Large Vessel Endothelial Cells Research Articles

Characterization of pulmonary vascular remodeling and MicroRNA-126-targets in COPD-pulmonary hypertension

BackgroundDespite causing increased morbidity and mortality, pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) patients (COPD-PH) lacks treatment, due to incomplete understanding of its pathogenesis. Hypertrophy of pulmonary arterial walls and pruning of the microvasculature with loss of capillary beds are known features of pulmonary vascular remodeling in COPD. The remodeling features of pulmonary medium- and smaller vessels in COPD-PH lungs are less well described and may be linked to maladaptation of endothelial cells to chronic cigarette smoking (CS). MicroRNA-126 (miR126), a master regulator of endothelial cell fate, has divergent functions that are vessel-size specific, supporting the survival of large vessel endothelial cells and inhibiting the proliferation of microvascular endothelial cells. Since CS decreases miR126 in microvascular lung endothelial cells, we set out to characterize the remodeling by pulmonary vascular size in COPD-PH and its relationship with miR126 in COPD and COPD-PH lungs.MethodsDeidentified lung tissue was obtained from individuals with COPD with and without PH and from non-diseased non-smokers and smokers. Pulmonary artery remodeling was assessed by ⍺-smooth muscle actin (SMA) abundance via immunohistochemistry and analyzed by pulmonary artery size. miR126 and miR126-target abundance were quantified by qPCR. The expression levels of ceramide, ADAM9, and endothelial cell marker CD31 were assessed by immunofluorescence.ResultsPulmonary arteries from COPD and COPD-PH lungs had significantly increased SMA abundance compared to non-COPD lungs, especially in small pulmonary arteries and the lung microvasculature. This was accompanied by significantly fewer endothelial cell markers and increased pro-apoptotic ceramide abundance. miR126 expression was significantly decreased in lungs of COPD individuals. Of the targets tested (SPRED1, VEGF, LAT1, ADAM9), lung miR126 most significantly inversely correlated with ADAM9 expression. Compared to controls, ADAM9 was significantly increased in COPD and COPD-PH lungs, predominantly in small pulmonary arteries and lung microvasculature.ConclusionBoth COPD and COPD-PH lungs exhibited significant remodeling of the pulmonary vascular bed of small and microvascular size, suggesting these changes may occur before or independent of the clinical development of PH. Decreased miR126 expression with reciprocal increase in ADAM9 may regulate endothelial cell survival and vascular remodeling in small pulmonary arteries and lung microvasculature in COPD and COPD-PH.

Abstract 884: Expression of Anti-mir-33a-5p in Cultured Endothelial Cells Increases Macrophage and Smooth Muscle Cell Cholesterol Efflux by Exosome-mediated Transfer of Anti-mir-33a-5p

Background: The microRNA miR-33a-5p impairs apoAI-mediated cholesterol efflux by silencing ABCA1 expression. Systemic injection of antagomirs to miR-33a-5p reduces atherosclerosis but can cause hepatic steatosis and hypertriglyceridemia. In contrast, local delivery of miR-33a-5p antagomirs to lipid-laden intimal cells [smooth muscle cells (SMC) and macrophages (Mϕ)] could potentially treat atherosclerosis without causing metabolic abnormalities. Helper-dependent adenoviral vectors (HDAd) provide durable transgene expression in endothelial cells (EC) in vivo; however, HDAd does not cross an intact large vessel EC layer and therefore cannot deliver antagomirs directly to SMC and Mϕ. We hypothesized that transducing EC with an HDAd expressing anti-miR-33a-5p (HDAdAnti) could result in packaging of anti-miR-33a-5p into EC-derived exosomes that, in turn, deliver anti-miR-33a-5p to neighboring SMC and Mϕ. Inhibition of miR-33a-5p in SMC and Mϕ would upregulate ABCA1, and enhance apoAI-mediated cholesterol efflux. Methods: We transduced cultured EC with either HDAdAnti or a control HDAd expressing a “scrambled” shRNA (HDAdScr), harvested conditioned medium (CM) from the EC, and treated SMC and Mϕ (some of which were cholesterol-loaded) with the CM. We measured levels of anti-miR-33a-5p and miR-33a-5p, ABCA1 protein, and apoAI-mediated cholesterol efflux in the treated SMC and Mϕ. Results: Exosomes containing anti-miR-33a-5p were present only in CM from HDAdAnti-transduced EC. Compared to Mϕ incubated with CM from EC transduced with HDAdScr, Mϕ incubated with CM from HDAdAnti-transduced EC had 80% lower levels of miR-33a-5p, a 2.2-fold increase in ABCA1 protein, and a 1.6-fold increase in apoAI-mediated cholesterol efflux (all P<0.001). Similar results were obtained with SMC: 65% lower levels of miR-33a-5p expression, 1.6-fold increase in ABCA1 protein, and 1.4-fold increase in apoAI-mediated cholesterol efflux (all P≤0.01). Conclusions: EC transduced with HDAdAnti release exosomes containing anti-miR-33a-5p. These exosomes transfer anti-miR-33a-5p to SMC and Mϕ, increasing cholesterol efflux. Application of this approach in vivo may accelerate vessel wall cholesterol efflux, preventing and reversing atherosclerosis.

