Prenatal profile of Pallister-Killian syndrome: Retrospective analysis of 114 pregnancies, literature review and approach to prenatal diagnosis.

Pallister-Killian syndrome (PKS) is a tissue limited mosaic disorder, characterized by variable degrees of neurodevelopmental delay and intellectual disability, typical craniofacial findings, skin pigmentation anomalies and multiple congenital malformations. The wide phenotypic spectrum of PKS in conjunction with the mosaic distribution of the i(12p) makes PKS an underdiagnosed disorder. Recognition of prenatal findings that should raise a suspicion of PKS is complicated by the fragmentation of data currently available in the literature and challenges in diagnosing a mosaic diagnosis on prenatal testing. Ultrasound anomalies, especially congenital diaphragmatic hernia, congenital heart defects, and rhizomelic limb shortening, have been related to PKS, but they are singularly not specific and are not present in all affected fetuses. We have combined prenatal data from 86 previously published reports and from our cohort of 114 PKS probands (retrospectively reviewed). Summarizing this data we have defined a prenatal growth profile and identified markers of perinatal outcome which collectively provide guidelines for early recognition of the distinctive prenatal profile and consideration of a diagnosis of PKS as well as for management and genetic counseling.

Angiotensin II modulates calponin gene expression in rat vascular smooth muscle cells in vivo.

OBJECTIVES: It has been shown that angiotensin II (Ang II) induces the expression of calponin, a 34 kD actin-binding protein, in vascular smooth muscle cells in vitro. The aim of this study was to investigate whether Ang II can modulate calponin gene expression in rat aorta in vivo. DESIGN: Aortic calponin gene expression was studied after chronic exogenous Ang II administration and in Goldblatt hypertension. METHODS: To investigate the effect of Ang II administration, Sprague Dawley rats were treated for 6 days with a continuous infusion of Ang II (200 ng/kg per min) or saline by osmotic minipumps. The effect of endogenous Ang II on aortic calponin mRNA expression was studied in Goldblatt hypertensive rats with (2K1C model), or without (1K1C model) activation of the renin-angiotensin system. In particular, calponin gene expression in 2K1C rats was studied both at 1 week (2K1C-HR, high renin) and 4 weeks after the onset of hypertension, when plasma renin activity (PRA) was returned to normal values (2K1C-NR, normal renin). Systolic blood pressure (SBP) was measured twice a week. At the end of the experimental period, PRA was measured by radioimmunoassay, and aortic calponin gene expression was measured by Northern hybridization. RESULTS: SBP was significantly higher (P < 0.01), whereas PRA was suppressed (P < 0.01), in Ang II versus saline-treated rats. Northern hybridization showed that the aortic calponin gene expression significantly increased (2.5-fold) in Ang II-treated rats (P = 0.01). In Goldblatt hypertensive rats, SBP was significantly higher in 2K1C-HR (P < 0.01), 2K1C-NR (P < 0.01) and 1K1C (P < 0.01) rats compared with the corresponding sham-treated rats. Activation of the renin-angiotensin system was present only in 2K1C-HR rats (P < 0.01), and Northern analysis showed that aortic calponin mRNA expression was significantly increased (2.2-fold) in this group of rats only (P < 0.01). CONCLUSIONS: Our data demonstrate that both exogenous and endogenous Ang II increase calponin gene expression in aortic smooth muscle cells, independently of the hemodynamic effect of Ang II.

Role of inflammation in the pathogenesis of unstable coronary artery disease.

In this article, the clinical, angiographic, and postmortem features of unstable angina are reviewed and its pathogenesis is discussed. Coronary plaque inflammation may play a key role in the pathogenesis of unstable angina and the evidence for this assertion is examined. Finally, the therapeutic implications of the involvement of inflammation in acute coronary syndromes are outlined.

Comparison of response to serotonin of radial artery grafts and internal mammary grafts to native coronary arteries and the effect of diltiazem.

