Development of strategies for vascular damage repair in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive and rare disease with so far unclear pathogenesis, limited treatment options and poor prognosis. Unbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is an important hallmark of PAH. In this research Sodium butyrate (BU) has been evaluated in vitro and in vivo models of PAH. This histone deacetylase inhibitor (HDACi) counteracted platelet-derived growth factor (PDGF)-induced ki67 expression in PASMCs, and arrested cell cycle mainly at G0/G1 phases. Furthermore, BU reduced the transcription of PDGFRbeta, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing and pulmonary artery ring assays indicated that BU inhibited PDGF-induced PASMC migration. BU strongly inhibited PDGF-induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculinA. In vivo, BU showed efficacy in monocrotaline-induced PAH in rats. Indeed, the HDACi reduced both thickness of distal pulmonary arteries and right ventricular hypertrophy. Besides these studies, Serial Analysis of Gene Expression (SAGE) has be used to obtain complete transcriptional profiles of peripheral blood mononuclear cells (PBMCs) isolated from PAH and Healthy subjects. SAGE allows quantitative analysis of thousands transcripts, relying on the principle that a short oligonucleotide (tag) can uniquely identify mRNA transcripts. Tag frequency reflects transcript abundance. We enrolled patients naïve for a specific PAH therapy (4 IPAH non-responder, 3 IPAH responder, 6 HeritablePAH), and 8 healthy subjects. Comparative analysis revealed that significant differential expression was only restricted to a hundred of down- or up-regulated genes. Interestingly, these genes can be clustered into functional networks, sharing a number of crucial features in cellular homeostasis and signaling. SAGE can provide affordable analysis of genes amenable for molecular dissection of PAH using PBMCs as a sentinel, surrogate tissue. Altogether, these findings may disclose novel perspectives in the use of HDACi in PAH and potential biomarkers.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.

Effects of glucocorticoids on vascular NADPH oxidases and endothelial NO synthase

Glukokortikoide (GCs) stellen wichtige Hormone in der Regulation der metabolischen Homöostase dar. Synthetische GCs, wie Dexamethasone (DEX), spielen eine essentielle Rolle in der Behandlung inflammatorischer Krankheiten. Jedoch sind unter einer Dexamethason-Therapie zahlreiche Nebenwirkungen bekannt, so z.B. auch die Entwicklung einer Hypertonie, in deren Pathogenese oxidativer Stress eine entscheidende Rolle spielt. Obwohl sich in den vergangenen Jahren zahlreiche Studien zum Ziel setzten die GC-induzierte Hypertonie (GC-HT) aufzuklären, sind die genauen Mechanismen bis heute unklar. Eine erhöhte Expression von NADPH Oxidasen (Nox) und eine Entkopplung der endothelialen NO Synthase (eNOS), die Hauptquellen reaktiver Sauerstoffspezies (ROS) im vaskulären System, tragen maßgeblich zur Pathogenese kardiovaskulärer Erkrankungen bei. Daher ist eine Beteiligung dieser Enzyme in GC-induziertem oxidativen Stress sehr wahrscheinlich. Folglich wurde die Hypothese aufgestellt, dass NADPH Oxidasen und eine entkoppelte eNOS die vielversprechendsten unter den zahlreichen involvierten pro- und anti-oxidativen Enzymen sind. Mit Fokus auf die oben genannten Systeme wurde in der vorliegenden Studie der Effekt von DEX mit Hilfe von in vivo (WKY Ratten) ebenso wie in vitro Experimenten (A7r5 und EA.hy 926 Zellen) untersucht. Dabei zeigte sich, dass Nox1, Nox4 und p22phox durch DEX unterschiedlich reguliert wurden. Nox1 wurde hoch-, Nox4 hingegen herunterreguliert, während p22phox unverändert blieb. Die Modufikation schien hierbei auf transkriptioneller und post-transkriptioneller Ebene stattzufinden. Durch die gegensätzliche Regulation von Nox1 und Nox4 bleibt die Nettowirkung der verschiedenen Nox Isoformen unklar. Immer mehr Studien bringen vaskulären oxidativen Stress mit der Pathogenese einer GC-HT in Zusammenhang, welche letztendlich zu einer verminderten Bioverfügbarkeit von Stickstoffmonoxid (NO) führt. Durch die eNOS produziertes NO stellt einen essentiellen Schutzfaktor der Blutgefäße dar. Eine verminderte NO-Bioverfügbarkeit könnte die Folge einer eNOS-Entkopplung darstellen, ausgelöst durch oxidativen Stress. Da die Verfügbarkeit von Tetrahydrobiopterin (BH4) entscheident ist für die Aktivität und enzymatische Kopplung der eNOS, beschäftigt sich die vorliegende Arbeit mit GC-induzierten Veränderungen in der BH4-Versorgung. Die Behandlung von EA.hy 926 Zellen mit DEX führte zu einer zeit- und konzentrationsabhängigen Herunterregulation von eNOS auf mRNA- und Proteinebene. Gleichzeitig wurde die Phosphorylierung an Serine1177 vermindert. Als maßgeblicher “Kopplungs-Schalter” kann BH4 endogen über zwei verschiedene Signalwege synthetisiert werden, welche durch die Enzyme GCH1 und DHFR reguliert werden. DEX führte zu einer zeit- und konzentrationsabhängigen Herunterregulation von BH4, BH2 und Biopterin, wobei ebenso das BH4 / BH2 -Verhältnis vermindert wurde. Beide Enzyme, GCH1 genauso wie DHFR, wurden auf mRNA- und Proteinebene herunterreguliert, was auf einen Effekt von GCs auf beide rnBH4-produzierenden Signalwege schließen lässt. Nach Behandlung mit DEX wurde die Produktion von NO in Endothelzellen maßgeblich vermindert. In ROS-Messungen zeigte sich eine Tendenz hin zu einer eNOS-Entkopplung, jedoch war es mit unserem experimentellen Aufbau nicht möglich, diese endgültig zu beweisen.rnZusammenfassend lässt sich sagen, dass die Behandlung mit GCs zu Veränderungen in beiden untersuchten Systemen, den NADPH Oxidasen ebenso wie dem eNOS-NO System, führte. DEX erhöhte die Expression von Nox1 in glatten Muskelzellen und reduzierte die Nox4-Expression in Endothelzellen. Gleichzeitig verminderte DEX die Verfügbarkeit von BH4 und inhibierte die Phosphorylierung / Aktivität von eNOS. Mithilfe weiterer Studien muss die endgültige Beteiligung von NADPH Oxidasen und einer eNOS-Entkopplung an oxidativem Stress in GC-HT abschließend aufgeklärt werden.rn

