Wild-type Measles Virus Infection Upregulates Poliovirus Receptor-Related 4 and Causes Apoptosis in Brain Endothelial Cells by Induction of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis.

Epigenetic Changes in Endothelial Progenitors as a Possible Cellular Basis for Glycemic Memory in Diabetic Vascular Complications

The vascular complications of diabetes significantly impact the quality of life and mortality in diabetic patients. Extensive evidence from various human clinical trials has clearly established that a period of poor glycemic control early in the disease process carries negative consequences, such as an increase in the development and progression of vascular complications that becomes evident many years later. Importantly, intensive glycemic control established later in the disease process cannot reverse or slow down the onset or progression of diabetic vasculopathy. This has been named the glycemic memory phenomenon. Scientists have successfully modelled glycemic memory using various in vitro and in vivo systems. This review emphasizes that oxidative stress and accumulation of advanced glycation end products are key factors driving glycemic memory in endothelial cells. Furthermore, various epigenetic marks have been proposed to closely associate with vascular glycemic memory. In addition, we comment on the importance of endothelial progenitors and their role as endogenous vasoreparative cells that are negatively impacted by the diabetic milieu and may constitute a "carrier" of glycemic memory. Considering the potential of endothelial progenitor-based cytotherapies, future studies on their glycemic memory are warranted to develop epigenetics-based therapeutics targeting diabetic vascular complications.

Reversal of Postprandial Endothelial Dysfunction by Cyclooxygenase Inhibition in Healthy Volunteers

The aim of this study was to evaluate the role of cyclooxygenase (COX) in venous vascular reactivity changes after an oral lipid overload (OLO). Venous endothelial function (dorsal hand vein technique) was evaluated in fasting, 30 minutes after COX inhibition (aspirin-fasting), 2 to 4 hours after an OLO (1000 kcal, 58% fat), and again after COX inhibition (aspirin-OLO, 600 mg/200 mL water) in 10 healthy adults (age, 28.1 +/- 1.3 years; body mass index, 22.3 +/- 0.6 kg/m(2)). Fasting, 2- to 4-hour post-OLO, and 60-minute postaspirin plasma glucose, insulin, and lipids were also evaluated. The OLO increased triglycerides and insulin, reduced low-density lipoprotein and high-density lipoprotein, but glycemia and total cholesterol remained unchanged. There were no metabolic differences between OLO and aspirin-OLO. In fasting, aspirin reduced acetylcholine-induced venodilation (107.0% +/- 14% versus 57.3% +/- 11%; P < 0.001). Vascular reactivity was blunted after the OLO (phenylephrine dose: 0.3 +/- 0.2 fasting versus 1.9 +/- 0.8 nmol/min after OLO; P < 0.001) and was partially corrected by aspirin (0.4 +/- 0.2; P < 0.001). Similar changes were observed in maximum venodilation after acetylcholine (107.0% +/- 14% fasting versus 60.4% +/- 9% after OLO, P < 0.001; aspirin-OLO: 95.9% +/- 6%; P < 0.001). The responses to sodium nitroprusside remained unchanged during the study. We conclude that the OLO reduction in the endothelium-dependent venoconstruction and venodilation is partially the result of the action of COX.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

Reduced allergic lung inflammation in rats following formaldehyde exposure: Long-term effects on multiple effector systems

Clinical and experimental evidences show that formaldehyde (FA) exposure has an irritant effect on the upper airways. As being an indoor and outdoor pollutant, FA is known to be a causal factor of occupational asthma. This study aimed to investigate the repercussion of FA exposure on the course of a lung allergic process triggered by an antigen unrelated to FA. For this purpose, male Wistar rats were subjected to FA inhalation for 3 consecutive days (1%, 90-min daily), subsequently sensitized with ovalbumin (OVA)-alum via the intraperitoneal route, and 2 weeks later challenged with aerosolized OVA. The OVA challenge in rats after FA inhalation (FA/OVA group) evoked a low-intensity lung inflammation as indicated by the reduced enumerated number of inflammatory cells in bronchoalveolar lavage as compared to FA-untreated allergic rats (OVA/OVA group). Treatment with FA also reduced the number of bone marrow cells and blood leukocytes in sensitized animals challenged with OVA, which suggests that the effects of FA had not been only localized to the airways. As indicated by passive cutaneous anaphylactic reaction, FA treatment did not impair the anti-OVA IgE synthesis, but reduced the magnitude of OVA challenge-induced mast cell degranulation. Moreover, FA treatment was associated to a diminished lung expression of PECAM-1 (platelet-endothelial cell adhesion molecule 1) in lung endothelial cells after OVA challenge and an exacerbated release of nitrites by BAL-cultured cells. Keeping in mind that rats subjected solely to either FA or OVA challenge were able to significantly increase the cell influx into lung, our study shows that FA inhalation triggers long-lasting effects that affect multiple mediator systems associated to OVA-induced allergic lung such as the reduction of mast cells activation, PECAM-1 expression and exacerbation of NO generation, thereby contributing to the decrease of cell recruitment after the OVA challenge. In conclusion, repeated expositions to air-borne FA may impair the lung cell recruitment after an allergic stimulus, thereby leading to a non-responsive condition against inflammatory stimuli likely those where mast cells are involved. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Somatic cell nuclear transfer is associated with altered expression of angiogenic factor systems in bovine placentomes at term

