MRJF4, a novel histone deacetylase inhibitor, induces p21 mediated autophagy in PC3 prostate cancer cells.

Autophagy is a cellular defense mechanism which occurs through degradation and recycling of cytoplasmic constituents and represents a caspase—independent alternative to cell death by apoptosis. It is generally accepted that the suppression of autophagy in many cancer cells is directly correlated to malignancy; hence, the control of autophagy genes could represent a target for cancer therapy. The inhibition of cell proliferation through autophagy activation could be an important mechanism for many anti—tumor drugs. Here we report the effects of a novel histone deacetylase inhibitor MRJF4 (racemic mixture) and of its two enantiomers [(+)—MRJF4 and (—)—MRJF4] on the morphological and molecular mechanisms causing death and migration of PC3 prostatic cancer cells. In particular, we investigated the occurrence of the autophagic process, both at morphological and molecular levels (LC3 expression), and its relationship with p21, a key molecule which regulates cell cycle and autophagy cell death. Moreover, pERK/Nf—kB driven intracellular signaling, the expression of MMP9 protein — a key component of cell migration — invasion, and metastasis were assayed. Our results showed that the anti—proliferative effects of MRJF4 due to autophagy occurrence, documented by LC3 increase and ultrastructural modifications, and the reduction of invasiveness seem to be mediated by the down—regulation of pERK/NF—kB signaling pathway, along with p21 up—regulation.

Limited hypotensive effect of sildenafil in a high-risk population: a preliminary report.

Erectile dysfunction (ED) is often associated with cardiovascular disease (CVD) and the risk of sildenafil-induced orthostatic hypotension (OH) in subjects with CVD is a matter of concern. We describe our experience in using the tilt test (TT) with continuous plethysmography to evaluate the occurrence of OH in patients with CVD and ED after a test dose of sildenafil. When sildenafil was added on top of their usual pharmacological treatment two patients out of 32 (6.2%) developed asymptomatic OH, with a maximum blood pressure fall of 40/20 mm Hg. The low prevalence and modest clinical relevance of OH in our high-risk population coupled with the known high sensitivity and reproducibility of the TT seem to suggest that sildenafil is haemodynamically safer than is generally believed even when added on top of vasoactive treatment. These findings should be put into perspective against the growing wealth of evidence that PDE5 inhibitors may have therapeutic potential for a number of CV conditions.

Direct visualization of neo-vessel formation following peripheral injection of bone marrow derived CD34+ cells in experimental myocardial damage.

The definitive fate of peripherally injected PKH26 labelled bone marrow mononuclear cells expressing the CD34+ antigen following experimental myocardial cryodamage in rats (n=10) has been examined by direct visualization on photoconverted light and electron microscopy images. One week after the injection in each rat of about 150,000 CD34+ cells early stage PKH26+ vascular structures were localized in the infarcted areas, suggesting that a potential benefit of this therapeutic approach consists in the regeneration of the vasculature.

Crohn's disease and Takayasu's arteritis: is this association difficult to find out?

Takayasu's arteritis is rarely reported associated with Crohn's disease in the English world literature. A case of Takayasu's arteritis type V in a young female affected by Crohn's disease is presented and the pathophysiology mechanism, and the clinical and diagnostic aspects are underlined. Surgical strategy is discussed.

Assessment of selective homing and contribution to vessel formation of cryopreserved peripherally injected bone marrow mononuclear cells following experimental myocardial damage.

