Heart Endothelial Cells Research Articles

Abstract 184: Pressure Overload Activates Left Ventricular (LV) Intramyocardial Vascular Endothelial Cells and induces T Cell Infiltration into the LV

Left ventricular dysfunction and Heart Failure (HF) are associated with systemic inflammation with clinical data showing that HF patients have higher levels of circulating pro-inflammatory cytokines. Recruitment of circulating T cells to tissues across the vascular endothelium is a key event in the inflammatory response, but whether it plays a role in the heart in HF is unknown. We hypothesized that pressure overload induced HF activates cardiac endothelial cells resulting in T cell recruitment into the left ventricle (LV). Using transverse aortic constriction (TAC), quantitative flow cytometry, immunohistochemistry, qPCR and real time live cell videomicroscopy, we examined mRNA and protein expression levels of endothelial cell adhesion molecules and the presence of T cell infiltrates in the LV in vivo , and also studied the T cell interactions with primary mouse heart endothelial cells (MHEC) under flow conditions in vitro , comparing Sham and TAC operated mice (6-10/group) during the course of HF. 48h after TAC, in the pre-hypertrophic state, no differences were observed in the recruitment of T cells in the LV. Interestingly, two and four weeks after TAC, when mice developed LVH and LV dysfunction (Fractional Shortening 25±13%), E-Selectin, VCAM-1 and ICAM-1 mRNA levels were significantly upregulated in the LV as compared to Sham mice (2.3, 2.8 and 4 fold, respectively), with notable enhancement of endothelial ICAM-1 protein levels in the LV intramyocardial vessels, and T cells infiltrated in the LV in response to TAC (P≤0.05 TAC vs Sham). Furthermore, T cells isolated from mice 2 and 4 weeks after TAC adhered to MHEC under flow conditions in significantly higher numbers than T cells from Sham mice (P≤0.01 TAC vs Sham). Systemically, the frequency of three different T cell subsets in the peripheral lymphoid organs was increased in TAC vs Sham mice, indicating activation of the adaptive immune response to pressure overload. Taken together, our studies indicate that activation of the heart vascular endothelium occurs in response to pressure overload resulting in T cell recruitment into the LV. Further studies will be needed to determine in the extent to which T cell recruitment into the heart contributes to the pathogenesis of HF.

HSPA12B and repairing the heart: beauty in simplicity

This editorial refers to ‘HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism’ by J. Li et al ., pp. 671–681, this issue. HSPA12B is a member of a newly identified subfamily of the Hsp70 family of heat shock proteins and is predominately expressed in endothelial cells.1,2 Blood pressure may play a role in its expression, as HSPA12B is expressed in endothelial cells in heart, adipose tissue, brain, kidney, and lung, but not in liver sinusoidal endothelial cells.1 In the heart and brain, vessels of all sizes express HSPA12B, whereas in lung and adipose tissue, expression is largely in capillaries. The cell-restricted expression of HSPA12B, which is otherwise not a feature of heat shock proteins, alone presages its importance to endothelial cell function and indeed studies have established that HSPA12B is required for angiogenesis, specifically in the processes of adhesion, migration, and tube formation.1,2 Endothelial cell HSPA12B expression seems to be dynamic and is increased in confluent HUVEC cultures kept in medium containing endothelial cell-specific growth factors, by heat shock, and during tubule formation.1–3 Protein levels are likely regulated by a post-transcriptional mechanism as well.1 Twenty-two putative client proteins have been identified for HSPA12B, including AKAP12 (A-kinase-anchoring protein 12) and hPODXL (human podocalyxin-like), both of which are implicated in cell adhesion, and aryl hydrocarbon receptor nuclear translocator (ARNT).1 HSPA12B also has protective actions in endothelial cells. Transgenic mice overexpressing the human hspa12b gene (including the endothelial cell-specific promoter) were found to be remarkably protected …

