Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 2006;114:2288-2297
Published online before print November 6, 2006, doi: 10.1161/CIRCULATIONAHA.106.639161
CLINICAL PERSPECTIVE
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
114/21/2288    most recent
CIRCULATIONAHA.106.639161v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Laake, L. W.
Right arrow Articles by Mummery, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Laake, L. W.
Right arrow Articles by Mummery, C. L.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Genetics Home Reference
Medline Plus Health Information
*Heart Attack
Related Collections
Right arrow Angiogenesis
Right arrow Animal models of human disease
Right arrow Genetically altered mice
Right arrow CT and MRI
Right arrow Acute myocardial infarction
Right arrow Endothelium/vascular type/nitric oxide

(Circulation. 2006;114:2288-2297.)
© 2006 American Heart Association, Inc.

Vascular Medicine

Endoglin Has a Crucial Role in Blood Cell–Mediated Vascular Repair

Linda W. van Laake, MD*; Sander van den Driesche, PhD*; Simone Post, MD; Alie Feijen, MS; Maurits A. Jansen, PhD; Mariette H. Driessens, PhD; Johannes J. Mager, MD; Repke J. Snijder, MD; Cornelius J. J. Westermann, MD, PhD; Pieter A. Doevendans, MD, PhD; Cees J. A. van Echteld, PhD; Peter ten Dijke, PhD; Helen M. Arthur, PhD; Marie-José Goumans, PhD; Franck Lebrin, PhD{dagger}; Christine L. Mummery, PhD{dagger}

From Hubrecht Laboratory (S.v.d.D., L.W.v.L., A.F., M.H.D., F.L., C.L.M.), Utrecht, the Netherlands; Laboratory of Experimental Cardiology, Department of Cardiology (L.W.v.L., S.P., P.A.D., M.J.G.) and NMR Laboratory (M.A.J., C.J.A.v.E.), Heart Lung Center, Utrecht, the Netherlands; St Antonius Hospital, Department of Pulmonary Disease (J.J.M., R.J.S., C.J.J.W.), Nieuwegein, the Netherlands; Leiden University Medical Center, Department of Molecular Cell Biology (P.t.D.), Leiden, the Netherlands; Institute of Human Genetics, International Centre for Life, University of Newcastle (H.M.A.), Newcastle Upon Tyne, UK; Interuniversity Cardiology Institute of the Netherlands (S.P., M.A.J., P.A.D., C.L.M.), Utrecht, the Netherlands; and INSERM Unit 525, Faculté de Médecine, Hôpital Pitié-Salpêtrière (F.L.), Paris, France.

Correspondence to C.L. Mummery, Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, Netherlands (e-mail Christin{at}niob.knaw.nl); or F. Lebrin, INSERM U525, Faculté de Médecine, Hôpital Pitié-Salpêtrière. Université Pierre et Marie Curie-Paris, 6. 91 Boulevard de l’Hôpital, 75013 Paris, France (e-mail franck.lebrin@chups.jussieu.fr).

Received October 24, 2005; de novo received May 9, 2006; revision received September 13, 2006; accepted September 15, 2006.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowConclusions
 down arrowReferences
 
Background— Endoglin, an accessory receptor for transforming growth factor-ß in vascular endothelial cells, is essential for angiogenesis during mouse development. Mutations in the human gene cause hereditary hemorrhagic telangiectasia type 1 (HHT1), a disease characterized by vascular malformations that increase with age. Although haploinsufficiency is the underlying cause of the disease, HHT1 individuals show great heterogeneity in age of onset, clinical manifestations, and severity.

Methods and Results— In situ hybridization and immunohistochemical analysis of mouse and human hearts revealed that endoglin is upregulated in neoangiogenic vessels formed after myocardial infarction. Microvascularity within the infarct zone was strikingly lower in mice with reduced levels of endoglin (Eng+/–) compared with wild-type mice, which resulted in a greater deterioration in cardiac function as measured by magnetic resonance imaging. This did not appear to be because of defects in host inflammatory cell numbers in the infarct zone, which accumulated to a similar extent in wild-type and heterozygous mice. However, defects in vessel formation and heart function in Eng+/– mice were rescued by injection of mononuclear cells from healthy human donors but not by mononuclear cells from HHT1 patients.

Conclusions— These results establish defective vascular repair as a significant component of the origin of HHT1. Because vascular damage or inflammation occurs randomly, it may also explain disease heterogeneity. More generally, the efficiency of vascular repair may vary between individuals because of intrinsic differences in their mononuclear cells.