In view of a potential clinical use we aimed this study to assess the selective homing to the injured myocardium and the definitive fate of peripherally injected labeled and previously cryopreserved Bone Marrow Mononuclear cells (BMMNCs). The myocardial damage (cryoinjury) was produced in 59 rats (45 treated, 14 controls). From 51 donor rats 4.4 x 10(9) BMMNCs were isolated and cryopreserved (slow-cooling protocols); the number of CD34+ and the viability of pooled cells was assessed by flow-cytometry analysis before and after cryopreservation and simulated delivery through a 23G needle. Seven days after injury, BMMNCs were thawed, labeled with PKH26 dye and peripherally injected (20 x 10(6) cells in 500 microl) in recipient rats. Two weeks after experimental injury, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. Except a small amount in the spleen, PKH26+ cells were found only in the infarcted myocardium of the treated animals. Typical vascular structures CD34+ were found in the infarcted areas of all animals; treated rats showed a significantly higher number of these structures if compared with untreated. Morphological ultra-structural examination of infarcted areas confirmed in treated rats the presence of early-stage PKH26+ vascular structures derived from injected BMMNCs. The estimated mean CD34+ cells loss due to the cryopreservation procedure and to the system of delivery was 0.24% and 0.1%, respectively, confirming the feasibility of the procedure. This study supports the possible therapeutic use of cryopreserved peripherally injecetd BMMNCs as a source of CD34+ independent vascular structures following myocardial damage.

High-altitude trekking in the Himalayas increases the activity of circulating endothelial cells.

Circulating endothelial progenitor cells (EPCs) are believed to contribute to vascular homeostasis; unfortunately, the response of EPCs in physiological conditions remains largely unknown. Herein we report our observations of a 44-year-old healthy subject after a trek in the Himalayas that support high-altitude hypoxia and exercise oxygen demands are strong stimuli for clonogenic endothelial cell activation and activity, as shown by the increase in the number of mature EPCs and in the endothelial colony-forming unit capacity. Both of these effects were completely reverted at sea level, 45 days after the subject's trek.

Evaluation of cardiac structure by echoreflectivity analysis in acromegaly: effects of treatment.

OBJECTIVES: Cardiac echoreflectivity is a noninvasive tool for evaluating cardiac fibrosis. The present paper aimed to study the modifications of cardiac echoreflectivity in a group of acromegalic patients before and after therapy, and to assess possible correlations with serum levels of procollagen III (PIIINP), a peripheral index of collagen synthesis. DESIGN AND METHODS: Cardiac echoreflectivity (as assessed by analyzing 2-D echocardiograms digitized off-line onto a personal computer) and PIIINP levels were evaluated in 16 acromegalic patients of new diagnosis not affected by arterial hypertension (10 males, six females, age+/-s.d.: 38+/-10 years), and in a group of 16 sex- and age-matched healthy subjects. All the patients were re-evaluated after surgical and/or medical therapy for acromegaly. The echo patterns were analyzed by software that supplies the derived collagen volume fraction (dCVF), an index of fibrosis. RESULTS: At baseline, acromegalic patients showed significantly higher dCVF values and PIIINP levels than healthy controls (3.1+/-0.5% vs 1.6+/-0.3%, P<0.01 and 8.7+/-2.2 vs 3.1+/-1.1 ng/ml, P<0.05, respectively, by unpaired Student's t-test). After therapy, dCVF and PIIINP levels normalized in the six controlled patients (that is, GH of <2.5 microg/l and IGF-I within normal range) (dCVF from 2.8+/-0.4% to 1.4+/-0.2%, P<0.001; PIIINP from 8+/-2.7 to 3.3+/-1.9 ng/ml, P<0.05), while no significant changes were found in noncontrolled patients (dCVF from 3.3+/-0.6% to 2.9+/-1.2% and PIIINP from 9.1+/-1.9 to 7.9+/-3.5 ng/ml, P=NS). A positive correlation between dCVF and PIIINP (r=0.75, P<0.001) and between IGF-I and both dCVF and PIIINP (r=0.65 and 0.61 respectively, P<0.05) was found in acromegalic patients. CONCLUSIONS: Cardiac echoreflectivity, which may be a reflection of heart collagen content, is increased in patients with active acromegaly and correlates with PIIINP concentrations. After cure or adequate control of the disease, both parameters revert to normal. Echoreflectivity analysis could be a useful adjuvant parameter in the assessment of the activity of acromegalic disease.

Effects of thyroid hormones on cardiac structure: a tissue characterization study in patients with thyroid disorders before and after treatment.