Biomarkers in Pulmonary Hypertension

Pulmonary hypertension (PH) is a hemodynamic condition that has a poor prognosis and can lead to right-sided heart failure. It may result from common diseases such as left-sided heart or lung disease or may present as the rare entity of idiopathic pulmonary arterial hypertension. Biomarkers that specifically indicate the pathologic mechanism, the severity of the disease, and the treatment response would be ideal tools for the management of PH. In this review, markers related to heart failure, inflammation, hemostasis, remodeling, and endothelial cell-smooth muscle cell interaction are discussed, and their limitations are emphasized. Anemia, hypocarbia, elevated uric acid, and C-reactive protein levels are unspecific markers of disease severity. Brain natriuretic peptide and N-terminal fragment of pro-brain natriuretic peptide have been recommended in current guidelines, whereas other prognostic markers, such as growth differentiation factor-15, osteopontin, and red cell distribution width, are emerging. Chemokines of the CC family and matrix metalloproteases have been linked to the vascular pathologic mechanisms, and new markers such as apelin have been described. Circulating endothelial and progenitor cells have received much attention as markers of disease activity, but with controversial findings. A lack of standards for cell isolation and characterization methods and differences in the pathologic mechanisms of the investigated patients may have contributed to the discrepancies. In conclusion, although several promising markers have been identified over the past few years, the development of more specific markers, standardization, and prospective validation are warranted.

Apelin elevates blood pressure in ICR mice with L-NAME-induced endothelial dysfunction

Apelin is the endogenous ligand of APJ, which belongs to the family of G protein-coupled receptors. Apelin and APJ are highly expressed in various cardiovascular tissues, including the heart, kidney and vascular endothelial and smooth muscle cells. Although apelin exerts hypotensive effects via activation of endothelial nitric oxide synthase (eNOS), the ability of apelin to regulate blood pressure under pathological conditions is poorly understood. In the current study, NG-nitro-L-arginine methyl ester (L-NAME), a potent NOS inhibitor, was administered chronically, to induce peripheral vascular damage in mice. L-NAME-treated mice exhibited hypertension, increased vascular cell adhesion molecule-1 and plasminogen activator inhibitor-1 mRNA levels in the aorta and impaired vasodilatation associated with decreased aortic eNOS expression, consistent with endothelial damage. Three days following withdrawal of L-NAME treatment, the blood pressure response to apelin stimulation was assessed. Although apelin reduced blood pressure in non-treated mice, it was found to transiently elevate blood pressure in L-NAME-treated mice. These results indicate that apelin functions as a vasopressor peptide under pathological conditions, including vascular endothelial dysfunction in mice.

Detection of EGHV-5 in a self-limiting papilloma-like lesion in the trunk of an Asian elephant (Elephas maximus)

HERPESVIRUS infections are widespread in captive populations of Asian elephants and result in major implications for elephant management. Molecular studies have thus far identified six distinct elephant gammaherpesviruses (EGHVs), and...

Cardiorenal syndrome: pathophysiology and potential targets for clinical management

Combined dysfunction of the heart and the kidneys, which can be associated with haemodynamic impairment, is classically referred to as cardiorenal syndrome (CRS). Cardiac pump failure with resulting volume retention by the kidneys, once thought to be the major pathophysiologic mechanism of CRS, is now considered to be only a part of a much more complicated phenomenon. Multiple body systems may contribute to the development of this pathologic constellation in an interconnected network of events. These events include heart failure (systolic or diastolic), atherosclerosis and endothelial cell dysfunction, uraemia and kidney failure, neurohormonal dysregulation, anaemia and iron disorders, mineral metabolic derangements including fibroblast growth factor 23, phosphorus and vitamin D disorders, and inflammatory pathways that may lead to malnutrition-inflammation-cachexia complex and protein-energy wasting. Hence, a pathophysiologically and clinically relevant classification of CRS based on the above components would be prudent. With the existing medical knowledge, it is almost impossible to identify where the process has started in any given patient. Rather, the events involved are closely interrelated, so that once the process starts at a particular point, other pathways of the network are potentially activated. Current therapies for CRS as well as ongoing studies are mostly focused on haemodynamic adjustments. The timely targeting of different components of this complex network, which may eventually lead to haemodynamic and vascular compromise and cause refractoriness to conventional treatments, seems necessary. Future studies should focus on interventions targeting these components.