We studied the response of radial artery (RA) or left internal mammary artery grafts to the intraluminal infusion of serotonin in 22 consecutive patients 1 year after the operation, subsequently evaluating the effect of diltiazem in 9 patients. Serotonin causes a significant vasoconstriction of the RA grafts, but not of the left internal mammary artery grafts, whereas oral diltiazem treatment does not prevent the effect of the higher dose of serotonin on RA grafts.

Use of the radial artery for myocardial revascularization.

BACKGROUND: The radial artery was first used as a coronary graft by Carpentier and associates in 1973 but, due to the disappointing results, it was abandoned. In 1992 its revival coincided with the widespread use of calcium-channel blockers in cardiovascular surgery, in the belief they could prevent spasm. METHODS: From January 1993 to October 1995 we operated on 109 patients for myocardial revascularization employing the radial artery with two different surgical techniques: in 95 patients (group 1) it was "pretreated" by opening its fascia after a gentle hydrostatic dilation and then anastomosed to the aorta; in 14 patients (group 2) it was branched to another conduit. We had two operative deaths (1.82%). RESULTS: At a mean interval of 532.42 days 105 patients are still alive, 2 (1.86%) having died of abdominal tumors. Fifty-six patients (52.33%) underwent angiography at a mean interval of 334.42 days: the patency of the radial artery was 88.88% in group 1 and 62.50% in group 2. Indications and contraindications are discussed. CONCLUSIONS: The radial artery is an easily manageable conduit whose early patency is very promising, although a longer follow-up is mandatory.

Calcium influx modulates DNA synthesis and proliferation in A7r5 vascular smooth muscle cells.

In A7r5 vascular smooth muscle cultures basal Ca2+ influx was higher in growing versus quiescent cells (P less than 0.05), due primarily to a five-fold increase in dihydropyridine-inhibitable Ca2+ influx (P less than 0.005). Verapamil decreased [3H]thymidine incorporation in a concentration dependent fashion with a significant 6 +/- 2% inhibition at 0.1 microM and a maximal inhibition of 67 +/- 2% at 100 microM. Similarly, nitrendipine inhibited fetal calf serum-stimulated [3H]thymidine incorporation with a threshold concentration of 1 nM and a maximal inhibition of 79 +/- 12% at 10 microM. In quiescent cells, verapamil (10 microM) inhibited the increases in [3H]thymidine incorporation stimulated by fetal calf serum, serotonin, vasopressin or 12-0-tetradecanoyl phorbol-13-acetate by 37-43% (P less than 0.001 vs. control for all). Finally, verapamil (100 microM) and nitrendipine (10 microM) inhibited proliferation by 39 +/- 10 and 20 +/- 6%, respectively (P less than 0.01 and 0.02 vs. control, respectively). Thus in A7r5 cells, proliferation is associated with increased Ca2+ influx via dihydropyridine-sensitive Ca2+ channels and organic Ca2+ channel antagonists inhibit DNA synthesis and cell proliferation.

Different Effects of Lipopolysaccharide on Plasminogen Activator Inhibitor-1 Production in Aortic Media in Vivo and in Culture.

Background: Lipopolysaccharide (endotoxin) has been shown to increase the expression of plasminogen activator inhibitor type-1 (PAI-1) in the vessel wall. Endotoxin is known to increase PAI-1 production in endothelial cells, but its action on smooth muscle cells (SMCs) is presently not clear. In this study we determined the effect of endotoxin on PAI-1 and tissue plasminogen activator (t-PA) production by aortic SMCs in vivo in two animal species, and in culture. Methods: The aortas of Sprague Dawley rats and of New Zealand White rabbits were rapidly excised after parenteral administration of endotoxin. Total RNA was extracted from the aortic media, and PAI-1 and t-PA mRNA levels were quantified after Northern blotting. In addition, cultured rat aortic SMCs were treated with endotoxin. PAI activity in the conditioned medium was determined with a spectrophotometric assay, and total RNA was extracted from the cells and analyzed. Results: A rapid and strong induction in the aortic medi a of PAI-1 mRNA was observed by endotoxin in both rat (50 mg/kg) and rabbit (1 mg/kg). t-PA mRNA was barely detectable and was not increased by endotoxin. Studies in cultured SMCs showed low expression of PAI-1 mRNA under serum-free conditions and little PAI activity in the cell-conditioned medium. Endotoxin did not increase the levels of PAI-1 mRNA nor PAI activity under serum-free conditions. The effect of endotoxin (10 mg/ml) in the presence of 10% (v/v) newborn calf serum on PAI-1 mRNA was negligible; PAI activity, however, increased by 50.3 +/- 7.3% compared with controls. mRNA levels of t-PA and low-density lipoprotein/receptor-related protein/alpha2-macroglobulin receptor also increased after endotoxin administration. PAI activity was identified as PAI-1 by immunoblotting. Fibrin zymography showed that t-PA was present only in complex with PAI-1. Conclusions: A strong increase in PAI-1 gene expression by endotoxin was observed in aortic SMCs in vivo but not in culture. Th is suggests that the effect of endotoxin on SMCs is indirect. The fibrinolytic/proteolytic potential of the SMCs in the vessel wall is likely to have important implications for the migration of cells during vessel wall remodeling, such as neointima formation, during tumor cell metastasis, and for the fate of intramural thrombi.