Experimental evidence suggests an involvement of thyroid hormones in myocardial nonmyocyte component growth. We evaluated the possible role of thyroid hormones in myocardial remodeling by ultrasonic tissue characterization (videodensitometry) in 8 hyperthyroid patients, in 10 hypothyroid patients, and in 2 patients with thyroid hormone resistance syndrome (RTH), before, 60, and 120 days after treatment (T0, T60, T120), and in 10 age-matched euthyroids. According to a previously described procedure, the derived collagen volume fraction (dCVF%, an echocardiographic index estimating the collagen content) was predicted from the pixel-level frequency distribution width (broadband, Bb) of the selected echocardiographic images. Thyrotropin (TSH), free thyroxine (FT4), and free triiodothyronine (FT3) were assessed by immunometric method. QT interval dispersion (QTd) on basal electrocardiogram was measured as a marker of dyshomogeneous ventricular repolarization. At T0, Bb and dCVF% were normal in hyperthyroid and euthyroid patients, and slightly increased in RTH patients, whereas significantly higher values were found in hypothyroids. At T60, a significant reduction in Bb was observed in hypothyroids, with nearly normal dCVF% values. This trend was confirmed at T120 with complete normalization of echoreflectivity. No echoreflectivity changes were observed in hyperthyroid and RTH patients during treatment. QTd was significantly increased in hypothyroids at T0, while no significant differences were found among groups at T60 and T120. Because the different videodeonsitometric myocardial properties observed in hypothyroid versus hyperthyroid patients correspond to an increase of dCVF%, this study suggests that thyroid hormones exert an inhibitory effect on myocardial collagen synthesis in humans.

Vascular network changes in the retina during ageing in normal subjects: a computerized quantitative analysis.

BACKGROUND: Direct ophthalmoscopic examination of fundus oculi is widely used for the qualitative evaluation of target organ damage in several pathological conditions. At present, there are no reliable techniques available to quantify retinal vascular damage. The aim of this study was to develop a computerized technique for the quantitative analysis of fundus oculi. METHODS: We studied 68 non-smoking, normotensive normal subjects, with a visual acuity > 8/10. From each subject retinal images were taken using a non-mydriatic ophthalmoscope and digitized onto a personal computer. In each image the area of analysis was set to a circular selection, corresponding to 25% of the whole fundus oculi, concentric with the optic disc. From each selection arterial and venous area and mean arteriolar bifurcation angle were obtained. Measurements were taken in a blinded way by two operators and, by the same operator, twice at different times. Parameters were correlated with age and body surface area. RESULTS: Retinal arterial density (arteries 7.44 +/- 1.25%; bifurcation angle 75 +/- 16 degrees) was significantly correlated with age, but not with body surface area. Intra- and interobserver coefficient of variation resulted 2.5 and 3.2%, respectively. CONCLUSIONS: Computerized analysis of the fundus oculi with the evaluation of vascular density indexes represents a simple and reproducible technique that could be useful in identifying changes in retinal vascular network during ageing.

Blood pressure-independent cardiac hypertrophy in acromegalic patients.

OBJECTIVE: Acromegaly is frequently associated with an increase in left ventricular mass, even in the absence of systemic hypertension. Pathological studies on acromegalic hearts have shown an extensive interstitial fibrosis, suggesting the existence of a specific acromegalic cardiomyopathy. The aim of this study was to assess left ventricular wall structure in acromegaly by ultrasonic tissue characterization. DESIGN AND METHODS: We studied 10 untreated acromegalic patients and 10 age-matched healthy control subjects. The echo patterns of two-dimensional long-axis end-diastolic echocardiograms were assessed by colour-scale analysis of the interventricular septum, with estimates of the mean colour scale value, the broad band (Bb) and the derived collagen volume fraction (dCVF). We also measured electrocardiographic QT interval dispersion (QTd) as a marker of dyshomogeneous ventricular repolarization. RESULTS: Seven patients had left ventricular hypertrophy according to the sex-independent criteria; of these, two had arterial hypertension. None of our patients had echocardiographic evidence of diastolic or systolic dysfunction. All patients showed significantly increased myocardial echoreflectivity (Bb = 106.4+/-12.1 versus 79.3+/-6.5; dCVF% = 2.78+/-0.53 versus 1.58+/-0.29; P < 0.0001) and QTd (66+/-13 ms versus 54+/-8 ms, P < 0.05). A significant correlation was found between dCVF and the duration of acromegaly (r = 0.80; P = 0.005). CONCLUSIONS: Left ventricular remodelling observed in acromegaly is not related to the presence of arterial hypertension; we hypothesize that the increased echoreflectivity and QTd are long-term consequences of cardiac hypertrophy and prolonged exposure to high levels of growth hormone and insulin-like growth factor-I.