Arginase in the vascular endothelium: friend or foe?

Arginase in the vascular endothelium: friend or foe?

0269: Vascular remodeling of the endocardium following cardiac infarction occured by arteriogenesis and angiogenesis

Coronary vasculature is required to maintain cardiomyocyte survival, via delivery of oxygen and nutrients, and consequently myocardial architecture and cardiac function. Ischemic heart disease following myocardial infarction causes irreversible cell loss and scaring and is a major cause of morbidity and mortality. Revascularization of injured, ischemic and regenerating organs is essential to restore organ function and requires the formation of new vessels by the mechanisms of vasculogenesis, angiogenesis or arteriogenesis. With the objective of studying vascular remodeling during myocardial infarction (MI), we have performed permanent left coronary ligation on Connexin40-GFP ( Cx40 GFP/ + ) mice. Cx40 encodes a gap junction protein and is expressed in endothelial cells of large vessels. In the heart, Cx40-GFP expression is detected in coronary arteries but not in veins, capillaries or endocardium. After two weeks of ligation, MI was detected in left ventricle by echocardiography and anatomical examination of these hearts revealed the presence of an extensive network of GFP-positive vasculature within the infarct area. These vessels follow a tortuous route in the remaining ventricular wall and some communicate with the left ventricular lumen forming a crater covered with GFP and VEGF-R2 positive endothelial cells at the endocardial surface. To determine whether these vessels result from neo-vascularization or coronary artery remodeling, we carried out genetic lineage tracing of coronary endothelial cells using an inducible Cx40-cre allele. Our results show that GFP positive endothelial cells forming the endocardial carters are not always derived from preexisting coronary arteries, suggesting that endocardium may also contribute to the generation of new vessels during vascular remodeling in the adult heart by angiogenesis.

DICAM inhibits angiogenesis via suppression of AKT and p38 MAP kinase signalling

Dual Ig domain-containing adhesion molecule (DICAM), a protein with homology to the junctional adhesion molecule family, has been demonstrated to interact with integrin αVβ3 that plays a critical role in angiogenesis. Here, we determined the role of DICAM during angiogenesis and the molecular mechanisms involved in the inhibition of angiogenesis. DICAM was expressed on the endothelial cells of large vessels to small capillaries. In human umbilical vein endothelial cells (HUVECs), DICAM was up-regulated by vascular endothelial growth factor (VEGF) through the MEK/ERK and PI3K/AKT pathways. Furthermore, the exogenous expression of DICAM in HUVECs suppressed angiogenesis in vitro Matrigel and in vivo plug assays, and conversely, DICAM knockdown enhanced angiogenesis. In addition, DICAM inhibited HUVEC migration and accelerated apoptosis via down-regulation of Bcl-2, but did not affect viability or proliferation of HUVEC. Mechanistically, the exogenous expression of DICAM suppressed VEGF-induced phosphorylarion of AKT and p38 MAP kinase. When integrin signalling was activated by vitronectin, a forced expression of DICAM attenuated integrin β3/FAK signalling and downstream AKT and p38 MAP kinase signalling in HUVECs. Collectively, DICAM suppressed angiogenesis by attenuating AKT and p38 MAP kinase signalling, which suggests that DICAM may be a novel negative regulator of angiogenesis.

Spleen endothelial cells from patients with myelofibrosis harbor the JAK2V617F mutation

AbstractIncreased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F+ ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.