Vascular and haemostatic gene polymorphisms associated with non-fatal myocardial infarction: a critical review.

The importance of genetics to the pathogenesis of myocardial infarction is suggested by the frequent familial clustering of premature disease. Yet, studies associating myocardial infarction with gene polymorphisms of vascular proteins (angiotensinogen, angiotensin converting enzyme, angiotensin II type 1 receptor, endothelial nitric oxide synthase) and haemostatic factors (fibrinogen, coagulation factors II, V, VII and XIII, plasminogen activator inhibitor-1, tissue-type plasminogen activator, platelet glycoproteins IIb/IIIa, Ia/IIa and Ib-IX-V, or methylenetetrahydrofolate reductase) have revealed conflicting results. This is hardly surprising, given: 1) the multigenic nature of myocardial infarction, whereby single polymorphisms are bound to play at best only a limited role in the global risk of disease; 2) the multiple pathogenetic mechanisms of infarction (e.g., atheromatous obstruction, plaque rupture, thrombosis, vasospasm), each of which is likely influenced by a number of genes and by several environmental factors. The simultaneous investigation of a set of polymorphisms--and of their interactions with environmental factors--in extremely homogeneous sets of patients should offer a better understanding of the contribution of specific genes to the risk of myocardial infarction.

Induction of interleukin-1beta and interleukin-6 gene expression in hypoperfused skeletal muscle of patients with peripheral arterial disease.

BACKGROUND: A growing amount of data supports the role of inflammation in the pathophysiology of atherosclerotic diseases but the cellular source of cytokines has not been clearly identified. Cytokines could be produced by inflammatory cells, activated endothelial and smooth muscle cells, and by the tissue exposed to recurrent ischemia. Accordingly, we evaluated whether hypoperfusion induces gene expression of interleukin (IL)-1beta and IL-6 in the skeletal muscle of patients with peripheral arterial disease and critical limb ischemia. METHODS: Skeletal muscle biopsies were obtained, during a femoral-distal bypass, from normoperfused (control) and hypoperfused skeletal muscles in 8 patients. Gene expression was assessed by semiquantitative reverse transcriptase-polymerase chain reaction, using glyceraldehyde-phosphate-deydrogenase mRNA levels as a normalization factor. RESULTS: In the hypoperfused biopsies, the level of IL-1beta gene expression was significantly higher in all but 2 patients (mean upregulation > 8.8 fold, p = 0.043), and the level of IL-6 gene expression was significantly higher in all but 1 patient (mean upregulation > 23.7 fold, p = 0.031). CONCLUSIONS: We report that IL-1beta and IL-6 gene expression is markedly upregulated in hypoperfused skeletal muscle of patients with critical lower limb ischemia. To our knowledge this is the first report of a local activation of the inflammatory cascade at the level of hypoperfused skeletal muscle. This activation, which could worsen symptoms and tissue viability and be involved in the pathophysiology of reperfusion injury, might be considered as a therapeutic target. It remains to be investigated whether our results may also apply to coronary artery disease.