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

BACKGROUND: Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases. METHODS: sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression. RESULTS: sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition. CONCLUSIONS: This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.

Prognosis of Atrial Fibrillation in Patients with Symptomatic Peripheral Arterial Disease: Data from the REduction of Atherothrombosis for Continued Health (REACH) Registry

BACKGROUND: Atrial fibrillation (AF) is a significant risk factor for cardiovascular (CV) mortality. This study aims to evaluate the prognostic implication of AF in patients with peripheral arterial disease (PAD). METHODS: The International Reduction of Atherothrombosis for Continued Health (REACH) Registry included 23,542 outpatients in Europe with established coronary artery disease, cerebrovascular disease (CVD), PAD and/or >/=3 risk factors. Of these, 3753 patients had symptomatic PAD. CV risk factors were determined at baseline. Study end point was a combination of cardiac death, non-fatal myocardial infarction (MI) and stroke (CV events) during 2 years of follow-up. Cox regression analysis adjusted for age, gender and other risk factors (i.e., congestive heart failure, coronary artery re-vascularisation, coronary artery bypass grafting (CABG), MI, hypertension, stroke, current smoking and diabetes) was used. RESULTS: Of 3753 PAD patients, 392 (10%) were known to have AF. Patients with AF were older and had a higher prevalence of CVD, diabetes and hypertension. Long-term CV mortality occurred in 5.6% of patients with AF and in 1.6% of those without AF (p<0.001). Multivariable analyses showed that AF was an independent predictor of late CV events (hazard ratio (HR): 1.5; 95% confidence interval (CI): 1.09-2.0). CONCLUSION: AF is common in European patients with symptomatic PAD and is independently associated with a worse 2-year CV outcome.

The Next 10 years in the Management of Peripheral Artery Disease: Perspectives from The 'PAD 2009' Conference

OBJECTIVES: To briefly inform on the conclusions from a conference on the next 10 years in the management of peripheral artery disease (PAD). DESIGN OF THE CONFERENCE: International participation, invited presentations and open discussion were based on the following issues: Why is PAD under-recognised? Health economic impact of PAD; funding of PAD research; changes of treatment options? Aspects on clinical trials and regulatory views; and the role of guidelines. RESULTS AND CONCLUSIONS: A relative lack of knowledge about cardiovascular risk and optimal management of PAD patients exists not only among the public, but also in parts of the health-care system. Specialists are required to act for improved information. More specific PAD research is needed for risk management and to apply the best possible evaluation of evidence for treatment strategies. Better strategies for funding are required based on, for example, public/private initiatives. The proportion of endovascular treatments is steadily increasing, more frequently based on observational studies than on randomised controlled trials. The role of guidelines is therefore important to guide the profession in the assessment of most relevant treatment.

