FOXC2 and NFATc1 control formation and maturation of collecting lymphatic vessels

The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. These data provide novel insights into the molecular program of lymphatic vascular specification and suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Preserved capillary density of dorsal finger skin in treated hypertensive patients with or without type 2 diabetes.

OBJECTIVES: Capillary rarefaction is a hallmark of untreated hypertension. Recent data indicate that rarefaction may be reversed by antihypertensive treatment in nondiabetic hypertensive patients. Despite the frequent association of diabetes with hypertension, nothing is known on the capillary density of treated diabetic patients with hypertension. METHODS: We enrolled 21 normotensive healthy, 25 hypertensive only, and 21 diabetic (type 2) hypertensive subjects. All hypertensive patients were treated with a blocker of the renin-angiotensin system, and a majority had a home blood pressure ≤135/85 mmHg. Capillary density was assessed with videomicroscopy on dorsal finger skin and with laser Doppler imaging on forearm skin (maximal vasodilation elicited by local heating). RESULTS: There was no difference between any of the study groups in either dorsal finger skin capillary density (controls 101 ± 11 capillaries/mm(2) , nondiabetic hypertensive 99 ± 16, diabetic hypertensive 96 ± 18, p > 0.5) or maximal blood flow in forearm skin (controls 666 ± 114 perfusion units, nondiabetic hypertensive 612 ± 126, diabetic hypertensive 620 ± 103, p > 0.5). CONCLUSIONS: Irrespective of the presence or not of type 2 diabetes, capillary density is normal in hypertensive patients with reasonable control of blood pressure achieved with a blocker of the renin-angiotensin system.

A fast 3D approach for coronary MRA.

Two-dimensional (2D)-breath-hold coronary magnetic resonance angiography (MRA) has been shown to be a fast and reliable method to depict the proximal coronary arteries. Recent developments, however, allow for free-breathing navigator gated and navigator corrected three-dimensional (3D) coronary MRA. These 3D approaches have potential for improved signal-to-noise ratio (SNR) and allow for the acquisition of adjacent thin slices without the misregistration problems known from 2D approaches. Still, a major impediment of a 3D acquisition is the increased scan time. The purpose of this study was the implementation of a free-breathing navigator gated and corrected ultra-fast 3D coronary MRA technique, which allows for scan times of less than 5 minutes. Twelve healthy adult subjects were examined in the supine position using a navigator gated and corrected ECG triggered ultra-fast 3D interleaved gradient echo planar imaging sequence (TFE-EPI). A 3D slab, consisting of 20 slices with a reconstructed slice thickness of 1.5 mm, was acquired with free-breathing. The diastolic TFE-EPI acquisition block was preceded by a T2prep pre-pulse, a diaphragmatic navigator pulse, and a fat suppression pre-pulse. With a TR of 19 ms and an effective TE of 5.4 ms, the duration of the data acquisition window duration was 38 ms. The in-plane spatial resolution was 1.0-1.3 mm*1.5-1.9 mm. In all cases, the entire left main (LM) and extensive portions of the left anterior descending (LAD) and right coronary artery (RCA) could be visualized with an average scan time for the entire 3D-volume data set of 2:57 +/- 0:51 minutes. Average contiguous vessel length visualized was 53 +/- 11 mm (range: 42 to 75 mm) for the LAD and 84 +/- 14 mm (range: 62 to 112 mm) for the RCA. Contrast-to-noise between coronary blood and myocardium was 5.0 +/- 2.3 for the LM/LAD and 8.0 +/- 2.9 for the RCA, resulting in an excellent suppression of myocardium. We present a new approach for free-breathing 3D coronary MRA, which allows for scan times superior to corresponding 2D coronary MRA approaches, and which takes advantage of the enhanced SNR of 3D acquisitions and the post-processing benefits of thin adjacent slices. The robust image quality and the short average scanning time suggest that this approach may be useful for screening the major coronary arteries or identification of anomalous coronary arteries. J. Magn. Reson. Imaging 1999;10:821-825.

A review of methods for assessment of coronary microvascular disease in both clinical and experimental settings.