The ranking of Italy's publications in the biomedical field: an objective evaluation.

To evaluate the scientific output of Italy compared to other countries in clinical and basic research, the twelve top ranking journals according to the impact factor in each group were considered. A total impact factor score of one country was the sum of the impact factor of all articles attributed to a certain country in all journals. Italy ranked sixth in clinical research but only ninth in basic research. According to our analysis, the Italian scientific output in the biomedical field is comparable to that of other countries. Further financial analysis of the correlation between research funding and scientific output could allow a more productive allocation of resources.

Phorbol ester-stimulated bidirectional transmembrane calcium flux in A7r5 vascular smooth muscle cells.

In A7r5 vascular smooth muscle cells, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) caused a marked increase in the rate of unidirectional Ca2+ influx and a 63 +/- 9% increase in net 30-min 45Ca2+ uptake. Maximal TPA-stimulated 45Ca2+ uptake occurred at concentrations less than or equal to 3 nM and was equivalent to the maximal uptake stimulated by 55 mM KCl or 1 microM Bay k 8644. TPA-stimulated Ca2+ uptake was not additive to KCl- or Bay k 8644-stimulated uptake, and was inhibited by verapamil (100 microM), nitrendipine (1 microM), or high concentrations of Bay k 8644 (greater than or equal to 10 microM). The biologically inactive phorbol ester 12-O-tetradecanoyl phorbol-13-acetate methyl ether (10 nM) had no effect on 45Ca2+ uptake. TPA caused a 43 +/- 12% increase in Ca2+ efflux in 30 min, and exposure to TPA (10 nM) for 5 and 30 min caused no significant change in net cellular Ca2+ content as determined by radioisotopic equilibration or atomic absorption spectroscopy. TPA caused no apparent change in intracellular free Ca2+ concentration as determined with quin 2. Thus, in A7r5 cells, TPA causes a marked increase in Ca2+ influx through channels with the pharmacological characteristics of dihydropyridine-sensitive, voltage-dependent channels. This TPA-stimulated Ca2+ uptake is associated with increased Ca2+ efflux and no significant change in net cellular Ca2+ content or intracellular free Ca2+ concentration.

[Treatment of refractory hypertension with combined minoxidil and captopril in low doses]. / Trattamento dell'ipertensione refrattaria con l'associazione di minoxidil e captopril a basse dosi.

Twelve patients with severe hypertension of different causes, resistant to conventional antihypertensive agents and also to maximum doses of either minoxidil or captopril added to other combinations, were effectively controlled with the association of small doses of minoxidil and captopril plus a diuretic and a beta-blocker. The untoward effects which were previously present in all the patients with maximum doses of minoxidil and in one of the patients treated with captopril disappeared or greatly diminished shortly after this association was started. The different mechanisms of action and opposite side effects of minoxidil and captopril provide the rationale for a combined treatment with the two drugs.

[Drug-induced orthostatic hypotension can be useful in antihypertensive therapy]. / L'ipotensione ortostatica da farmaci può essere utile nella terapia antiipertensiva.

The evaluation of the efficacy of antihypertensive therapy may be misleading if it is based on office blood pressures which are usually higher than ambulatory or home pressures. An erroneous evaluation may also derive from the presence of orthostatic hypotension induced by antihypertensive therapy. In seven patients with moderate to severe hypertension who were treated with different antihypertensive agents and who presented drug-induced orthostatic hypotension we studied 24 hour blood pressures by means of the Oxford system. The mean of the blood pressures recorded with the patients up and about were lower than with the patients in the supine position. Consequently the physiological fall in blood pressure which is present in untreated patients during sleep was not observed. The following conclusions may be drawn: a) drug-induced orthostatic hypotension may be useful in antihypertensive therapy; b) drugs which are capable of reducing blood pressure particularly in the supine position should be preferred during the night; c) for the evaluation of antihypertensive therapy it is necessary to take into account both supine and standing blood pressure.

Inflammation and acute coronary syndromes.