An improved method for coronary sinus blood flow monitoring by intravascular Doppler technique.

In order to obtain accurate measurements of coronary sinus blood flow (CSBF), a new catheter (7 French) with a radiopaque, flexible, and basket-shaped tip was developed for guiding a standard 3 Fr Doppler catheter in the coronary sinus (CS) in man. The radiopaque "basket" tip of the catheter allows the operator to stabilize the position of the Doppler transducer in the center of the CS and to accurately measure the CS internal diameter radiologically. CSBF was calculated as the product of CS cross-sectional area by mean CSBF velocity. Doppler-derived CSBF values at rest and during handgrip were compared with those obtained by the local thermodilution technique in 16 patients undergoing diagnostic coronary angiography. During handgrip, mean CSBF increased from 154+/-23 (rest) to 299+/-34 mL/min by the Doppler method and from 148+/-22 to 288+/-32 mL/min by the thermodilution technique. A good correlation (r = 0.86) between the CSBF values with the two techniques was observed. The authors conclude that the intravascular Doppler technique associated with the use of the basket guide catheter provides an accurate and simple tool for monitoring CSBF in patients.

[A multimedia echocardiographic archive]. / Archivio ecocardiografico multimediale.

The widespread diffusion of echocardiography requires to rationalize clinical examination archives; the recent improvement in computer processing speed and the addition of image processing capabilities on standard personal computer by using multimedia technology provide a low-cost solution to improve video digital acquisition and base management. Furthermore, the diffusion of computer networks supports the possibility of sending images in digital format from a work station to another. In this study we describe the setting-up of a system for echocardiographic image acquisition, storage, base management and analysis based on a standard multimedia Macintosh personal computer using readily available not-dedicated software. We tested the overall efficiency of this system in terms of time required to perform hardware and software procedures, storage capacity of the archive and possibility to exchange information with other wire-linked computer work-stations or via modem. This system has proven to require an acceptable time to perform all the procedures showing a high level of connectivity with other standard personal computer work-stations; however, some limitations with regard to time required in sending via modem long movie files need to be pointed out. In conclusion, in our experience new multimedia personal computer could offer to every physician with a minimum informatic knowledge the well known advantages of digital-video, at a reasonable cost.

Echocardiographic patterns of myocardial fibrosis in hypertensive patients: endomyocardial biopsy versus ultrasonic tissue characterization.

Echocardiographic image texture has been demonstrated to reflect the physical properties of the tissue under examination. To evaluate the role of collagen in determining the echo pattern of the left ventricular wall, we studied nine hypertensive patients with left ventricular hypertrophy (left ventricular mass index > 125 gm/m2) and biopsy-proven different degrees of myocardial fibrosis by analyzing the echocardiographic examinations performed before the biopsy. Myocardial tissue was sampled under fluoroscopy and two-dimensional echo guidance in the interventricular septum. Collagen volume fraction (CVF; normal range up to 2%) was taken as an index of fibrosis. The echo patterns were assessed by analyzing standard two-dimensional parasternal long-axis echocardiograms recorded on videotape. Images were color-coded at 256 levels (0 = yellow, 256 = black) and digitized off-line onto a personal computer. The region of analysis was set using a selection tool (20 x 10 mm) in the general area of septum where the specimen was taken. For each selection a color-level histogram, representing the frequency distribution, was derived with estimates of the average pixel intensity (mCS), skewness (SK), kurtosis (K), and the broad band (Bb) of the echoes about the distribution. Echo-derived parameters in each patient were compared with corresponding CVF values. CVF was out of range in all patients, ranging from 2.6% to 7.6% (mean 4.3% +/- 1.6%). No correlation was found between CVF and mCS, whereas a significant correlation was found at end diastole between CVF and the parameters describing histogram morphology, respectively, SK (r = 0.73), K (r = 0.69), Bb (r = 0.72). These findings for the first time demonstrate in vivo in hypertensive patients with left ventricular hypertrophy an agreement between echo amplitude and histologically assessed collagen volume. Thus in our studied patients collagen content appears to be the major determinant of regional echo intensity, its increase resulting in a significant and progressive wider asymmetrical left shift (yellow) of the color histogram.