Obstructive disease of the large coronary arteries is the prominent cause for angina pectoris. However, angina may also occur in the absence of significant coronary atherosclerosis or coronary artery spasm, especially in women. Myocardial ischaemia in these patients is often associated with abnormalities of the coronary microcirculation and may thus represent a manifestation of coronary microvascular disease (CMD). Elucidation of the role of the microvasculature in the genesis of myocardial ischaemia and cardiac damage-in the presence or absence of obstructive coronary atherosclerosis-will certainly result in more rational diagnostic and therapeutic interventions for patients with ischaemic heart disease. Specifically targeted research based on improved assessment modalities is needed to improve the diagnosis of CMD and to translate current molecular, cellular, and physiological knowledge into new therapeutic options.

Reproducibility of twitch mouth pressure, sniff nasal inspiratory pressure, and maximal inspiratory pressure.

Twitch mouth pressure (Pmo,tw) during magnetic phrenic nerve stimulation and sniff nasal inspiratory pressure (SNIP) were recently proposed as alternative noninvasive methods for assessing inspiratory muscle strength. This study aimed to compare their reproducibility with maximal inspiratory pressure (MIP) in normal subjects. Ten healthy subjects were studied at functional residual capacity in semirecumbent position. Cervical magnetic phrenic nerve stimulation was performed during gentle expiration against an occlusion incorporating a small leak. Constancy of stimulation was controlled by recording diaphragmatic electromyogram. Within and between-session reproducibility of pressure were studied for Pmo,tw, SNIP, and MIP. The subjects were studied during a session of 10 manoeuvres repeated after 1 day and 1 month. The mean values were 16 cmH2O for Pmo,tw, 118 cmH2O for SNIP, and 115 cmH2O for MIP. For the three tests, the within subject variation was small in relation to between-subject variation, with the intraclass correlation coefficient ranging 0.79-0.90 for Pmo,tw, 0.85-0.92 for SNIP, and 0.88-0.92 for MIP. At 1 day interval, the coefficient of repeatability (2 SD of differences) was 3.6 cmH2O for Pmo,tw, 32 cmH2O for SNIP and 28 cmH2O for MIP. At 1 month interval, the coefficient of repeatability was 5.8 cmH2O for Pmo,tw, 23 cmH2O for SNIP and 21 cmH2O for MIP. We conclude that the within session reproducibility of the new tests twitch mouth pressure and sniff nasal inspiratory pressure is sufficient to be clinically useful. For sniff nasal inspiratory pressure, the between session reproducibility established after 1 day was maintained after 1 month. For twitch mouth pressure, the between session reproducibility declined slightly after 1 month. These characteristics should be considered when using these methods to follow an individual patient over time.

Mediastinal reinforcement after induction therapy and pneumonectomy: comparison of intercostal muscle versus diaphragm flaps.

OBJECTIVE: Prospective non-randomised comparison of full-thickness pedicled diaphragm flap with intercostal muscle flap in terms of morbidity and efficiency for bronchial stump coverage after induction therapy followed by pneumonectomy for non-small cell lung cancer (NSCLC). METHODS: Between 1996 and 1998, a consecutive series of 26 patients underwent pneumonectomy following induction therapy. Half of the patients underwent mediastinal reinforcement by use of a pedicled intercostal muscle flap (IF) and half of the patients by use of a pedicled full-thickness diaphragm muscle flap (DF). Patients in both groups were matched according to age, gender, side of pneumonectomy and stage of NSCLC. Postoperative morbidity and mortality were recorded. Six months follow-up including physical examination and pulmonary function testing was performed to examine the incidence of bronchial stump fistulae, gastro-esophageal disorders or chest wall complaints. RESULTS: There was no 30-day mortality in both groups. Complications were observed in one of 13 patients after IF and five of 13 after DF including pneumonia in two (one IF and one DF), visceral herniations in three (DF) and bronchopleural fistula in one patient (DF). There were no symptoms of gastro-esophageal reflux disease (GERD). Postoperative pulmonary function testing revealed no significant differences between the two groups. CONCLUSIONS: Pedicled intercostal and diaphragmatic muscle flaps are both valuable and effective tools for prophylactic mediastinal reinforcement following induction therapy and pneumonectomy. In our series of patients, IF seemed to be associated with a smaller operation-related morbidity than DF, although the difference was not significant. Pedicled full-thickness diaphragmatic flaps may be indicated after induction therapy and extended pneumonectomy with pericardial resection in order to cover the stump and close the pericardial defect since they do not adversely influence pulmonary function.