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reduced allergic lung inflammation in rats following formaldehyde exposure: Long-term effects on multiple effector systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of diethylpropion treatment and withdrawal on aorta reactivity, endothelial factors and rat behavior

Diethylpropion (DEP) is an amphetamine-like compound used as a coadjutant in the treatment of obesity and which presents toxicological importance as a drug of abuse. This drug causes important behavioral and cardiovascular complications; however, the vascular and behavioral alterations during DEP treatment and withdrawal, have not been determined. We evaluated the effects of DEP treatment and withdrawal on the rat aorta reactivity to noradrenaline, focusing on the endothelium, and the rat behavior during DEP treatment and withdrawal. DEP treatment caused a hyporreactivity to noradrenaline in aorta, reversible after 2 days of withdrawal and abolished by both the endothelium removal and the presence of L-NAME, but not by the presence of indomethacin. Furthermore, DEP treatment increased the general activity of rats. Contrarily, DEP withdrawal caused a decrease in the locomotor activity and an increase in grooming behavior, on the 2nd and 7th days after the interruption of the treatment, respectively. DEP treatment also caused an adaptive vascular response to noradrenaline that seems to be dependent on the increase in the endothelial nitric oxide system activity, but independent of prostaglandins synthesis. The data evidenced chronological differences in the adaptive responses of the vascular and central nervous systems induced by DEP treatment. Finally, a reversion of the adaptive response to DEP was observed in the vascular system during withdrawal, whereas a neuroadaptive process was still present in the central nervous system post-DEP. These findings advance on the understanding of the vascular and behavioral pathophysiological processes involved in the therapeutic and abusive uses of DEP. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Applying the National Institute for Clinical Excellence criteria to patients treated with the Genous (TM) Bio-engineered R stent (TM): a sub-study of the e-HEALING (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth) worldwide registry

The National Institute for Clinical Excellence (NICE) guidelines recommend the use of bare-metal stents (BMS) in non-complex lesions with a low risk of restenosis (diameter a parts per thousand yen3 mm and lesion length a parts per thousand currency sign15 mm) and the use of drug-eluting stents (DES) in more complex lesions with a high risk of restenosis (diameter < 3.0 mm or lesion length > 15 mm). However, the guidelines were created based on studies evaluating BMS and DES only. We performed an analysis of patients undergoing non-urgent percutaneous coronary intervention with the novel endothelial cell capturing stent (ECS). The ECS is coated with CD34(+) antibodies that attract circulating endothelial progenitor cells to the stent surface, thereby accelerating the endothelialization of the stented area. We analyzed all patients enrolled in the worldwide e-HEALING registry that met the NICE criteria for either low-risk or high-risk lesions and were treated with a parts per thousand yen1 ECS. The main study outcome was target vessel failure (TVF) at 12-month follow-up, defined as the composite of cardiac death or MI and target vessel revascularization (TVR). A total of 4,241 patients were assessed in the current analysis. At 12-month follow-up, TVF occurred in 7.0% of the patients with low-risk lesions and in 8.8% of the patients with high-risk lesions (p = 0.045). When evaluating the diabetic patients versus the non-diabetic patients per risk group, no significant differences were found in TVF, MI or TVR in either risk group. The ECS shows good clinical outcomes in lesions carrying either a high or a low risk of restenosis according to the NICE guidelines with comparable rates of cardiac death, myocardial infarction, and stent thrombosis. The TVF rate with ECS was slightly higher in patients with high-risk lesions, driven by higher clinically driven TLR. The risk of restenosis with ECS in patients carrying high-risk lesions needs to be carefully considered relative to other risks associated with DES. Furthermore, the presence of diabetes mellitus did not influence the incidence of TVF in either risk group.

Mechanisms of endothelial dysfunction in obesity-associated hypertension

Obesity is strongly associated with high blood pressure, dyslipidemia, and type 2 diabetes. These conditions synergistically increase the risk of cardiovascular events. A number of central and peripheral abnormalities can explain the development or maintenance of high blood pressure in obesity. Of great interest is endothelial dysfunction, considered to be a primary risk factor in the development of hypertension. Additional mechanisms also related to endothelial dysfunction have been proposed to mediate the development of hypertension in obese individuals. These include: increase in both peripheral vasoconstriction and renal tubular sodium reabsorption, increased sympathetic activity and overactivation of both the renin-angiotensin system and the endocannabinoid system and insulin resistance. The discovery of new mechanisms regulating metabolic and vascular function and a better understanding of how vascular function can be influenced by these systems would facilitate the development of new therapies for treatment of obesity-associated hypertension.