Coronary blood flow and myocardial ischaemia in hypertension.

The aim of this investigation was to study the coronary pressure-flow relationship in 60 patients with chronic arterial hypertension of diverse aetiologies and in 14 normotensive subjects (control group). The hypertensive cohort included 6 patients with isolated systolic hypertension (ISH), 7 renovascular hypertensive patients with abnormally elevated angiotensin II plasma levels but without electrocardiographic and/or echocardiographic evidence of left ventricular hypertrophy (LVH) and 47 subjects with essential hypertension (EH), 21 of whom had LVH by electrocardiogram and/or echocardiogram. In the hypertensive cohort a Frank-Starling-like curve was found to describe the coronary pressure-flow relationship when the baseline values for coronary sinus blood flow (CBF, intravascular Doppler technique) were plotted against mean aortic pressure (intra-arterial blood pressure). In particular, the descending limb of such a curve represented a critical region where CBF was "inappropriately" low with respect to perfusion pressure. It was thus concluded that this inability of the heart to adapt CBF to its needs might account for the higher propensity to develop myocardial ischaemia encountered in severe essential hypertensive subjects with concomitant LVH and renovascular hypertensive patients.

Coronary hemodynamics and the renin angiotensin system.

Coronary hemodynamics were investigated invasively at rest and during handgrip exercise in two groups of mild essential hypertensive subjects and in one group of renovascular hypertensive patients. The former subjects received either furosemide (50 mg/day for one week) to ensure activation of the renin-angiotensin system or an intravenous infusion of angiotensin II (AngII) at a subpressor dose (3 ng/kg/min for 15 minutes) and at a pressor dose (13 ng/kg/min for 15 minutes). Furosemide induced a significant reduction in coronary blood flow (CBF), a significant increase in coronary vascular resistance (CVR) and also blunted the increase in CBF during handgrip exercise. Captopril restored CBF and CVR to pretreatment values. Infusion of the subpressor dose of AngII decreased myocardial oxygen supply, both at rest and during exercise; the pressor dose increased myocardial oxygen supply at rest and blunted the expected increase in myocardial oxygen supply during exercise. Converting-enzyme inhibition in renovascular hypertension caused mean arterial pressure to decrease and CBF to increase significantly. The performance of handgrip exercise after cilazapril resulted in higher increases in CBF for a given increase in myocardial oxygen requirements. These data suggest that there is a negative interference by abnormally high plasma levels AngII with myocardial perfusion and that the AngII-induced effects on coronary hemodynamics are reversed by converting enzyme inhibition.

Prolonged water immersion. Effects on blood pressure maturation in normotensive rats.

The purpose of this experiment was to study the impact of simulated microgravity and of chronic removal of hydrostatic pressure gradients on blood pressure maturation and body growth in rats. A special device was developed in our laboratory to transfer prolonged "dry" water immersion (a technique that has been used for training astronauts under hypogravic conditions) to six Sprague-Dawley test rats (immersion-G group). The time course of heart rate, systolic blood pressure, urinary output, and body weight was monitored from weaning to maturity and then compared with those responses from six sex- and age-matched Sprague-Dawley rats grown in a gravity environment (group G). A downward shift in systolic blood pressure and body weight maturation curves was observed in immersion-G rats from the age of 60 days. Cessation of dry water immersion produced a gradual, significant rise in systolic blood pressure but not in body weight to control values. No marked changes in heart rate and urinary output between G and immersion-G rats were noticed throughout the investigation. Our results provide indirect evidence that an interference in the natural history of blood pressure maturation was introduced by immersion, which dissociated the effects of body weight increase during growth from the effects of ageing per se. It is concluded that the physiological increase in systolic blood pressure during growth is partly gravity-dependent.