Extracellular matrix rigidity controls podosome induction in microvascular endothelial cells

Podosomes are actin-based structures involved in cell adhesion, migration, invasion and extracellular matrix degradation. They have been described in large vessel endothelial cells, but nothing is known concerning microvascular endothelial cells. Here, we focussed on liver sinusoidal endothelial cells (LSECs), fenestrated microvascular cells that play major roles in liver physiology. Liver fibrosis induces a dedifferentiation of LSECs leading notably to a loss of fenestrae. Because liver fibrosis is associated with increased matrix stiffness, and because substrate stiffness is known to regulate the actin cytoskeleton, we investigated the impact of matrix rigidity on podosome structures in LSECs. Using primary LSECs, we demonstrated that microvascular endothelial cells are able to form constitutive podosomes. Podosome presence in LSECs was independent of cytokines such as transforming growth factor-β or vascular endothelial growth factor, but could be modulated by matrix stiffness. As expected, LSECs lost their differentiated phenotype during cell culture, which was paralleled by a loss of podosomes. LSECs however retained the capacity to form active podosomes following detachment/reseeding or actin-destabilising drug treatments. Finally, constitutive podosomes were also found in primary microvascular endothelial cells from other organs. Our results show that microvascular endothelial cells are able to form podosomes without specific stimulation. Our data suggest that the major determinant of podosome induction in these cells is substrate rigidity.

Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity, and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin-10) in large vessel endothelial cells (EC) at levels as high as obtained with the CMV immediate-early promoter, while retaining EC-specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC-specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely 4 copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large vessel EC, while minimizing systemic effects.

Thrombospondin-1 Inhibits VEGF Receptor-2 Signaling by Disrupting Its Association with CD47

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.

Critical Role for GATA3 in Mediating Tie2 Expression and Function in Large Vessel Endothelial Cells

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

Anti-angiogenic activity of sesterterpenes; natural product inhibitors of FGF-2-induced angiogenesis

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. We have investigated the anti-angiogenic potential of two naturally occurring sesterterpenes, leucosesterterpenone (compound 1) and leucosterlactone (compound 2) isolated from the Himalayan plant Leucosceptrum canum and identified as having biological activity in preliminary screening. Compound 1 inhibited fibroblast growth factor-2-induced proliferation, migration in a wounding assay, chemotaxis and tube formation with small vessel (human dermal) and large vessel (bovine aortic) endothelial cells while compound 2 was largely inactive. Both compounds were also active in an in vivo angiogenic model using the chick chorioallantoic membrane. Neither compounds showed inhibitory activity in the absence of fibroblast growth factor-2. We were able to demonstrate in a binding assay that compounds 1 and 2 bound to the fibroblast growth factor-2 receptor-1 with IC(50) values of 1.4 +/- 0.956 and 132.47 +/- 7.90 muM, respectively, with a concomitant down regulation of phosphorylated ERK1/2 but did not bind to receptor-2. Compound 1 was less hydrophobic than compound 2 and this may contribute to its increased activity. Compound 1 is a new addition to the small number of inhibitors of fibroblast growth factor-2-induced angiogenesis. The compound was a specific inhibitor in that it had no effect on vascular endothelial growth factor or epithelial growth factor-induced angiogenesis. Since angiogenesis is essential for tumour development we conclude that these compounds may have potential as anti-tumour agents.

Abstract 1505: Sunitinib-Induced Cardiotoxicity is Mediated in Part via Direct Effects on Cardiac Myocytes and Smooth Muscle Cells

Tyrosine kinase inhibitors (TKI) have revolutionized cancer therapy, spawning over 500 clinical trials, yet cardiotoxicities are emerging. Sunitinib, a multi-targeted TKI, prolongs life in renal cell and gastrointestinal cancer patients, but hypertension and heart failure have required treatment adjustments. Objective: Sunitinib cardiotoxicity may be due, in part, to direct effects on smooth muscle and cardiac myocytes. Methods: We used C57Bl/6J mice, rat neonatal cardiomyocytes (NCM), and human coronary artery smooth muscle cells (SMC) treated with Sunitinib to measure drug-induced cardiovascular toxicity. Structural (transmission electron microscopy, immunohistochemistry (IHC)/confocal microscopy) and functional [apoptosis/survival, nitric oxide synthesis (intracellular and extracellular), signaling] effects were assessed [real-time PCR, Western blotting, receptor phosphorylation (immunoprecipitation/Western blotting)]. Results: We show mitochondrial injury in hearts from Sunitinib treated mice which persisted off of the drug. In vitro , Sunitinib accumulated in myocytes, caused myofibrillar disorganization, mitochondrial injury, and localized to the lysosomes. Sunitinib caused reductions in akt, MAPK, and AMPK signaling, and activated pro-apoptotic caspase-9 in NCM and SMC with similar effects in large and small vessel endothelial cells. In hearts of Sunitinib-treated mice, we found similar changes in signaling pathways. Conclusion: Sunitinib causes direct cardiac myocyte and smooth muscle cell toxicities, which may contribute to the cardiovascular side effects seen in patients. Further understanding of these mechanisms will guide monitoring, intervention and future rational drug design.