The presence of inflammatory infiltrates in unstable coronary plaques suggests that inflammatory processes may contribute to the pathogenesis of these syndromes. In patients with unstable angina, coronary atherosclerotic plaques are characterized by the presence of macrophages, and to a lesser extent, T-lymphocytes, at the immediate site of either plaque rupture or superficial erosion; moreover, the rupture-related inflammatory cells are activated, indicating ongoing inflammation at the site of plaque disruption. These observations are confirmed by clinical studies demonstrating activated circulating neutrophils, lymphocytes and monocytes, and increased concentrations of pro-inflammatory cytokines, such as interleukin (IL) 1 and 6, and of acute phase reactants in patients with unstable angina and myocardial infarction. In particular elevated levels of C-reactive protein are associated with an increased risk of in-hospital and 1 to 2 years new coronary events in patients with unstable angina, but are also associated with an increased long-term risk of death and myocardial infarction in apparently normal subjects. Thus, accumulating evidence suggests that inflammation may cause local endothelial activation and, possibly, plaque fissure, leading to unstable angina and infarction. Although no information is yet available on the causes of inflammation and on its localization, these novel lines of research may open the way to a different approach to the patient with acute coronary syndromes.

Angiotensin II increases plasminogen activator inhibitor type 1 and tissue-type plasminogen activator messenger RNA in cultured rat aortic smooth muscle cells.

BACKGROUND: The role of angiotensin as a vasoconstrictor is well established. Lately, several other actions of this hormone on vascular smooth muscle (VSM) cells have been recognized including the induction of hypertrophy and/or DNA synthesis. Platelet-derived growth factor (PDGF), a mitogen recently shown to increase plasminogen activator inhibitor type 1 (PAI-1) synthesis in VSM cells, shares with angiotensin II (Ang II) several steps of its intracellular signaling pathway. METHODS AND RESULTS: The expression of PAI-1 and tissue-type plasminogen activator (TPA) mRNA in cultured rat VSM cells was studied. Northern blot analysis demonstrated a severalfold increase in the PAI-1 mRNA 3 to 8 hours after stimulation with 300 nmol/L Ang II. A similar response for TPA mRNA was observed. This induction did not require the synthesis of an intermediate protein or peptide because it was not affected by cycloheximide. In the cell-conditioned supernatant, the net result was an increase in PAI-1 activity from 4.18 +/- 1.8 to 13.2 +/- 6.8 IU/mL 6 hours after the addition of 300 nmol/L Ang II (mean +/- SD, P < or = .008, n = 6). The Ang II-induced increase in PAI activity was dose related, with a maximal effect at a concentration of 23 nmol/L (n = 3) and an ED50 of 3.3 +/- 1.5 nmol/L (n = 3). [Sar1-Ile8]angiotensin II, a specific competitive antagonist of Ang II, blocked 90 +/- 9% (n = 3) of the PAI activity induced by 10 nmol/L Ang II. In basal conditions, fibrin overlay zymography demonstrated the presence of free TPA. After stimulation with Ang II, lysis caused by the in situ dissociation of TPA was also present in the region of the TPA/PAI-1 complex. Angiotensin I (Ang I) elicited an increase in PAI activity similar to that obtained with equivalent doses of Ang II. Captopril (5 micrograms/mL), an inhibitor of the angiotensin-converting enzyme (ACE), completely prevented the Ang I effect, demonstrating that VSM cells display an ACE-like activity. CONCLUSIONS: Recent research has demonstrated the existence of a localized vascular renin-angiotensin system. The finding that Ang II can potentially modulate the plasminogen activation in the arterial wall has important biological and therapeutical implications for the evolution of arterial wall thrombi and the migration of cells through the vessel wall in the genesis of atherosclerotic lesions. We speculate that the reduction in thrombotic events observed in patients with a previous myocardial infarction and in high-renin, hypertensive patients treated with ACE inhibitors could be due at least in part to the decreased production of PAI-1 by VSM cells caused by these agents.

Cultured rat aortic vascular smooth muscle cells digest naturally produced extracellular matrix. Involvement of plasminogen-dependent and plasminogen-independent pathways.

