, ACE inhibitors seem to be effective because the growth of fibroblasts was found to be stimulated by angiotensin II. Recently, clinical studies have confirmed previous experimental data that an improvement of the impaired coronary vasodilator reserve can be realized by long-term antihypertensive therapy. An antihypertensive treatment strategy which fully restores myocardial structure and completely repairs coronary microcirculation has to be considered as a causative treatment of hypertensive heart disease.

Evidence of endothelial dysfunction in coronary resistance vessels in patients with angina pectoris and normal coronary angiograms.

This study determines whether an impaired endothelium-mediated vasodilation in coronary resistance vessels exists in patients with microvascular angina. In 23 patients with clinically suspected coronary artery disease and smooth coronary arteries in the angiogram, coronary flow in response to an endothelium-related (acetylcholine) and endothelium-unrelated (dipyridamole) vasodilation was measured. Coronary flow was determined by the gas-chromatographic argon method (1) before, (2) with intracoronary acetylcholine infusion, and (3) after dipyridamole administered intravenously. In 8 patients, acetylcholine did not significantly increase coronary flow (from 91 +/- 28 to 118 +/- 37 ml/min.100 g), whereas flow was greatly increased after administration of dipyridamole (258 +/- 97 ml/min.100 g), indicating an endothelium-related vasodilator defect. In 6 patients, neither acetylcholine nor dipyridamole caused a significant increase in coronary flow, indicating an impaired coronary vasodilation on the vascular site. In 6 patients, coronary flow increased markedly after both administration of both acetylcholine and dipyridamole (from 81 +/- 26 to 191 +/- 68 and 234 +/- 87 ml/min.100 g). In 3 patients given acetylcholine, coronary artery constriction occurred. No significant correlation was found between the response to acetylcholine and that to dipyridamole (r = 0.40, p = not significant). The results indicate that in a subgroup of patients with smooth coronary arteries angina can be caused by an abnormality of the endothelial function in the microcirculation.

Coronary circulation in arterial hypertension.

Arterial hypertension is the most common cause of congestive heart failure and an important risk factor in coronary artery disease (CAD). However, even in the absence of CAD, coronary reserve is frequently impaired in hypertensive patients. To study whether the reduced coronary reserve is due to the degree of left ventricular hypertrophy (LVH) or is a consequence of primary vascular alterations, coronary reserve was determined in 31 hypertensive patients (age of 56 +/- 10 years; systolic/diastolic blood pressure of 167 +/- 18/98 +/- 9 mm Hg) with angina pectoris and normal coronary angiogram. Coronary reserve was determined by measuring coronary resistance before and after dipyridamole (0.5 mg/kg of body weight i.v.). Coronary blood flow was measured quantitatively by the gas chromatographic argon method. LV muscle mass was measured by ventriculography. Twelve normotensive patients (age of 52 +/- 8 years) were studied for comparison. Coronary resistance was 20% higher in hypertensive than in normotensive patients, whereas coronary blood flow at rest was not significantly different. The maximal coronary blood flow after dipyridamole was 40% lower in hypertensive than in normotensive patients; accordingly, minimal coronary resistance was significantly increased by 112% in hypertensive patients (p less than 0.0005). Coronary reserve was reduced by 37% (p less than 0.001) in hypertensive patients compared with normotensive patients. Between the impairment in coronary reserve and left ventricular muscle mass, no significant correlation (r = 0.024, n.s.) was found. The impaired coronary vasodilator reserve is reflected by episodes of transient myocardial ischemia during ST-segment monitoring.

Disorders of coronary microcirculation and arrhythmias in systemic arterial hypertension.