Sinusoidal endothelial cell repopulation following ischemia/reperfusion injury in rat liver transplantation

We evaluated the kinetics by which rat liver sinusoidal endothelial cells (LSECs) are repopulated in the reperfused transplanted liver after 18 hours of cold ischemic storage. We found that the majority of LSECs in livers cold-stored for 18 hours in University of Wisconsin solution are seriously compromised and often are retracted before transplantation. Sinusoids rapidly re-endothelialize within 48 hours of transplantation, and repopulation is coincident with up-regulation of hepatocyte vascular endothelial growth factor expression and vascular endothelial growth factor receptor-2 expression on large vessel endothelial cells and repopulating LSECs. Although re-endothelialization occurs rapidly, we show here, using several high-resolution imaging techniques and 2 different rat liver transplantation models, that engraftment of bone marrow-derived cells into functioning LSECs is routinely between 1% and 5%. Bone marrow plays a measurable but surprisingly limited role in the rapid repopulation and functional engraftment of bone marrow-derived LSECs after cold ischemia and warm reperfusion.

Thrombospondin-1 Expression Is Increased during Follicular Atresia in the Primate Ovary

Thrombospondin (TSP)-1 is an antiangiogenic extracellular matrix glycoprotein that modulates several aspects of cellular function. The aim of this study was to determine the pattern of TSP-1 mRNA and protein expression as well as expression of its receptor CD36 in the marmoset ovary and to investigate the effects of inhibition of gonadotropins or VEGF activity on TSP-1 and CD36 expression in vivo. GnRH antagonist or VEGF Trap, a soluble decoy receptor, was administered on d 0 of the follicular phase of the cycle, and ovaries were collected at the end of the follicular phase (d 10). TSP-1 mRNA and protein were present in granulosa cells of preantral and antral follicles, with the highest staining at the late secondary and tertiary stages. Moreover, expression of TSP-1 mRNA and protein was significantly increased in tertiary follicles undergoing atresia. CD36 protein was detected in granulosa cells of preantral and antral follicles as well as in endothelial cells of large vessels. Inhibition of gonadotropin secretion or VEGF activity had no effect on TSP-1 expression; however, expression of CD36 protein was inhibited by the VEGF Trap. In conclusion, TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia; alternatively, TSP-1 may act on granulosa and/or endothelial cells to promote follicular atresia in the ovary. Angiogenesis is likely to involve a balance between pro- and antiangiogenic factors. Our results suggest that loss of VEGF activity does not regulate TSP-1 expression directly but may influence TSP-1 activity via down-regulation of the CD36 receptor.

A PRACTICAL QUESTION BASED ON CROSS-PLATFORM MICROARRAY DATA NORMALIZATION: ARE BOEC MORE LIKE LARGE VESSEL OR MICROVASCULAR ENDOTHELIAL CELLS OR NEITHER OF THEM?

Since the available microarray data of BOEC (human blood outgrowth endothelial cells), large vessel, and microvascular endothelial cells were from two different platforms, a working cross-platform normalization method was needed to make these data comparable. With six HUVEC (human umbilical vein endothelial cells) samples hybridized on two-channel cDNA arrays and six HUVEC samples on Affymetrix arrays, 64 possible combinations of a three-step normalization procedure were investigated to search for the best normalization method, which was selected, based on two criteria measuring the extent to which expression profiles of biological samples of the same cell type arrayed on two platforms were indistinguishable. Next, three discriminative gene lists between the large vessel and the microvascular endothelial cells were achieved by SAM (significant analysis of microarrays), PAM (prediction analysis for microarrays), and a combination of SAM and PAM lists. The final discriminative gene list was selected by SVM (support vector machine). Based on this discriminative gene list, SVM classification analysis with best tuning parameters and 10,000 times of validations showed that BOEC were far from large vessel cells, they either formed their own class, or fell into the microvascular class. Based on all the common genes between the two platforms, SVM analysis further confirmed this conclusion.