Key Words: angiogenesis • genetics • magnetic resonance imaging • myocardial infarction • vessels


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowConclusions
 down arrowReferences
 
Revascularization of injured, ischemic and regenerating organs is essential to restore organ function. Neovascularization resulting from the proliferation, migration, and remodeling of terminally differentiated endothelial cells (ECs) from preexisting blood vessels is called angiogenesis.1 Evidence suggests that mononuclear cells (MNCs), which include endothelial progenitor cells, circulating ECs, and bone marrow monocytic lineages, home to sites of ischemic damage and contribute to new blood vessel formation through vasculogenesis, transdifferentiation into ECs, and secretion of cytokines that stimulate neoangiogenesis.2,3

Clinical Perspective p 2297

Hereditary hemorrhagic telangiectasia (HHT; or Rendu-Osler-Weber syndrome) is an autosomal-dominant disorder with low prevalence, estimated to be 1 in 10 000.4 It is characterized by epistaxis, telangiectases, and multiorgan vascular dysplasia.4,5 Two variants of HHT, HHT1 and HHT2, have been linked to mutations in the ENG (endoglin) and ACVRL1 (activin receptor-like kinase 1 or ALK1) genes, respectively,6,7 although recently, 2 more genes have been implicated.8,9 Endoglin is highly expressed by active ECs from most blood vessels.10 Deletion of endoglin in mice revealed its critical role during cardiovascular development. Mutant endoglin (Eng–/–) mice die at embryonic day 10.5 as a result of defects in vessel and heart development. Vasculogenesis in the Eng–/– mice is normal but angiogenesis is impaired, along with the remodeling of the primary vascular plexus.11,12 Endoglin is an accessory receptor for transforming growth factor-ß (TGF-ß),13,14 a cytokine controlling proliferation, migration, adhesion, and apoptosis of many cell types.15 The activated form of TGF-ß binds to type II receptors (TßRIIs), which recruit TGF-ß type I serine/threonine kinase receptors to the complex and propagate signals to the nucleus by phosphorylating intracellular effectors called Smads.16,17 Two type I receptors mediate TGF-ß signaling: ALK1, expressed mainly in endothelium, and ALK5, expressed widely in most cell types. In ECs, TGF-ß/ALK5 signaling via Smad2/3 leads to inhibition of cell migration and proliferation, whereas TGF-ß/ALK1 signaling via Smad1/5/8 promotes cell migration and proliferation.18 Endoglin and ALK1 are thought to act in a common pathway and inhibit TGF-ß/ALK5 signaling indirectly. The combined effect promotes the activation phase of angiogenesis during which vascular permeability increases, basement membrane is degraded, and ECs proliferate and migrate.19,20

Several studies have implied haploinsufficiency as the mechanism responsible for HHT and indicated that disease heterogeneity is not related to the position or type of mutation21,22; all mutations in the ENG gene result in reduced functional cell surface protein and deregulation of TGF-ß signaling pathways.4,23 Individuals with HHT1 show great variability in age of disease onset, clinical manifestations, and severity, however, both within and between families with the same mutation. In addition, epigenetic factors, including exposure to ultraviolet light and local inflammation, have been implied as contributing to its diversity, suggesting that HHT is a complex genetic disorder.24

Here, we used experimental myocardial infarction (MI) in wild-type and endoglin-heterozygous mice to investigate the effects of the HHT1 mutation on angiogenesis and vasculogenesis that are integral components of the remodeling after MI.25 Thus, experimental MI represents a useful model for studying these processes in normal and mutant adult mice. In addition, because cardiac biopsies are available from adult humans after MI, there are opportunities to translate the findings in mice to human disease.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowConclusions
 down arrowReferences
 
Mice and Coronary Artery Ligation
Endoglin mice contain a ß-galactosidase reporter cassette in the disrupted locus.11 Analyses were carried out on wild-type (Eng+/+) and endoglin-heterozygous transgenic mice (Eng+/–) from a C57Bl/6J genetic background. Balb/c mice also were used for injection of human MNCs via the tail vein.26 MI was induced as described.27 Briefly, adult mice bred in our laboratory (Hubrecht Laboratorium, Utrecht, The Netherlands; 20 to 35 g) were intubated and ventilated with 2% isoflurane/98% oxygen. The left coronary artery was exposed via a left thoracotomy and opening of the pericardium and occluded just below the inferior border of the left auricle with a 7-0 prolene ligature. Sham-operated mice underwent similar operations without coronary artery ligation. Animal experiments were approved by the Animal Care Committee.