Vascular smooth muscle (VSM) cell migration and proliferation play a major role in the development of atherosclerotic lesions, graft occlusion, and restenosis after angioplasty. Cell migration implies the digestion of the surrounding extracellular matrix. Cell-associated proteolysis has been extensively studied in neoplastic and inflammatory cells, but very little is known about the proteolytic properties of VSM. We have evaluated the ability of rat cultured VSM cells to solubilize [3H]amino acid-labeled extracellular matrices produced by bovine VSM. When plated at a density of 30,000 cells per well in 24 multiwell plates, VSM cells were able to solubilize 63.3 +/- 7.0% of the extracellular matrix after 10 days in culture. Extracellular matrix digestion occurred also when the cells were cultured in plasminogen-depleted serum but was higher in the presence of 10 micrograms/ml purified plasminogen (net percent digestion after the subtraction of the appropriate control, 8.6 +/- 3.0% versus 21.2 +/- 3.5% after 3 days in culture, p less than 0.005, respectively). The involvement of other enzymes in addition to plasmin is confirmed by the ability of VSM cells to degrade extracellular matrices from which the plasmin-sensitive component was removed with plasmin pretreatment. Rat VSM cells were able to solubilize 52.3 +/- 2.0% of this residual extracellular matrix-associated radioactivity after 6 days in culture versus 26.1 +/- 1.5% in the control dishes (p less than 0.01, n = 5). Cell contact was required for extracellular matrix degradation: cell-conditioned medium did not have any effect on extracellular matrix digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol ester increases the dihydropyridine-sensitive calcium conductance in a vascular smooth muscle cell line.

In vascular smooth muscle, phorbol esters cause a slowly developing contraction and an associated transmembrane calcium flux, both of which are inhibited by dihydropyridine calcium channel antagonists. In the A7r5 cultured vascular cell line, we used the whole-cell voltage-clamp technique to identify voltage-dependent calcium conductances and investigate the effect of phorbol esters on that conductance having characteristic dihydropyridine sensitivity (slowly inactivating, high-threshold, "L-type"). With barium as the charge carrier, large-amplitude (100-800 pA) inward currents of two types were characterized by their kinetics and voltage dependence. With holding potential--80 mV, a rapidly inactivating, low-threshold current ("T-type") was activated by depolarizations above-40 mV and was maximal at -10 mV. With holding potential -30 mV, this component was inactivated, and a second slowly inactivating, high-threshold current was activated above -10 mV and was maximal at +10 to +20 mV. These currents are similar to the T-type and L-type currents previously described in vascular smooth muscle cells. When added to the bath, the active phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (100 nM) increased the slowly inactivating (L-type) current by 32 +/- 20% (n = 8, +/- SD). Phorbol-12,13-dibutyrate (100 nM) caused a similar effect, but the inactive phorbol, 4-alpha-phorbol (100 nM), did not. We conclude that at least two distinct calcium conductances are expressed in A7r5 vascular smooth muscle cells, and that the dihydropyridine-sensitive calcium conductance is acutely modulated by phorbol esters, presumably acting through stimulation of protein kinase C. Such modulation may play a role in increasing transmembrane calcium influx mediated by agonist-receptor interactions that lead to activation of protein kinase C and may help to sustain or amplify calcium-dependent cell responses.

Prevalence and possible mechanisms of ventricular arrhythmias in athletes.

To investigate the prevalence and nature of ventricular arrhythmias in athletes, 40 well trained healthy endurance athletes (20 cyclists and 20 runners) and 40 healthy sedentary subjects as controls were studied. Twenty four-hour continuous ECG monitoring showed a higher, even though not significant prevalence of ventricular ectopy in athletes (70% vs 55%). Complex forms of ventricular ectopy were also more prevalent in the athletes than in the controls (25% vs 5%; p less than 0.05). A prolongation of both measured QT interval and QT corrected for heart rate was observed at the ECG. Echocardiograms showed higher dimensional indices in the athletes, while functional indices were similar in athletes and controls. No correlation was found between any ECG or echocardiographic finding and the grade of ventricular arrhythmia. The comparison between the cyclists and the runners did not show any significant difference in the prevalence of ventricular arrhythmias or in the QT interval duration.