325 PRODUCTION OF GAL KNOCKOUT/hA20 TRANSGENIC PIGS WITH IMPROVED XENOPROTECTIVE PROPERTIES

Pig-to-human xenotransplantation is promising for overcoming the shortage of suitable human donor organs, but is hampered by immunological barriers. The next immunological hurdle is the acute vascular rejection (AVR), which is associated with activation of the endothelium and the coagulation system. Recently, we demonstrated that transgenic expression of the zinc finger protein A20 protects porcine cells against apoptotic and inflammatory stimuli (Oropeza et al. 2009 Xenotransplantation 16, 522–534). Compared with other anti-apoptotic proteins, A20 also has immune-modulatory potential as shown in an CD95(Fas)Ligand assay. However, in that study, hA20 was only expressed in skeletal muscle, heart and porcine aortic endothelial cells of transgenic pigs. For use in xenotransplantation, it is critical to produce pigs with ubiquitous expression of hA20. Here, we constructed a new vector based on the Sleeping Beauty transposon plasmid pT2/HB containing the hA20 cDNA driven by the ubiquitously and strongly expressing CAGGS-promoter and co-transfected gal–/– porcine fibroblasts (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010) together with the SB transposase 100X plasmid (pT2/HB and SB transposase both kindly provided by Dr. Zoltan Ivics). Cells were selected with 400 µg of G418/mL of medium for 14 days. Subsequently, cells were screened by PCR. Transfected cell clones were pooled and used as donor cells in somatic cell nuclear transfer. Reconstructed embryos were transferred to 2 synchronized sows. Both remained pregnant on Day 25 of gestation. One recipient is expected to deliver in September 2012. The second sow, which received 104 embryos, was sacrificed on Day 26 of pregnancy and 2 fetuses could be obtained (cloning efficiency 1.92%). The fetuses had integrated the transgene into their genome as shown by PCR. Real-time PCR results from fetal fibroblasts indicated similar hA20 mRNA expression levels in both fetuses, whereas wild type controls were negative. The hA20 expression level was 2.5 and 3.1 times lower than in the original pooled of transfected cells. Fetus #1, which showed a slightly higher hA20 mRNA expression, was used for recloning. In total, 3 more recipients received an average of 104 hA20 transgenic embryos each. Currently, the hA20 protein level in fetal fibroblasts is determined by fluorescence activated cell sorting analysis. Once pigs are born, the tissue distribution of hA20 will be analysed. In parallel, the function of the transgene will be studied in the CD95(Fas)Ligand assay. Hearts and kidneys of the hA20-transgenic pigs will be further tested in ex vivo perfusion assays and in a pig-to-baboon xenotransplantation. This approach is promising for advancing pig-to-human xenotransplantation to preclinical application.

Abstract 255: The Kidney and Heart are Hemogenic Organs During Embryonic Life.