Isolation, Culture, and Injection of Human MNCs
Blood samples from healthy human volunteers or HHT1 patients were collected in potassium/ethylenediaminetetraacetic acid tubes (Monovette, Sarstedt, Nümbrecht, Germany). MNCs were isolated by density gradient centrifugation with histopaque-1077 (Sigma-Aldrich, St Louis, Mo) following the manufacturer’s protocol and washed twice with phosphate-buffered saline (PBS) containing 2 mmol/L ethylenediaminetetraacetic acid. Cells (10x106) were plated in fibronectin-coated plates and cultured in EGM2 (Clonetics, Cambrex, East Rutherford, NJ) with 20% fetal calf serum and incubated in 5% CO2 at 37°C. Patient procedures were approved by the Medical Ethics Committee at St Antonius Hospital. The investigation conformed to principles in the Declaration of Helsinki.

For MNC injection, mice underwent MI as above, received 5x106 human MNCs in 40 µL PBS via a tail vein injection 1 to 3 hours later, and were immunosuppressed with tacrolimus (5 mg · kg–1 · d–1) subcutaneously. Mice were divided randomly into groups for follow-up at 4, 7, 14, or 30 days to assess homing of human MNCs to the infarct area, angiogenesis, and cardiac function.

Analyses of Myocardial Function by Magnetic Resonance Imaging
Cardiac- and respiratory-triggered cine magnetic resonance images were acquired on a 9.4-T scanner (Bruker Biospin GmbH, Rheinstetten, Germany) using a birdcage radiofrequency coil. A gradient-echo pulse sequence was used to acquire data with a repetition time of 9.8 ms, an echo time of 1.9 ms, a matrix of 256x256, a field of view of 3.0x3.0 cm, a slice thickness of 1 mm, a flip angle 18°, and 4 signal averages. The number of phases was 11 to 13, depending on heart rate. Seven to 8 short-axis slices were needed to image the entire left ventricle. Images were processed with dedicated imaging software (CAAS-MRV, Pie Medical Imaging BV, Maastricht, the Netherlands).

Isolation and Processing of Hearts
The hearts were dissected from euthanized mice 7, 14, or 30 days after MI, fixed overnight in 4% paraformaldehyde in PBS, washed twice in 0.83% NaCl, and washed once each in 0.42% NaCl, 50% ethanol, and 70% ethanol, all overnight at 4°C. After paraffin embedding, hearts were sectioned (6 µm) onto coated slides (Klinipath, Duiven, the Netherlands) and stored at 4°C. For MNC homing experiments, hearts were isolated 4 days after MI and processed for cryosections as described.28 Human fetal hearts were collected after elective abortion and with informed consent as previously.29 Biopsies from adult human cardiac tissue were obtained from the Pathology Department after autopsy. Formalin-fixed samples were embedded in paraffin, and 6-µm sections were used for immunohistochemical analysis.

RNA Isolation and Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA from dissected atria and ventricles was isolated using TRIzol Reagent (Invitrogen, Carlsbad, Calif) following manufacturer’s instructions. Samples were treated with DNAse I to eliminate genomic DNA, and 1 µg RNA was reversed transcribed. Real-time polymerase chain reaction (PCR) was performed in an MyiQ single-color real-time detection system (Bio-Rad Laboratories, Hercules, Calif). Samples were normalized with glyceraldehyde phosphate dehydrogenase.

Immunohistochemistry and Immunofluorescence on Sections
Paraffin sections were stained with the tyramide signal amplification biotin system (Perkin Elmer, Life Science, Wellesley, Mass). Briefly, sections were treated with 0.25% trypsin in 9 mmol/L CaCl2/50 mmol/L Tris-HCl, pH 7.8, for 30 minutes at room temperature for antigen retrieval. Primary antibodies were rat anti-mouse platelet endothelial cell adhesion molecule (PECAM) (Clone MEC13.3, dilution 1:100, BD Biosciences, San Jose, Calif), rat anti-mouse endoglin (Clone 2Q1707, dilution 1:100, USBiological, Swampscott, Mass), rat anti-mouse CD68/macrosialin (Clone FA-11, dilution 1:100, Serotec Ltd, Kidlington, Oxfordshire, UK), rat anti-mouse Mac-3 (Clone M3/40, dilution 1:100, BD Pharmingen, San Diego, Calif), and rat anti-CD45 (Clone 30F11.1, dilution 1:100, BD Pharmingen) incubated overnight at 4°C. Biotin-conjugated goat anti-rat IgG (1:250, Dako, Glostrup, Denmark) was used as a secondary antibody incubated for 1 hour at room temperature. Peroxidase activity was detected with 3,3'-diaminobenzidine tablet set (Fast DAB, Sigma) following manufacturer’s instructions.