Preliminary experimental evaluation of the immediate angiographic occlusion time with use of the AMPLATZER vascular plug II for carotid artery occlusion

Delayed occlusion time in parent artery occlusion of brain-supplying vessels might carry risk for thromboembolic complications. Vascular plug devices are successfully used in cardiopulmonary and peripheral interventions to occlude high-flow lesions and have been adapted for use in neurointerventions. The purpose of the present study was to experimentally evaluate the immediate occlusion time of the AMPLATZER vascular plug (AVP) II-a second-generation cylindrical, self-expandable, resheathable nitinol wire mesh consisting of three lobes-in the carotid artery.

Results of the randomized, placebo-controlled clopidogrel and acetylsalicylic acid in bypass surgery for peripheral arterial disease (CASPAR) trial

OBJECTIVE: Dual antiplatelet therapy with clopidogrel plus acetylsalicylic acid (ASA) is superior to ASA alone in patients with acute coronary syndromes and in those undergoing percutaneous coronary intervention. We sought to determine whether clopidogrel plus ASA conferred benefit on limb outcomes over ASA alone in patients undergoing below-knee bypass grafting. METHODS: Patients undergoing unilateral, below-knee bypass graft for atherosclerotic peripheral arterial disease (PAD) were enrolled 2 to 4 days after surgery and were randomly assigned to clopidogrel 75 mg/day plus ASA 75 to 100 mg/day or placebo plus ASA 75 to 100 mg/day for 6 to 24 months. The primary efficacy endpoint was a composite of index-graft occlusion or revascularization, above-ankle amputation of the affected limb, or death. The primary safety endpoint was severe bleeding (Global Utilization of Streptokinase and Tissue plasminogen activator for Occluded coronary arteries [GUSTO] classification). RESULTS: In the overall population, the primary endpoint occurred in 149 of 425 patients in the clopidogrel group vs 151 of 426 patients in the placebo (plus ASA) group (hazard ratio [HR], 0.98; 95% confidence interval [CI], 0.78-1.23). In a prespecified subgroup analysis, the primary endpoint was significantly reduced by clopidogrel in prosthetic graft patients (HR, 0.65; 95% CI, 0.45-0.95; P = .025) but not in venous graft patients (HR, 1.25; 95% CI, 0.94-1.67, not significant [NS]). A significant statistical interaction between treatment effect and graft type was observed (P(interaction) = .008). Although total bleeds were more frequent with clopidogrel, there was no significant difference between the rates of severe bleeding in the clopidogrel and placebo (plus ASA) groups (2.1% vs 1.2%). CONCLUSION: The combination of clopidogrel plus ASA did not improve limb or systemic outcomes in the overall population of PAD patients requiring below-knee bypass grafting. Subgroup analysis suggests that clopidogrel plus ASA confers benefit in patients receiving prosthetic grafts without significantly increasing major bleeding risk.

Antibodies recognising sulfated carbohydrates are prevalent in systemic sclerosis and associated with pulmonary vascular disease

Glycosylation represents an important modification that regulates biological processes in tissues relevant for disease pathogenesis in systemic sclerosis (SSc), including the endothelium and extracellular matrix. Whether patients with SSc develop antibodies to carbohydrates is not known.

Systemic vascular dysfunction in patients with chronic mountain sickness

Chronic mountain sickness (CMS) is a major public health problem characterized by exaggerated hypoxemia and erythrocytosis. In more advanced stages, patients with CMS often present with functional and structural changes of the pulmonary circulation, but there is little information on the systemic circulation. In patients with diseases associated with chronic hypoxemia at low altitude, systemic vascular function is altered. We hypothesized that patients with CMS have systemic vascular dysfunction that may predispose them to increased systemic cardiovascular morbidity.

Effect of a single, oral, high-dose vitamin D supplementation on endothelial function in patients with peripheral arterial disease: a randomised controlled pilot study

Apart from its role in bone metabolism, vitamin D may also influence cardiovascular disease. The objective of this study was: (1) to determine the effect of a single, oral, high-dose vitamin D supplementation on endothelial function and arterial stiffness in patients with peripheral arterial disease (PAD) and (2) to investigate the impact of this supplementation on coagulation and inflammation parameters.