During early embryonic life, the heart starts to beat before an effective circulation is established, and the kidney starts to form its vasculature before it connects to the general circulation. We and others have shown a close lineage relationship between endothelial cells (ECs) and hematopoietic cells. In fact, during embryonic development erythroblasts bud from the endothelium of developing vessels, a process we termed hemovasculogenesis. Those studies suggested the possibility that embryonic organs may have hemogenic potential. To test this hypothesis, we performed lineage studies and colony forming unit (CFC) assays to trace the fate of hematopoietic stem cells (HSCs), erythroid cells, and ECs in heart and kidney from embryonic mice. Using ER-GFPcre mice that express both GFP and cre under control of the erythropoietin receptor locus in the erythroid cells, we identified hematopoietic progenitors (Hb+Nanog+) within nascent vessels in the early embryonic kidney and heart. Using EC-SCL-Cre-ERT transgenic mice that specifically express tamoxifen inducible Cre in EC progenitors, we found both circulating and non-circulating cells from the EC lineage in the early embryonic heart and kidney. CFC assays using HSC-SCL-Cre-ERT; mTmG mice (which express GFP in the cells from the HSC lineage upon tamoxifen induction) showed that both the embryonic kidney and heart possess HSCs. Further, transplantation studies of pre-vascular embryonic kidneys from EC-SCL-Cre-ERT;R26R mice under the kidney capsule of WT adult mice showed blood cells derived from the embryonic kidney suggesting that the embryonic kidney also possesses HSCs that originate in situ. These studies indicate that the embryonic kidney and heart function as hematopoietic organs during early embryogenesis. In addition to solve an important scientific controversy in our understanding of lineage/fate relationships in the developing embryo, these findings are relevant for tissue repair/regeneration and may help explain why under pathological circumstances, hematopoiesis occurs in extramedullary organs.

The relative contributions of cardiomyocytes versus endothelial cells to cardiac BH4 production; studies in novel cardiomyocyte- and endothelial-specific GTPCH I-deficient mice

BH4 is a critical cofactor for the NOS enzymes and is essential for NOS coupling and NO production. NO modulates cardiac function, although its exact role remains unclear. Recent advances have shown that both eNOS and nNOS are present in cardiomyocytes, yet evidence suggests that BH4 synthesis in myocytes is suboptimal. Currently, little is known about cardiac BH4, yet this information is essential to understand the roles of NOSs/BH4 in the heart. Using 2 novel floxed mouse models of cell-specific GTPCH I deficiency, the rate-limiting enzyme in de novo BH4 biosynthesis, we aimed to elucidate the relative contributions of myocytes and endothelial cells to cardiac BH4 levels. PCR confirmed cardiomyocyte- and endothelial-specific GCH1 excision in GCHfl/flMHCcre and GCHfl/flTie2cre mice, respectively, concomitant with depleted GTPCH1 protein and BH4 production in these cells. Whereas BH4 and total biopterins were significantly reduced in GCHfl/flTie2cre hearts compared to GCHfl/fl (0.55 ± 0.17 versus 1.88 ± 0.28 pmol BH4/mg protein, respectively), levels remained unchanged in GCHfl/flMHCcre hearts, indicating that myocytes are not a major contributor of cardiac biopterins. Furthermore, BH4 levels in isolated wild-type heart endothelial cells were >4-fold higher than in isolated cardiomyocytes (2.7 ± 0.97 versus 0.53 ± 0.23 mol/mg protein). However, despite low BH4 levels in wild-type cardiomyocytes, reducing this further in GCHfl/flMHCcre mice significantly increased heart:body weight. These studies demonstrate that endothelial cells, not cardiomyocytes, are a major source of cardiac biopterins. However, depleting BH4 specifically in myocytes elicits cardiac morphometric changes. Future studies will explore the impact of BH4 deficiency in these cell types on cardiovascular diseases.

Nerve growth factor: Does this have the potential to become a miraculous treatment for diabetic heart?

Nerve growth factor: Does this have the potential to become a miraculous treatment for diabetic heart?

A restricted subset ofvargenes mediates adherence ofPlasmodium falciparum-infected erythrocytes to brain endothelial cells

Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.

Effect of HSP65 on the Expression of Adhesion Molecules in Mice Heart Endothelial Cells

This study aims to research the effect of HSP65 on the expression of adhesion molecules in activated mice heart endothelial cells (MHECs), which were from myocardial tissue of newborn animals. We used different concentrations of LPS as potent inducers to stimulate MHECs, adhesion molecule expression in vitro, including intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), E-, and P-selectins, then compared the mRNA and protein levels of adhesion molecules expression with or without HSP65 treatment at different levels. The optimal concentration of LPS to induce MHECs adhesion molecule expression is 100ng/ml; HSP65 treatment significantly reduced the mRNA and protein levels of MHECs' ICAM-1, VCAM-1, E-, and P-selectins expression (p < 0.05), and the optimal concentration of HSP65 in inhibiting MHECs activation is 0.8ng. HSP65 has the inhibitory effect on adhesion molecules expression in activated MHECs.