Paraffin sections used for Ki67 immunohistochemistry were treated as described previously.30 Primary antibody was mouse anti-Ki67 (Clone MM1, dilution 1:500, Monosan, Uden, the Netherlands) incubated overnight at 4°C. Secondary antibody was goat anti-mouse PowerVision Poly-HRP-Conjugates (ImmunoVision Technologies, Springdale, Ark) incubated for 30 minutes at room temperature. Peroxidase activity was detected with 3,3'-diaminobenzidine tablet set (Fast DAB, Sigma) following manufacturer’s instructions. Sections were counterstained with hematoxylin, dehydrated, and mounted in DePex.

Cryosections of hearts containing human MNC-derived ECs were fixed in acetone for 10 minutes at 4°C, dried for 30 minutes at room temperature, permeabilized for 5 minutes with 0.2% Triton X-100 in PBS, and blocked with 2% bovine serum albumin in PBS at room temperature for 1 hour. Slides were then incubated with rat anti-mouse PECAM antibody (Clone MEC 13.3, dilution 1:100, BD Biosciences) overnight at 4°C, washed 4 times in PBS, and incubated 1 hour simultaneously with goat anti-rat Cy3 (Jackson ImmunoResearch Laboratories, West Grove, Pa) and Ulex europaeus agglutinin-1 (UEA-1) lectin coupled to fluorescein isothiocyanate (1:100 dilution from 1 mg/mL stock, Sigma) diluted in 2% bovine serum albumin in PBS. Cryosections of human fetal hearts were incubated with UEA-1 and with goat anti-human PECAM antibody (Clone M-20, 1:100 dilution, Santa Cruz Biotechnology, Inc, Santa Cruz, Calif). The slides were then washed 4 times in PBS and mounted in Mowiol before confocal laser microscope analysis.

In Situ Hybridization
The endoglin probe was generated from a BamHI fragment (266 to 1039 bp) from full-length mouse endoglin (clone pCDNA1-7/18) cloned into Bluescript and linearized with XmaI. The antisense RNA probe was generated by transcription of the T7 RNA polymerase in the presence of [{alpha}35S]-UTP (Amersham, Uppsala, Sweden). Autoradiography was performed with Ilford photo emulsion. The slides were exposed for 1 to 2 weeks at 4°C. Photography consisted of combining bright-field (blue filter) and dark-field (red filter) images.

Statistical Analysis
Statistical significance was evaluated with the Mann-Whitney U test for comparison between 2 groups and the median test for multiple group comparisons using SPSS version 11.5 for Windows. Results are expressed as median±interquartile range. A value of P<0.05 or P<0.01 denoted statistical significance.

The authors had full access to the data and take responsibility for their integrity. All authors have read and agree to the manuscript as written.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowConclusions
 down arrowReferences
 
We examined endoglin expression by in situ hybridization and immunohistochemistry in hearts of normal adult mice (Figure 1A through 1E) and 1 week after coronary artery ligation–induced MI (Figure 1F through 1I). Endoglin mRNA was expressed predominantly in the atria but was detectable at low levels in the ventricles of normal hearts (Figure 1B and 1C). One week after MI, expression was strongly increased in the infarcted area compared with healthy tissue in sham-operated hearts (Figure 1D and 1H). Staining of comparable sections from sham-operated and MI hearts with anti-PECAM and anti-endoglin antibodies 1 week postoperatively revealed overlapping protein expression, indicating that endoglin is expressed only in ECs in the infarct zone and not in myofibroblasts (Figure 1I), as previously described,30 nor in vessels in the noninfarcted zone (Figure 1E). Neoangiogenesis within the scar is an integral component of the remodeling process occurring after MI.31,32 To compare the prevalence of activated blood vessels in hearts from sham-operated mice, sections from healthy noninfarcted zones and corresponding infarcted zones of adult mice 1 week after MI were stained with antibodies against Ki67, a marker for cycling cells, PECAM, and endoglin. In both the sham-operated hearts and the noninfarcted zone 1 week after MI, 5% to 8% of blood vessels were Ki67+. In contrast, {approx}50% of blood vessels were Ki67+ in the infarct zone (Figure 2A). Staining of sections from biopsies of human hearts with MI taken from within and outside the infarct zone showed similar differences in the prevalence of activated vessels (Figure 2B).