Role of outgrowth endothelial cells for applications in tissue engineering

Co-culture systems, consisting of outgrowth endothelial cells (OEC) and primary osteoblasts (pOB), represent a prom¬ising instrument to mimick the natural conditions in bone repair processes and provide a new concept to develop constructs for bone replacement. Furthermore, co-culture of OEC and pOB could provide new insights into the molecular and cellular mechanisms that control essential processes during bone repair. The present study described several advantages of the co-culture of pOB and OEC for bone tissue engineering applications, including beneficial effects on the angiogenic activation of OEC, as well as on the assembly of basement membrane matrix molecules and factors involved in vessel maturation and stabilization. The ongoing angiogenic process in the co-culture system proceeded during the course of co-cultivation and correlated with the upregulation of essential angiogenic factors, such as VEGF, angiopoietins, basement membrane molecules and mural cell-specific markers. Furthermore the co-culture system appeared to maintain osteogenic differentiation capacity.rnrnAdditional treatment of co-cultures with growth factors or morphogens might accelerate and improve bone formation and furthermore could be useful for potential clinical applications. In this context, the present study highlights the central role of the morphogen, sonic hedgehog, which has been shown to affect angiogenic activation as well as osteogenic differentiation in the co-culture model of OEC and pOB. Treatment of co-cultures with sonic hedgehog resulted in an increased formation of microvessel-like structures as early as after 24 hours. This proangiogenic effect was induced by the upregulation of the proangiogenic factors, VEGF, angiopoietin1 and angiopoietin 2. In contrast to treatment with a commonly used proangiogenic agent, VEGF, Shh stimulation induced an increased expression of factors associated with vessel maturation and stabilization, mediated through the upregulation of growth factors that are strongly involved in pericyte differentiation and recruitment, including PDGF-BB and TGFbeta. In addition, Shh treatment of co-cultures also resulted in an upregulation of osteogenic differentiation markers like alkaline phosphatase, osteocalcin, osteonectin and osteopontin, as well as an increased matrix calcification. This was a result of upregulation of the osteogenic differentiation regulating factors, BMP2 and RUNX2 which could be assessed in response to Shh treatment. rn

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

Characterisation of human co-culture systems for applications in bone tissue engineering

For the successful integration of bone tissue engineering constructs into patients, an adequate supply with oxygen and nutrients is critical. Therefore, prevascularisation of bone tissue engineering constructs is desirable for bone formation, remodelling and regeneration. Co-culture systems, consisting of human endothelial cells and primary osteoblasts (pOB) as well as osteosarcoma cell lines, represent a promising method for studying the mechanisms involved in the vascularisation of constructs in bone tissue en- gineering and could provide new insights into the molecular and cellular mechanisms that control essential processes during angiogenesis. The present study demonstrated the im- portant components of co-culture systems with a focus on bone tissue replacement and the angiogenic effects of pOB and osteosarcoma cell lines on human endothelial cells. Furthermore, the studies emphasised an overall approach for analysis of signal molecules that are involved in the angiogenic activation of human endothelial cells by the regulation of VEGF-related pathways at the transcriptional and translational levels. The osteosarcoma cell lines Cal-72, MG-63 and SaOS-2, as well as pOB from several donors, differed in their angiogenesis-inducing potential in 2-D and 3-D co-culture systems. SaOS-2 cells appeared to have a high osteogenic differentiation level with no detectable angiogenesis-inducing potential in co-culture with human endothelial cells. The angiogenic potential of the osteoblast-like cells is mainly correlated with the upregulation of essential angiogenic growth factors, such as VEGF, bFGF and HGF and the downregulation of the angiogenesis inhibitor, endostatin. However, other factors involved in angiogenic regulation were found to differ between SaOS-2 cells, compared to Cal-72 and MG-63. The present study focuses on VEGF pathway-effecting genes as key players in the regulation of angiogenesis. The levels of VEGF and VEGF-effecting genes, such as TGF-α and TIMP-2 are down-regulated in SaOS-2 cells. In contrast, direct regulators of VEGF, such as IL6, IL8 and TNF are strongly upregulated, which indicates disruptions in growth factor regulating pathways in SaOS-2 cells. Potential pathways, which could be involved include MEK, PI3K, MAPK, STAT3, AKT or ERK. Additional treatment of co-cultures with single growth factors did not accelerate or improve the angiogenesis-inducing potential of SaOS-2 cells. Knowledge of the detailed molecular mechanisms involved in angiogenesis control will hopefully allow improved approaches to be developed for prevascularisation of bone tissue engineering constructs.

Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.