Comparative studies of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1: evidence for a eutherian mammalian origin for the GPIHBP1 gene from an LY6-like gene

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) functions as a platform and transport agent for lipoprotein lipase (LPL) which functions in the hydrolysis of chylomicrons, principally in heart, skeletal muscle and adipose tissue capillary endothelial cells. Previous reports of genetic deficiency for this protein have described severe chylomicronemia. Comparative GPIHBP1 amino acid sequences and structures and GPIHBP1 gene locations were examined using data from several mammalian genome projects. Mammalian GPIHBP1 genes usually contain four coding exons on the positive strand. Mammalian GPIHBP1 sequences shared 41–96% identities as compared with 9–32% sequence identities with other LY6-domain-containing human proteins (LY6-like). The human N-glycosylation site was predominantly conserved among other mammalian GPIHBP1 proteins except cow, dog and pig. Sequence alignments, key amino acid residues and conserved predicted secondary structures were also examined, including the N-terminal signal peptide, the acidic amino acid sequence region which binds LPL, the glycosylphosphatidylinositol linkage group, the Ly6 domain and the C-terminal α-helix. Comparative and phylogenetic studies of mammalian GPIHBP1 suggested that it originated in eutherian mammals from a gene duplication event of an ancestral LY6-like gene and subsequent integration of exon 2, which may have been derived from BCL11A (B-cell CLL/lymphoma 11A gene) encoding an extended acidic amino acid sequence.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-011-0026-4) contains supplementary material, which is available to authorized users.

470 Hypoxia-induced pulmonary hypertension in mice modifies the expression pattern of von willebrand factor gene in lung endothelial cells

470 Hypoxia-induced pulmonary hypertension in mice modifies the expression pattern of von willebrand factor gene in lung endothelial cells

Endothelial protein C receptor in renal tubular epithelial cells and influencing factors

The endothelial protein C receptor (EPCR) plays an important role within the protein C pathway in regulating coagulation and inflammation. It was reported that EPCR was expressed in large vessels, placenta, heart, liver and lung endothelial cell. However, there are a few studies concerned about renal epithelial cells. This study aims to investigate EPCR expression in renal tubular epithelial cells and related influencing factors. EPCR expression was assessed by flow cytometry in renal tubular epithelial cells. The effects of some reagents (high glucose, tumor necrosis factor–α and interleukin-1β) were measured by RT-PCR. The results showed that renal tubular epithelial cells had the high expression of EPCR level. High glucose, tumor necrosis factor–α and interleukin-1β might reduce EPCR expression. And troglitazone could significantly improve the inhibition. In conclusion, we found EPCR expression in renal tubular epithelial cells in vitro . Some factors such as high glucose, tumor necrosis factor–α and interleukin-1β can impact on EPCR. However, troglitazone had protective effects of EPCR on injured cells. Key words: Endothelial protein C receptor, renal tubular epithelial cell, troglitazone, tumor necrosis factor-α, interleukin-1β; high glucose.

Detection of EGHV-5 in a self-limiting papilloma-like lesion in the trunk of an Asian elephant (Elephas maximus)

HERPESVIRUS infections are widespread in captive populations of Asian elephants and result in major implications for elephant management. Molecular studies have thus far identified six distinct elephant gammaherpesviruses (EGHVs), and...