Figure 1179369
View larger version (85K):
[in this window]
[in a new window]
  		Figure 1. Upregulation of endoglin expression after MI. A–E, Endoglin expression in normal (sham-operated) adult mouse heart. A, Paraffin section of mouse heart. B, Real-time PCR for endoglin using left atrium (LA), right atrium (RA), left ventricle (LV), and right ventricle (RV) cDNA. Samples were normalized with glyceraldehyde phosphate dehydrogenase primers. Note the relatively low expression of endoglin in the LV and RV. C, Radioactive in situ hybridization for endoglin showing enhanced endoglin expression in atrium (arrow). D–E, High magnification of the LV. D, Top, AZAN-Mallory staining showing viable tissue (red/magenta). D, Bottom, Radioactive in situ hybridization (endoglin mRNA red) and (E) endoglin are not detectable immunohistochemically in the LV of adult heart. F–I, Endoglin expression upregulated in the infarcted zone after MI. F, Macroscopic view of the heart 1 week after MI. G, Paraffin section showing dilation of LV 1 week after MI. H, Top, AZAN-Mallory staining showing infarcted area (blue). H, Bottom, Radioactive in situ hybridization and (I) immunohistochemistry showed increased endoglin expression (arrow) in infarcted zone of adult heart 1 week after MI. E, I, PECAM staining as a marker for capillaries and blood vessels.


Figure 2179369
View larger version (89K):
[in this window]
[in a new window]
  		Figure 2. Endoglin is upregulated in active endothelium. A, Upregulation of endoglin associated with neoangiogenesis after MI. PECAM in all vessels (top); endoglin specifically in the infarct zone (right). Ki67+ ECs associated with endoglin-positive vessels of the infarct zone (bottom). B, Endoglin and Ki67 immunohistochemistry of infarcted human hearts shows upregulation of endoglin associated with neoangiogenesis. Black arrows show Ki67+ cells.

Because our data indicated that endoglin expression was highly associated with sites of active neoangiogenesis in both mice and humans, we used endoglin-heterozygous (Eng+/–) mice to investigate its function in adult neovascularization. Eng+/– mice survive into adulthood but have reduced endoglin levels and can develop HHT symptoms.33 Hearts from Eng+/+ and Eng+/– mice were stained for endoglin after MI. This staining revealed that expression was increased in both groups in the infarct zone but more in Eng+/+ mice as previously reported (Figure 3A).34 We also characterized the inflammatory cell accumulation in the infarct zones 1 week after MI by staining with antibodies against CD68 and Mac-3 for macrophages or with anti-CD45 for polymorphonuclear leukocytes (Figure 3B). This revealed no significant differences in the number of immunoreactive cells in the infarct zones of Eng+/– mice compared with Eng+/+ mice (Figure 3C through 3E). In contrast, although we found a slight increase in the basal number of vessels in Eng+/– mice (online Data Supplement, Figure IA), the number of vessels in the infarct zone of Eng+/– mice was significantly lower than in Eng+/+ mice 1 week after MI (657±39 versus 1138±86 vessels per 1 mm2; Figure 3F), suggesting that defects in angiogenesis occur during cardiac remodeling as a result of reduced endoglin levels.


Figure 3179369
View larger version (93K):
[in this window]
[in a new window]
  		Figure 3. Inflammatory cell accumulation and angiogenesis in Eng+/– mice. A, Representative examples of AZAN-Mallory and endoglin staining of Eng+/+ (left) and Eng+/– (right) adult mouse heart 1 week after MI. Endoglin protein expression was reduced in Eng+/– hearts. B, Paraffin sections immunostained for PECAM, CD68, Mac-3, and CD45. Accumulation of immunoreactive cells, quantified as number (+) of cells per 1 mm2, did not significantly change between wild-type and Eng+/– hearts (C–E). The total number of vessels per 1 mm2 was reduced in the Eng+/– vs Eng+/+ hearts 1 week after MI in the infarct area (blue area of AZAN staining).

We next analyzed heart function in these mice by magnetic resonance imaging (MRI). There were no differences between Eng+/+ and Eng+/– mice before MI or 1 week after MI (Figure 4B, supplementary Figure IB and IC). The survival curves of the Eng+/+ and Eng+/– mice also were identical (65% versus 67%, respectively; P=0.979; supplementary Figure IIA). However, MRI analysis 1 month after MI showed that stroke volume index, cardiac index, and ejection fraction were significantly lower in Eng+/– mice compared with Eng+/+ littermates (0.914±0.286 versus 1.348±0.556 mL/kg, P=0.001; 0.426±0.097 versus 0.7±0.219 L · min–1 · kg–1, P=0.001; 16.7±14.6% versus 35.3±16.0%, P=0.001, respectively; Figure 4A through 4E). The number of vessels also was lower in Eng+/– compared with Eng+/+ mice (260153 versus 476.7±75.2 vessels per 1 mm2, P=0.004; Figure 4F), indicating that neoangiogenesis defects in Eng+/– mice were associated with markedly greater deterioration in cardiac function after MI.