56: In vitro generation of human microvessel networks

Introduction: Successful incorporation of bioengineered tissues requires vascularization. Attempts at de novo vascular generation or angiogenic induction have had little success in improving tissue perfusion. An alternate approach to neovascularization is to form a prevascularized construct that is capable of rapid inosculation and rapid cellular perfusion. Human parathyroid (PTH) tissue is uniquely capable of a rapid angiogenic induction, with restoration of calcium homeostasis within 6 weeks of PTH autografting. We hypothesized that human PTH cells could induce human microvessel fragments to form networks of capillary microvessels in vitro. Because PTH cells release VEGF and Angiopoietin-2 (Ang-2) after devascularization, we further hypothesized that PTH angiogenic induction is driven by PTH release of VEGF and Ang-2. Methods: Human subcutaneous fat was partially digested with collagenase to yield microvessel fragments. After sequential filtering, 30-350 micron fragments were embedded in DMEM + 10% FBS-containing collagen I matrix in 3 dimensional culture. PTH cells were cocultured on the matrix surface to induce an angiogenic response. Other matrix cultures were treated with VEGF (50 ng/ml) or VEGF + Ang-2 (50 ng/ml) and microvessel responses noted. Because Stat3 phosphorylation has been identified with endothelial cell (EC) angiogenic responses, we also tested for Stat3 activation in human dermal microvessel EC (HDMEC) after treatment with VEGF or VEGF + Ang-2. Results: Human microvessel fragments in culture are completely quiescent. Coculture with PTH cells induces the rapid development of neovascular networks. Treatment of the human microvessels in vitro with VEGF failed to induce an angiogenic event. VEGF + Ang-2 induced EC migration but no capillary formation. Both VEGF and Ang-2 induced Stat3 phosphorylation in HDMEC, but not in iliac vein EC. Conclusions: Human PTH cells release or induce release of all necessary angiogenic factors for capillary network angiogenic induction of human microvessels in vitro. The mechanism of angiogenic induction is more complex than VEGF stimulation alone. Stat 3 activity is upregulated in microvessel EC after VEGF or Ang-2 stimulation, but not in large vessel EC, suggesting a possible role in microvessel angiogenic processes. PTH cells have the angiogenic potential to induce capillary network development in vitro. This system may be capable of providing vascularization of bioengineered tissues for therapeutic intervention.

1A.4 The protein C anticoagulant pathway: new insights

Protein C is part of an important anticoagulant mechanism that down regulates blood coagulation. It is activated on the endothelial cell surface by thrombin/thrombomodulin complex. Activated protein C (APC), together with its cofactor protein S, proteolytically inactivates factor Va and factor VIIIa. Optimum activation of protein C to APC requires presentation of protein C to its activation complex. On large blood vessels this is achieved by specific interaction of protein C with its receptor, EPCR, an interaction mediated via its Gla domain. A number of specificity issues surrounding activation of protein C and the function of APC have been clarified recently: By what mechanism is EPCR selectively expressed in large blood vessel endothelial cells? Preliminary in vitro and transgenic studies have been reported that have focussed on the proximal promoter of the human (h) and murine (m) EPCR genes. Clusters of SP1 sites have been shown to be important in directing EPCR expression in vitro. However, it has been unclear whether the proximal promoter alone is sufficient to direct cell specific expression in vivo. In order to clarify this, we examined 39 kb of the hEPCR locus for regulatory elements. Using a combination of in vitro and in transgenic approaches, we identified a critical enhancer element in a hypersensitive site upstream of hEPCR that can act in an autonomous manner to direct gene expression to large vessel endothelial cells in vivo [1]. How does protein C specifically recognise its receptor, EPCR? The published crystal structure of the EPCR/protein C Gla domain complex demonstrates binding between the alpha helices of EPCR and the w loop of the Gla domain of protein C. Because w loops of the Gla domains of the vitamin K dependent proteins are largely conserved (to enable phospholipid binding), it has been unclear what determines the specificity of binding to EPCR. We have approached this by making a series of protein C/prothrombin chimaeras. One of these chimaeras, containing a single residue change at position 8 (Leu to Val) abolished binding to EPCR, while preserving phospholipid binding [2]. This has suggested that Leu8 of protein C is the critical specificity determinant controlling EPCR recognition. How does APC specifically recognise protein S? The interaction by which protein S enhances APC function has remained obscure, possibly because direct binding between APC and protein S is difficult to demonstrate. Using the above chimaeras, we have identified a 4 residue motif, 36 39, within the sequence 33 39, that appears essential for interaction with protein S Protein C Gla domain (residues 1-45) Residues 33-39 required for protein S cofactor activity