Quantification de la perfusion rénale par échographie de contraste, une étude pilote

Mise en perspective Le rein est un organe vital dont la fonction dépend en grande partie d'une perfusion tissulaire adéquate. Les techniques actuellement utilisées pour étudier la microcirculation rénale sont soit invasives soit très dispendieuses. L'échographie de contraste est une nouvelle technologie, non invasive, facile à réaliser au lit du malade et pour laquelle certaines techniques récemment présentées semblent permettre de quantifier la perfusion d'un organe. Une telle technique pourrait avoir des applications dans l'étude de l'insuffisance rénale aiguë. Buts de l'étude Notre étude vise à tester le potentiel de l'échographie de contraste pour détecter des modifications de la perfusion rénale, corticale et médullaire chez l'être humain. Plan de l'étude Nous avons étudié la microcirculation rénale par échographie de contraste chez 12 sujets sains. Les mesures ont été réalisées en baseline puis sous perfusion de doses croissantes d'angiotensine II (un puissant et spécifique vasoconstricteur rénal) et enfin après la prise orale d'un comprimé de Captopril (un puissant vasodilatateur rénal). Les valeurs obtenues par échographie de contraste ont été comparées à la perfusion globale rénale évaluée par la clairance du PAH, qui constitue le gold standard en physiologie rénale. Résultats principaux Le protocole a pu être réalisé sans complication. Les perfusions vasoactives ainsi que les différentes administrations de Sonovue ont été parfaitement tolérées. Les séquences vidéos ont été analysées par deux lecteurs et une bonne concordance a été trouvée. Nous avons trouvé une diminution statistiquement significative et dose dépendante de l'index de perfusion rénale lors de la perfusion d'angiotensine II: -45% (p<0.02) entre baseline et Ang II faible dose et -65% (p<0.01) entre baseline et Ang II à haute dose. Enfin, nous avons observé une hausse significative de cet indice après administration orale de Captopril (+35%, p>0.2). Ces changements étaient parallèles aux changements de flux plasmatique rénal mesurés par calcul de la clairance du PAH. Une corrélation moyenne mais significative (r=0.57, p=0.002) a été observée entre ces deux mesures. Conclusions et perspectives L'échographie de contraste est capable de détecter des modifications dans la microcirculation rénale induites par une perfusion d'angiotensine et ou une administration de Captopril. Il s'agit d'une technique rapide sensible et fiable qui pourrait participer à l'évaluation des patients souffrant d'insuffisance rénale aiguë. D'autres études sont nécessaires pour démontrer son utilité dans un contexte clinique.

2

Alveolar haemorrhage (AH) is a rare and potentially life-threatening condition characterised by diffuse blood leakage from the pulmonary microcirculation into the alveolar spaces due to microvascular damage. It is not a single disease but a clinical syndrome that may have numerous causes. Autoimmune disorders account for fewer than half of cases, whereas the majority are due to nonimmune causes such as left heart disease, infections, drug toxicities, coagulopathies and malignancies. The clinical picture includes haemoptysis, diffuse alveolar opacities at imaging and anaemia. Bronchoalveolar lavage is the gold standard method for diagnosing AH. The lavage fluid appears macroscopically haemorrhagic and/or contains numerous haemosiderin-laden macrophages. The diagnostic work-up includes search for autoimmune disorders, review of drugs and exposures, assessment of coagulation and left heart function, and search for infectious agents. Renal biopsy is often indicated if AH is associated with renal involvement, whereas lung biopsy is only rarely useful. Therapy aims at correction of reversible factors and immunosuppressive therapy in autoimmune causes, with plasmapheresis in selected situations.

Respiratory distress in a one-month-old child suffering brachial plexus palsy.

This paper describes a one-month-old girl presenting with respiratory and growth failure due to diaphragmatic paralysis associated with left brachial plexus palsy after forceps delivery. Despite continuous positive pressure ventilation and nasogastric feeding, the situation did not improve and a laparoscopic diaphragmatic plication had to be performed. When dealing with a child born with brachial plexus palsy, one must think of this possible association and if necessary proceed to the complementary radiological examinations. The treatment must avoid complications like feeding difficulties and failure to thrive, respiratory infections or atelectasis. It includes intensive support and a good evaluation of the prognosis of the lesion to decide the best moment for a surgical therapy.