Figure 4179369
View larger version (46K):
[in this window]
[in a new window]
  		Figure 4. Injection of healthy donor but not HHT1 MNCs into Eng+/– mice with acute MI rescues neoangiogenesis and deterioration in cardiac function associated with reduced endoglin. A, MRI analysis of Eng+/+ and Eng+/– mice 4 weeks after MI. B, Analysis of heart function of Eng+/+ and Eng+/– mice without and 1 month after MI. C–E, Cardiac functions of Eng+/– mice are reduced vs Eng+/+ mice. E, F, After healthy donor but not HHT1 MNC injection, ejection fraction is improved (E) and neoangiogenesis is rescued (F) in Eng+/– mice. HR indicates heart rate; EDV, end-diastolic volume; ESV, end-systolic volume; EDVI, end-diastolic volume index; ESVI, end-systolic volume index; SV, stroke volume; SVI, stroke volume index; CO, cardiac output; CI, cardiac index; MM, myocardial mass; and EF, ejection fraction.

In the light of evidence that MNCs contribute to the formation of new blood vessels, we investigated whether MNCs might contribute to these angiogenesis defects by injecting MNCs from healthy human donors and MNCs from HHT1 patients into the tail veins of Eng+/– mice after MI. For ENG mutations of the patients, see supplementary Tables I and II. Previous analysis of endoglin protein levels in affected patients strongly supported haploinsufficiency and associated reduced levels of functional protein as the underlying cause of HHT1.4 This suggested that disease heterogeneity cannot be explained by the position and type of mutations.21,22 The survival curves of the injected and noninjected groups were identical (supplementary Figure IIA). MRI analysis 1 month later revealed that MNCs from healthy human donors significantly improved heart function of Eng+/– mice (ejection fraction, 16.7±14.6% versus 34.9±3.4%; P=0.004; Figure 4B and 4E) and stimulated neoangiogenesis (260±153 versus 460±89.5 vessels per 1 mm2; P=0.003; Figure 4F), whereas MNCs from HHT1 patients had no effect (ejection fraction, 16.7±14.6% versus 18.8±10.8%; P=0.749; Figure 4B and 4E; and 260±153 versus 305.8±55.0 vessels/mm2; P=0.317; respectively; Figure 4F). To confirm the difference in the ability of MNCs from healthy donors and HHT1 patients to contribute to vascular repair, we injected MNCs intravenously into wild-type mice 1 to 3 hours after MI. We found that 75% of mice receiving PBS vehicle alone, 89% receiving healthy donor MNCs, and 81% receiving HHT1 MNCs recovered normally from the procedure (supplementary Figure IIB). The number of vessels in the infarct zone was determined 14 days after MI. Vessel formation was efficiently stimulated by intravenous injection of MNCs from healthy donors (271.8±106.5 versus 418±265.8 vessels per 1 mm2; P=0.028; Figure 5A). However, MNCs from HHT1 patients showed consistently impaired ability to stimulate neoangiogenesis (269.8±181.2 versus 271.8±106.5 vessels per 1 mm2; P=0.884; Figure 5A), even though fluorescent-activated cell sorter analysis of blood from the patients and control subjects used for injection into the mice indicated no difference in the number of CD34+ cells or the number of CD34+/KDR+ cells. In this respect, the composition of control and patient blood samples was identical. In addition, there was no difference in cell numbers in MNC cultures from patients versus controls after 8 days. Because endoglin is expressed in vascular and various hematopoietic lineages, MNC-derived ECs homing to the infarct zone were traced by costaining with mouse-specific anti-PECAM antibody and human UEA-1 lectin, a marker commonly used for human cells with endothelial characteristics (Figure 5B).35 MNCs homed only to the infarcted zone, as previously reported (Figure 5C).25,26 The number of UEA-1+ cells that accumulated in the infarct zone of mice receiving cells from HHT1 patients was significantly lower than in mice receiving cells from healthy donors (4.01±12.33 versus 32.4±26.7 MNC-derived ECs per 1 mm2; P=0.003; Figure 5D). These MNC defects did not seem to depend on obvious differences in their ability to attach (Figure 5E), survive, or proliferate (S. Post, M.J. Goumans, unpublished observations), at least as determined in vitro on fibronectin-plated wells. In addition, we found no changes in expression of any major components of the TGF-ß signaling pathway (Figure 5E).