Coronary haemodynamic effects of angiotensin II in mild essential hypertension in man.

1. The present investigation was carried out to elucidate the possible role of the renin-angiotensin system in modulating coronary vasomotor responses in eight patients with uncomplicated mild essential hypertension with no electrocardiographic-echocardiographic evidence of left ventricular hypertrophy. 2. Systemic and coronary haemodynamics were monitored at baseline and during intravenous infusion of angiotensin II at a subpressor dose (3 ng min-1 kg-1 for 15 min) and at a pressor dose (13 ng min-1 kg-1 for 15 min) both at rest and during handgrip exercise. Infusion of the subpressor dose of angiotensin II decreased coronary sinus blood flow at rest (207 +/- 10 versus 182 +/- 9 ml/min, P less than 0.05) without a significant change in mean arterial pressure, heart rate or mean right atrial pressure. The performance of handgrip at baseline and during infusion of the subpressor dose of angiotensin II resulted in 55% (321 +/- 13 versus 207 +/- 10 ml/min) and 44% (263 +/- 16 versus 182 +/- 9 ml/min) increases in coronary sinus blood flow, respectively, in response to comparable increments in the rate-pressure product. At rest, infusion of the pressor dose of angiotensin II increased both coronary sinus blood flow (235 +/- 11 versus 207 +/- 10 ml/min, P less than 0.01) and the rate-pressure product (134 +/- 5 versus 111 +/- 8 mmHg beats/min, P less than 0.01). The increase in coronary sinus blood flow during isometric exercise was less than control (309 +/- 18 versus 321 +/- 13 ml/min, P less than 0.01). 3. It is thus concluded that (1) the opposite effects of angiotensin II on coronary blood flow are dose-dependent, and that (2) angiotensin II competes with the ability of the coronary arteries to dilate during handgrip exercise.

The effects of a microgravitational environment on the blood pressure maturation curve in normotensive rats.

In order to determine whether gravity influences the growth of terrestrial mammals, systolic blood pressure, heart rate, urinary output and body weight were measured at 10-day intervals from the age of 30 days (weaning) to 150 days in 12 male Sprague-Dawley rats. Six rats were grown in a gravitational environment. 'Dry' water immersion was used to subject six rats to a microgravitational environment between the ages of 30 and 90 days. A downward shift in systolic blood pressure and body weight maturation curves was observed in the microgravitational group from the age of 60 days. After the 'dry' water immersion was stopped, the fully developed rats showed a significant increase in systolic blood pressure, which returned to control values, but not in body weight. We conclude that the physiological increase in systolic blood pressure taking place in rats during growth is partly dependent on gravity.

Cardiac responses to head up tilt during early extrauterine life: relevance of active acquisition of erect posture.

Mammals must adapt to gravity on passing from the intrauterine to the extrauterine environment. In order to evaluate the cardiovascular effects of gravity in the first phases of extrauterine life, the effects of passive orthostatism on the cardiac filling volume were investigated through longitudinal haemodynamic studies in 14 normal healthy males before (6 months) and after (18 months) acquiring the ability to stand. Left ventricular diameter (by echocardiographic measurement), arterial blood pressure (by sphygmomanometry) and heart rate were measured in the supine and upright position at both ages. At 6 months the left ventricular end diastolic volume was not modified by a change in posture [supine 6(SEM 3) ml, upright 6(3) ml], so heart rate was minimally altered [supine 128(9), upright 130(11) beats.min-1] and blood pressure remained stable [supine 74(6), upright 73(5) mm Hg]. After the acquisition of the erect posture (18 months) left ventricular end diastolic volume was reduced [supine 14(3), upright 8(2) ml], heart rate increased [supine 110(11), upright 127(12) beats.min-1] and blood pressure remained constant [supine 80(6), upright 79(7) mm Hg]. The assumption of the erect posture therefore represents a phase when, for the first time in the natural history of the cardiovascular system, translocation of intravascular volume from the cardiopulmonary area to the periphery stimulates nervous and humoral responses to control the dynamics of body fluids and arterial blood pressure in a gravitational environment.