Arterial hypertension is associated with an increased cardiovascular mortality and an increased risk of sudden cardiac death. Therefore, the prevalence of ventricular arrhythmias, identified on 24-hour ambulatory electrocardiographic monitoring, was analyzed in 54 patients (aged 56 +/- 10 years) with arterial hypertension and normal coronary angiograms with respect to left ventricular muscle mass, mass-to-volume ratio, systolic wall stress and coronary microangiopathy. Ventricular ectopic beats (VEBs) were assessed according to a score (modified Lown classification: 1 = no VEB, 2 = less than 30 VEB/hour, 3 = greater than 30 VEB/hour, 4 = polymorphic VEB, 5 = bigeminys, 6 = couplets, 7 = nonsustained ventricular tachycardia). Coronary blood flow was measured by the gas chromatographic argon method. Coronary reserve was determined by measuring coronary resistance before (Rcor) and after (Rmin) administration of intravenous dipyridamole (0.5 mg/kg body weight) (Cor Res = Rcor/Rmin). The frequency (p less than 0.05) and severity (p less than 0.025) of VEBs was higher in normotensive than in hypertensive patients. Although the score of VEBs did not correlate with left ventricular muscle mass (r = 0.20, difference not significant), a slight correlation with mass-to-volume ratio (r = 0.32; p less than 0.05) and systolic wall stress (r = 0.30; p less than 0.05) was found. Hypertensives with a severely reduced Cor Res (less than 2.0) had a significantly higher score of VEBs than those with a nearly normal Cor Res (greater than 3.0) (5.0 +/- 1.5 vs 3.5 +/- 1.7; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Transient myocardial ischemia in hypertensive heart disease.

This study determined whether episodes of myocardial ischemia occur in hypertensive patients with normal coronary angiograms. ST-segment analysis during 24-hour Holter electrocardiography was determined in 48 patients (24 men and 24 women, mean age 54.6 +/- 10.4 years) with essential arterial hypertension (systolic/diastolic blood pressure 189.7 +/- 29/99.5 +/- 15 mm Hg). The thickness of left ventricular posterior wall and septum were measured with echocardiography. Stenosis of coronary vessels were excluded on angiography in all patients. In 24 of 48 patients, 12.8 +/- 13.8 episodes of transient myocardial ischemia (ST-segment depression greater than or equal to 1 mm, duration of the episode greater than or equal to 1 minute) were observed. The duration of the episodes was 48.1 +/- 69.93 minutes and the maximal ST-segment depression was 1.91 +/- 0.82 mm. In 95% of the episodes the patients did not experience any angina pectoris. The degree of left ventricular wall thickness did not differ in hypertensive patients with and without transient myocardial ischemia (septum thickness 11 +/- 2 mm). It is concluded that transient myocardial ischemia often occurs in hypertensive patients. Thus, left ventricular hypertrophy does not appear to play any important role. The underlying cause appears to be the impaired coronary dilation capacity, i.e., vascular alterations.

[Transient myocardial ischemia in hypertensive patients]. / Transiente Myokardischämien bei Hypertonikern.

We wanted to determine whether there are episodes of myocardial ischemia in hypertensive patients with a normal coronary angiogram. ST-segment analysis on 24-h-Holter ECG was performed in 35 patients (18 males/17 females; mean age 54.6 +/- 10.4 years) with essential arterial hypertension (systolic/diastolic blood pressure 189.7 +/- 29/99.5 +/- 15 mm Hg). Left ventricular muscle mass (LVMM), enddiastolic volume (EDV), and the relation of mass to volume (M/V) were measured by ventriculography. Stenosis of coronary vessels was excluded by angiography in all patients. In 16 of 35 patients we observed 6.63 +/- 6.73 episodes of transient myocardial ischemia (ST-segment depressions greater than or equal to 1 mm, duration of the episode greater than or equal to 1 min). The duration of the episodes was 29.3 +/- 58.1 min, the maximal ST-segment depression 1.6 +/- 0.6 mm. In 95% of the episodes the patients did not experience any angina pectoris ("silent ischemia"). The degree of left ventricular muscle mass did not differ in hypertensive patients with and without transient myocardial ischemia (185.2 +/- 48.3 vs 227.1 +/- 71.5 g/m2). Systolic wall stress i.e. afterload was significantly higher in hypertensive patients with ST-segment depressions than in those without. In conclusion, these results demonstrate that transient myocardial ischemia often occurs in hypertensive patients. It seems that left ventricular hypertrophy by itself (myocardial factor) does not play a major role. Transient myocardial ischemia occurs mainly in hypertensive patients with eccentric myocardial hypertrophy i.e. low mass-volume ratio and high systolic wall stress. Accordingly, the occurrence of transient myocardial ischemia in hypertensive patients seems to be dependent on the myocardial energy demand.