Figure 5179369
View larger version (27K):
[in this window]
[in a new window]
  		Figure 5. MNCs from HHT1 patients fail to stimulate neoangiogenesis. A, Number of vessels in the infarcted zone of hearts of Balb/c mice 14 days after MI was determined by counting PECAM+ vessels after immunohistochemical staining. B, UEA-1 lectin–coupled fluorescein isothiocyanate stains ECs of human fetal heart and not other cell types. C, MNC-derived ECs were only in the infarcted zone. D, MNCs from HHT1 patients show impaired homing to infarcted mouse hearts. The number of MNC-derived ECs per 1 mm2 in hearts of Balb/c mice 4 days after MI was determined by counting UEA-1+ cells. E, MNCs from healthy donors and HHT1 patients showed no differences in the number of UEA-1+ and Ac Dil-LDL+ cells attached to fibronectin-coated plates after 8 days. Expression of TGF-ß signaling pathway components in HHT1 MNCs were comparable to those in healthy donor MNCs.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowConclusions
 down arrowReferences
 
Neovascularization is a normal component of remodeling that occurs after MI. Its promotion has been proposed as an important target for therapeutic improvement of heart function.36 However, the mechanisms underlying neovascularization after MI are still not fully understood, although signaling pathways activated by TGF-ß, vascular endothelial growth factor, and insulin-like growth factor are thought to be involved.37 Here, we examined the function of endoglin, a TGF-ß receptor involved in angiogenesis during development. We demonstrated that endoglin has a crucial role in normal remodeling after MI that coincides with upregulation of its major ligand, TGF-ß, in myofibroblasts in the infarct zone.38 Specifically, upregulation of endoglin in ECs, possibly stimulated by local TGF-ß,39 correlates with neoangiogenesis. Furthermore, Eng+/– mice showed impaired angiogenesis after MI, which resulted in enhanced deterioration of cardiac function; this confirmed recent findings using an ischemic hind-limb injury model.34 Our results are consistent with reports demonstrating that endoglin is a marker of angiogenesis,40 that endoglin activates TGF-ß/ALK1 signaling and inhibits TGF-ß/ALK5 signaling to promote EC proliferation,20 and that downregulation of endoglin expression induces EC apoptosis.41 More generally, impaired angiogenesis in Eng+/– mice may be mediated at least in part by EC defects.

Compelling evidence indicates that recruitment of circulating vascular and hematopoietic cells contributes to the revascularization of ischemic tissues.42 Because endoglin is expressed in various cell lineages making up the MNC fraction, we investigated the effects of reduced endoglin expression in these circulating cells using experimental MI in mice as a model system. With respect to the disease HHT1, the present study provides the first evidence that MNCs derived from patients are impaired in their capacity to stimulate vessel formation. Moreover, injection of healthy MNCs into Eng+/– mice was sufficient to restore vessel formation and to improve heart function defects associated with reduced levels of endoglin, but MNCs from HHT1 patients were not, demonstrating that the origin of HHT1 is possibly associated with defective ability of MNCs to repair local vessel damage.

It is unclear which cell populations are affected by decreased endoglin expression. Previous studies have shown that blood outgrowth ECs from HHT have abnormalities that would be compatible with a role in vascular lesions,43 that endoglin functions to support lineage-specific hematopoietic development from Flk-1 precursors44 and defines long-term repopulating hematopoietic populations,45,46 and that endoglin is expressed in activated monocytes.47,48 We demonstrated that MNC-derived ECs from HHT1 patients have a reduced ability to accumulate in the infarct zone in vivo and stimulate vessel formation in mice that had undergone MI. One possible explanation is that there are differences in the MNC populations between patients and control subjects. We found no differences, however, in the numbers of CD34+ or CD34+/KDR+ cells in the MNC population nor any differences in adhesion and differentiation to endothelial-like cells in culture. Therefore, the differential behavior in the heart rescue experiments may be because of defective homing, transdifferentiation, proliferation, or secretion of angiogenic factors, which will require investigation in future studies. Although we found no differences in TGF-ß, TßRII, ALK1, or ALK5 expression in HHT1 MNCs in vitro, we have shown previously that in mouse development, ECs lacking endoglin entirely are defective in their ability to process and secrete active TGF-ß,49 which in turn affects their ability to differentiate and recruit smooth muscle cells to the vessel wall. Defective processing of TGF-ß or other cytokines could play a role in patients and also directly or indirectly affect neoangiogenesis.