Photodynamic therapy for enhanced drug delivery to tumor

Photodynamic therapy (PDT) has been used as an adjunct to cytoreductive surgery in patients with malignant pleura mesothelioma (MPM). However, it was associated with substantial side effects and found to be only of modest clinical benefit. In contrast, Visudyne®-mediated low-dose PDT has been shown to selectively increase the concentration of macromolecular cytostatic compounds in various tumors grown subpleurally on rodent lungs. Consequently, it was thought that PDT-assisted enhanced tumor penetration for cytostatic agents might be better suited to achieve additional tumor control after cytoreductive surgery for mesothelioma. This effect seems to be mainly related to PDT-mediated modulations of tumor vessels which improve the distribution of circulating, systemically administered chemotherapeutic macromolecular agents. However, the mechanisms involved and the optimization of this effect for therapeutic implications remain to be solved. By using the dorsal skin fold chamber method we demonstrated that both angiogenesis and microcirculation of human mesothelioma xenografts can be continuously assessed in vivo by intravital microscopy. We described a new, simple, reproducible and reliable scoring system for the assessment of tumor angiogenesis and microcirculation in this model, thereby allowing the quantitative description of the neo-vascular network development while avoiding a complicated technical setup. This method can serve as a useful tool for the assessment of novel vessel-targeted therapies against MPM. We then applied this newly established model so as to elucidate the underlying mechanisms of PDT-induced extravasation of macromolecular compounds across the endothelial barrier in tumors and surrounding normal tissue. We found that low-dose PDT selectively enhanced the uptake of macromolecular compounds in human mesothelioma xenografts compared to surrounding normal tissue. Interestingly, this increase of effective permeability of tumor vasculature was not related to the inflammatory stimuli generated by PDT such as the mobilization of leucocytes and their adhesion and penetration of the injured vessel wall. We then used the model for optimizing the drug-light conditions of low- dose PDT in order to obtain maximal leakage of the macromolecular compounds in the tumor with minimal uptake in normal surrounding tissue and we were able to identify such a therapeutic window. With these optimized PDT treatment conditions, we assessed the therapeutic effect of this new treatment concept in vivo by measuring tumor growth rates on subcutaneously grown mesothelioma xenografts in nude mice after low-dose PDT of the tumors following systemically administered liposomal (macromolecular) cisplatin, a cytostatic compound commonly used in clinical practice. We were able to demonstrate that low-dose PDT with optimized drug-light conditions combined with systemic chemotherapy indeed resulted in a reduction in tumor growth compared to chemotherapy or PDT alone. In conclusion, our work demonstrates that low-dose PDT may selectively enhance the uptake of macromolecular cytostatic drugs in superficially growing tumors such as mesotheliomas and opens new perspectives for the treatment of these diseases. - Les effets cytotoxiques de la thérapie photodynamique (PDT) sur le mésothéliome pleural malin (MPM) n'ont pas apporté de bénéfice clinique significatif. Toutefois, une application innovante non cytotoxique de la PDT serait la bienvenue en supplément des chimiothérapies pour améliorer le contrôle local de la tumeur. Le prétraitement des néovaisseaux tumoraux par une PDT à bas régime, qui améliorerait la distribution d'une chimiothérapie administrée par voie systémique de façon concomitante, a attiré une attention particulière pour de futures applications cliniques. Toutefois, les mécanismes impliqués dans cet événement et les implications thérapeutiques de ces changements physiopathologiques restent non résolus. Dans cette thèse, nous avons observé en premier que l'angiogenèse et la microcirculation dans les xénogreffes de mésothéliomes humains peuvent être observées et analysées in vivo par microscopie intravitale. Le nouveau système de score appliqué pour l'évaluation de l'angiogenèse et de la microcirculation tumorale dans cette étude est une méthode simple, reproductible et fiable servant à décrire de manière quantitative le réseau néo-vasculaire en développement, tout en évitant d'utiliser une installation technique compliquée. Ce modèle sert de nouvel outil pour l'évaluation des thérapies anti-vasculaires dirigées contre le MPM. Le modèle animal nouvellement établi a alors été utilisé pour élucider les mécanismes sous-jacents de Γ extravasation d'agents macromoléculaires induite par PDT dans les vaisseaux tumoraux et normaux. Nous avons trouvé que la PDT à fable dose améliore la distribution ciblée de drogues macromoléculaires dans des greffes de mésothéliome humain, de manière sélective pour la tumeur. La perméabilité vasculaire tumorale n'est pas influencée par les stimuli inflammatoires générés par la PDT, ce qui joue un rôle important dans la sélectivité de notre photodynamic drug delivery. Ensuite, nous avons recherché la fenêtre thérapeutique optimale de la PDT pour obtenir une accumulation sélective du colorant macromoléculaire dans le tissu tumoral ainsi qu'une efficacité de la PDT combinée avec une chimiothérapie macromoléculaire sur la croissance tumorale. Nous avons démontré que la PDT à faible dose combinée avec une administration systémique de cisplatine liposomale mène à un ralentissement de la croissance tumorale dans notre modèle de mésothéliome malin humain. En conclusion, l'utilisation de la PDT comme prétraitement pour améliorer sélectivement la distribution d'agents thérapeutiques dans des tumeurs poussant superficiellement est prometteuse. Cette observation fourni une preuve du concept remarquable et garanti la suite des investigations, éventuellement ayant pour but de développer de nouveaux concepts de thérapie pour les patients atteints de mésothéliome. Une PDT intra cavitaire à faible dose après pleuro- pneumonectomie pourrait améliorer la pénétration des agents cytostatiques administrés de façon concomitante par voie systémique dans les îlots tumoraux résiduels, et ainsi améliorer le contrôle local.