Semi-supervised learning via penalized mixture model with application to microarray sample classification

It is biologically interesting to address whether human blood outgrowth endothelial cells (BOECs) belong to or are closer to large vessel endothelial cells (LVECs) or microvascular endothelial cells (MVECs) based on global expression profiling. An earlier analysis using a hierarchical clustering and a small set of genes suggested that BOECs seemed to be closer to MVECs. By taking advantage of the two known classes, LVEC and MVEC, while allowing BOEC samples to belong to either of the two classes or to form their own new class, we take a semi-supervised learning approach; for high-dimensional data as encountered here, we propose a penalized mixture model with a weighted L1 penalty to realize automatic feature selection while fitting the model. We applied our penalized mixture model to a combined dataset containing 27 BOEC, 28 LVEC and 25 MVEC samples. Analysis results indicated that the BOEC samples appeared to form their own new class. A simulation study confirmed that, compared with the standard mixture model with or without initial variable selection, the penalized mixture model performed much better in identifying relevant genes and forming corresponding clusters. The penalized mixture model seems to be promising for high-dimensional data with the capability of novel class discovery and automatic feature selection.

Flow Perturbation Is Linked to Endothelial PAR Signaling

The vascular endothelium integrates a variety of humoral stimuli and biophysical forces to maintain patent microvascular and macrovacular beds. At the interface between the blood and vessel wall, endothelial cells communicate in both directions by the release of growth factors, cytokines, and bioactive mediators to maintain vascular homeostasis. Endothelial dysfunction is central for acute systemic inflammatory syndromes including sepsis and chronic inflammatory disorders such as atherosclerosis. Early atherosclerotic lesions preferentially develop at sites where laminar flow patterns are disturbed, and endothelial cells sense changes in biomechanical forces that result from flow perturbation. Exposure to shear forces rapidly activates G proteins, ion fluxes, and mechanosensing receptors, including integrins and a complex involving PECAM-1, VEGFR-2, and vascular endothelial cell cadherin.1 Activation of downstream signaling pathways results in cytoskeletal reorganization and a flow-adapted endothelial phenotype.2 Endothelial cells under laminar flow have an atheroprotected phenotype, but perturbation of laminar flow patterns induces a complex transcriptional response that favors the induction of adhesion molecules, proinflammatory cytokines, and procoagulant genes. See page 1185 Dekker et al profiled the repertoire of laminar flow-regulated genes in endothelial cells and identified lung Krueppel-like factor (LKLF or KLF-2) that was selectively induced on flow adaptation and suppressed by the inflammatory mediator tumor necrosis factor (TNF)α.3 KLF2 was indeed tonically expressed in vivo in noninflamed large vessel endothelial cells. KLFs are zinc finger transcription factors similar to Sp1 and Egr-1 families that act as transcriptional repressors or activators by high affinity binding to a CACCC promoter/enhancer element. KLF2 was originally identified …

Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo

The vascular endothelium in vivo is a remarkably quiescent cell layer that displays a highly differentiated and tissue-specific phenotype. Once established in culture, endothelial cells (EC) are phenotypically different from their in situ counterparts, displaying altered gene expression, increased mitotic index, and decreased cell density. To determine whether manipulating the microenvironment of cells in vitro would lead to a more differentiated phenotype, we cultured bovine aortic EC on floating collagen gels. EC cultured to confluence on floating gels for 24 or 48 hr display mitotic indices nearly identical to those of quiescent endothelium in vivo, nearly two log orders lower than that of EC cultured to confluence on plastic, and cell density on floating gels also resembles that observed for endothelium in vivo. Culture of EC on floating gels leads to decreased expression of platelet-derived growth factor-B, fibronectin, and fibronectin isoform ED-B, and increased levels of connexin40, relative to cells cultured on plastic. We conclude that culture of bovine aortic EC under standard culture conditions results in a phenotype reminiscent of development and/or wound healing, and that culturing them on a floating collagen gel leads to a more differentiated phenotype, reminiscent of that observed for large vessel EC in vivo.