Endothelial C-reactive protein increases platelet adhesion under flow conditions

While data regarding the pathogenetic role of C-reactive protein (CRP) in atherothrombosis are accumulating, it is still controversial whether local CRP secretion is of any pathobiological significance. The present study examined whether endothelial-derived CRP modulates autocrine prothrombotic activity. Endothelial cells were isolated from hearts of mice transgenic to human CRP and grown in primary cultures. Human CRP expression was confirmed in these cells compared with no expression in cultures derived from wild-type congenes. Adhesion of human platelets to endothelial cells was studied in the "cone and plate" flow system. Platelet adhesion to cells expressing CRP was significantly increased compared with that in controls (n = 6, P < 0.01). The proadhesive effect of CRP was significantly suppressed in mouse heart endothelial cells and in human umbilical vein endothelial cells following treatment with small interfering RNA for human CRP. Adhesion was modulated by an increase in P-selectin. P-selectin expression correlated with a proadhesive phenotype, and blocking P-selectin with neutralizing antibody significantly decreased the adhesion of platelets to CRP-expressing cells (40.4 ± 10.5 to 9.4 ± 6.9 platelets/high-power field, n = 5 to 6, P < 0.01). In conclusion, human CRP that is locally produced in endothelial cells increases platelet adhesion to endothelial cells under normal shear flow conditions. These findings indicate that CRP exerts a local effect on endothelial cells via P-selectin expression, which promotes platelet adhesion and subsequent thrombus formation.

Regulation of Endothelial Cell Adhesion Molecule Expression by Mast Cells, Macrophages, and Neutrophils

BackgroundLeukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined.Methods and ResultsUsing bone marrow-derived mast cells from wild-type, Tnf−/−, Ifng−/−, Il6−/− mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC.ConclusionMast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases.

O22. Cardiovascular derivatives of embryonic stem cells in cardiac repair and drug discovery

Derivation of heart and vascular endothelial cells from human pluripotent stem cells (embryonic stem cells or HESCs and induced pluripotency stem cells or hiPS cells) is an area of growing interest both as a route to cell therapy for the heart and as a platform for drug discovery and toxicity. Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to cardiac progenitors and their derivatives and mimicking these in defined culture conditions in vitro is now essential for moving the field forward. Culture conditions have now been sufficiently refined that cardiomyocyte and vascular differentiation is a fairly efficient and reproducible process. Genetically marked HESCs have been produced in which expression of the green fluorescent protein marker is expressed ubiquitously or driven by specific lineage markers. We have used these tagged lines to trace cardiomyocytes following transplantation into a mouse heart after myocardial infarction and select the progenitors of cardiomyocytes, endothelial cells and smooth muscle cells. Long term survival of the cells and integration into the host heart has been observed and early improvements in cardiac function but these are not sustained. Cardiac repair using stem cell derived cardiomyocytes will likely require more than efficient cardiomyocytes production. More immediate applications of hESC- and hiPSC derived cardiomyocytes and vascular endothelial cells in drug discovery and disease are now close to implementation. Results of these studies, in particular drug responses of hESC-derived cardiomyocytes and an hiPSC model for vascular disease in which Thalidomide has a therapeutic effect, will be shown.

Repressors NFI and NFY Participate in Organ-Specific Regulation of von Willebrand Factor Promoter Activity in Transgenic Mice

To determine the role of repressors in cell type and organ-specific activation of von Willebrand factor (VWF) promoter sequences -487 to 247 in vivo. Activation patterns of wild-type and mutant VWF promoters (sequences -487 to 247) containing mutations in repressors nuclear factor-I (NFI)- and nuclear factor Y (NFY)-binding sites were analyzed in transgenic mice. Mutation of the NFI-binding site activated the promoter in heart and lung endothelial cells, whereas mutation of the NFY-binding site activated the promoter in kidney vasculature. Immunofluorescence analyses showed that NFIB was predominant in heart and lung endothelial cells, whereas NFIX was predominantly detected in kidney endothelial cell nuclei. By using chromatin immunoprecipitation, we demonstrated that the distal lung-specific enhancer (containing a YY1 site) of the VWF gene is brought in proximity to the NFI binding site. The NFI and NFY repressors contribute differentially to organ-specific regulation of the VWF promoter, and the organ-specific action of NFI may reflect its organ-specific isoform distribution. In addition, the lung-specific enhancer region of the endogenous VWF gene may inhibit NFI repressor function through chromatin looping, which can approximate the 2 regions.