Rationale, scope, and 20-year experience of vascular surgical training with lifelike pulsatile flow models

Vascular surgical training currently has to cope with various challenges, including restrictions on work hours, significant reduction of open surgical training cases in many countries, an increasing diversity of open and endovascular procedures, and distinct expectations by trainees. Even more important, patients and the public no longer accept a "learning by doing" training philosophy that leaves the learning curve on the patient's side. The Vascular International (VI) Foundation and School aims to overcome these obstacles by training conventional vascular and endovascular techniques before they are applied on patients. To achieve largely realistic training conditions, lifelike pulsatile models with exchangeable synthetic arterial inlays were created to practice carotid endarterectomy and patch plasty, open abdominal aortic aneurysm surgery, and peripheral bypass surgery, as well as for endovascular procedures, including endovascular aneurysm repair, thoracic endovascular aortic repair, peripheral balloon dilatation, and stenting. All models are equipped with a small pressure pump inside to create pulsatile flow conditions with variable peak pressures of ~90 mm Hg. The VI course schedule consists of a series of 2-hour modules teaching different open or endovascular procedures step-by-step in a standardized fashion. Trainees practice in pairs with continuous supervision and intensive advice provided by highly experienced vascular surgical trainers (trainer-to-trainee ratio is 1:4). Several evaluations of these courses show that tutor-assisted training on lifelike models in an educational-centered and motivated environment is associated with a significant increase of general and specific vascular surgical technical competence within a short period of time. Future studies should evaluate whether these benefits positively influence the future learning curve of vascular surgical trainees and clarify to what extent sophisticated models are useful to assess the level of technical skills of vascular surgical residents at national or international board examinations. This article gives an overview of our experiences of >20 years of practical training of beginners and advanced vascular surgeons using lifelike pulsatile vascular surgical training models.

Use of endovascular therapy for peripheral arterial lesions: an analysis of the national trauma data bank from 2007 to 2009

An endovascular approach is increasingly used for the treatment of peripheral arterial trauma (PAT), but evidence supporting this approach is lacking. The objective of our study was to assess outcomes for endovascular repair (ER) versus operative repair (OR) in PAT.

Long-term effects of endovascular angioplasty on orthostatic vasocutaneous autoregulation in patients with peripheral atherosclerosis

OBJECTIVE: To test the hypothesis that endovascular revascularization of femoropopliteal lesions improves the impaired venoarteriolar response (VAR) in patients with atherosclerosis. METHODS: We prospectively compared VARs in 15 healthy controls (18 legs) and 14 patients (17 legs) with mild to moderate peripheral arterial disease before and after successful peripheral endovascular angioplasty of femoropopliteal lesions. In all subjects, foot skin blood flow was assessed by laser Doppler flowmetry in the horizontal (HBF) and sitting (SBF) positions. VAR was calculated as (HBF - SBF)/HBF x 100. RESULTS: In patients with peripheral arterial disease, mean HBF (in arbitrary units [AU]; mean +/- SD) was similar before (25.6 +/- 15.3 AU) and after (27.0 +/- 16.4 AU) angioplasty (P = .67), whereas SBF was significantly lower after than before the endovascular procedure (11.6 +/- 7.7 AU to 18.4 +/- 14.1 AU; P < .05). Intragroup differences between SBF and HBF were significant before and after angioplasty (P < .001). VAR was higher after angioplasty (55.1% +/- 21.2%) compared with VAR before intervention (33.4% +/- 20.2%; P = .015). Although VAR increased after the intervention, VAR was still lower than in healthy controls (68.4% +/- 20.5%; P = .025). During the 6 months of follow-up, the ankle-brachial index and VAR remained unchanged (P > .05). CONCLUSIONS: Patients with mild to moderate peripheral arterial disease have an impaired orthostatic autoregulation that improves after successful endovascular revascularization of femoropopliteal obstructive lesions. The effect on VAR is sustained in the absence of restenosis.

A modified calculation of ankle-brachial pressure index is far more sensitive in the detection of peripheral arterial disease

BACKGROUND: Ankle-brachial pressure index (ABI) is a simple, inexpensive, and useful tool in the detection of peripheral arterial occlusive disease (PAD). The current guidelines published by the American Heart Association define ABI as the quotient of the higher of the systolic blood pressures (SBPs) of the two ankle arteries of that limb (either the anterior tibial artery or the posterior tibial artery) and the higher of the two brachial SBPs of the upper limbs. We hypothesized that considering the lower of the two ankle arterial SBPs of a side as the numerator and the higher of the brachial SBPs as the denominator would increase its diagnostic yield. METHODS: The former method of eliciting ABI was termed as high ankle pressure (HAP) and the latter low ankle pressure (LAP). ABI was assessed in 216 subjects and calculated according to the HAP and the LAP method. ABI findings were confirmed by arterial duplex ultrasonography. A significant arterial stenosis was assumed if ABI was <0.9. RESULTS: LAP had a sensitivity of 0.89 and a specificity of 0.93. The HAP method had a sensitivity of 0.68 and a specificity of 0.99. McNemar's test to compare the results of both methods demonstrated a two-tailed P < .0001, indicating a highly significant difference between both measurement methods. CONCLUSIONS: LAP is the superior method of calculating ABI to identify PAD. This result is of great interest for epidemiologic studies applying ABI measurements to detect PAD and assessing patients' cardiovascular risk.