Real-time full field laser Doppler imaging.

We present a full field laser Doppler imaging instrument, which enables real-time in vivo assessment of blood flow in dermal tissue and skin. This instrument monitors the blood perfusion in an area of about 50 cm(2) with 480 × 480 pixels per frame at a rate of 12-14 frames per second. Smaller frames can be monitored at much higher frame rates. We recorded the microcirculation in healthy skin before, during and after arterial occlusion. In initial clinical case studies, we imaged the microcirculation in burned skin and monitored the recovery of blood flow in a skin flap during reconstructive surgery indicating the high potential of LDI for clinical applications. Small animal imaging in mouse ears clearly revealed the network of blood vessels and the corresponding blood perfusion.

Dysfonction microvasculaire et ischémie du myocarde [Microvascular dysfunction and myocardial ischemia]

Une lésion fonctionnelle ou structurale des artérioles intramurales influence le seuil ischémique du myocarde. Le diagnostic de dysfonction microvasculaire est retenu en présence d'une diminution du flux coronaire maximal et de coronaires angio-graphiquement normales ou presque normales. Un trouble de la microcirculation peut traduire une dysfonction endothéliale chez le sujet diabétique ou hyperlipidémique, ou une lésion structurale ou fonctionnelle dans le cadre de la cardiomyopathie hypertrophique, la sténose aortique ou l'hypertension artérielle. Après recanalisation de l'artère responsable d'un infarctus, la mesure de la fonction microcirculatoire permet d'estimer la qualité de la reperfusion myocardique. L'appréciation de la fonction microvasculaire est un enjeu majeur dans l'évaluation de l'ischémie du myocarde en l'absence de sténose coronaire. Functional or structural lesions in intramural arterioles influence the ischemic threshold of the myocardium. Microvascular dysfonction is evidenced by a decrease in coronary blood flow during maximum hyperemia in the presence of angiographically normal or near-normal coronary arteries. Microvascular dysfonction may reflect endothelial dysfonction in diabetic or hyperlipidemic patients, as well as structural and functional changes in patients with hypertrophic cardiomyopathy, aortic stenosis or hypertension. Assessing microvascular fonction after thrombolysis or primary angioplasty for acute myocardial infarction allows to estimate the quality of myocardial reperfusion. Assessing microvascular fonction is a major component of the evaluation of myocardial ischemia in the absence of coronary artery stenoses.