*    Conclusions
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *Conclusions
down arrowReferences
 
Because vessel damage may occur randomly in HHT patients as a result of trauma, ultraviolet light exposure, inflammation, or multiple individual pathological or physiological differences, this defect in normal repair may contribute to explanations of why families or even individual family members with the same mutation present with highly variable symptoms of the disease. Second, and more generally, in light of ongoing trials investigating the use of autologous bone marrow in vascular repair after MI, the results suggest that HHT1 patients and others with intrinsic defects in their capacity to form endothelial progenitors may derive greater benefit from transplantation with matched heterologous bone marrow.


*    Acknowledgments
 
We thank J. Korving, R. van de Nagel, K. den Ouden, and all blood donors.

Sources of Funding

This work was supported by the Netherlands Heart Foundation (NHS 1999B46), EU (QLG1-CT-2001–01032 and IP "Angiotargeting" contract No. 504743), Janivo Stichting Zeist, the Netherlands Organization for Scientific Research (NWO-VIDI grant 016.056.319), BSIK (Dutch Programme for Tissue Engineering), and the British Heart Foundation (EX/03/002 to H.M.A.).

Disclosures

None.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 up arrowConclusions
 *References
 

  1. Risau W. Mechanisms of angiogenesis. Nature. 1997; 386: 671–674.[CrossRef][Medline] [Order article via Infotrieve]
  2. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.[Abstract/Free Full Text]
  3. Rehman J, Li J, Orschell CM, March KL. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation. 2003; 107: 1164–1169.[Abstract/Free Full Text]
  4. Abdalla SA, Letarte M. Hereditary haemorrhagic telangiectasia: current views on genetics and mechanisms of disease. J Med Genet. 2006; 43: 97–110.[Abstract/Free Full Text]
  5. Van den Driesche S, Mummery CL, Westermann CJ. Hereditary hemorrhagic telangiectasia: an update on transforming growth factor-ß signaling in vasculogenesis and angiogenesis. Cardiovasc Res. 2003; 58: 20–31.[Abstract/Free Full Text]
  6. Johnson DW, Berg JN, Gallione CJ, McAllister KA, Warner JP, Helmbold EA, Markel DS, Jackson CE, Porteous ME, Marchuk DA. A second locus for hereditary hemorrhagic telangiectasia maps to chromosome 12. Genome Res. 1995; 5: 21–28.[Abstract/Free Full Text]
  7. McAllister KA, Grogg KM, Johnson DW, Gallione CJ, Baldwin MA, Jackson CE, Helmbold EA, Markel DS, McKinnon WC, Murrell J, et al. Endoglin, a TGF-ß binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet. 1994; 8: 345–351.[CrossRef][Medline] [Order article via Infotrieve]
  8. Gallione CJ, Repetto GM, Legius E, Rustgi AK, Schelley SL, Tejpar S, Mitchell G, Drouin E, Westermann CJ, Marchuk DA. A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet. 2004; 363: 852–859.[CrossRef][Medline] [Order article via Infotrieve]
  9. Cole SG, Begbie ME, Wallace GM, Shovlin CL. A new locus for hereditary haemorrhagic telangiectasia (HHT3) maps to chromosome 5. J Med Genet. 2005; 42: 577–582.[Abstract/Free Full Text]
  10. Gougos A, Letarte M. Identification of a human endothelial cell antigen with monoclonal antibody 44G4 produced against a pre-B leukemic cell line. J Immunol. 1988; 141: 1925–1933.[Abstract]
  11. Arthur HM, Ure J, Smith AJ, Renforth G, Wilson DI, Torsney E, Charlton R, Parums DV, Jowett T, Marchuk DA, Burn J, Diamond AG. Endoglin, an ancillary TGF-ß receptor, is required for extraembryonic angiogenesis and plays a key role in heart development. Dev Biol. 2000; 217: 42–53.[CrossRef][Medline] [Order article via Infotrieve]
  12. Li DY, Sorensen LK, Brooke BS, Urness LD, Davis EC, Taylor DG, Boak BB, Wendel DP. Defective angiogenesis in mice lacking endoglin. Science. 1999; 284: 1534–1537.[Abstract/Free Full Text]
  13. Barbara NP, Wrana JL, Letarte M. Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-ß superfamily. J Biol Chem. 1999; 274: 584–594.[Abstract/Free Full Text]
  14. Cheifetz S, Bellon T, Cales C, Vera S, Bernabeu C, Massague J, Letarte M. Endoglin is a component of the transforming growth factor-ß receptor system in human endothelial cells. J Biol Chem. 1992; 267: 19027–19030.[Abstract/Free Full Text]
  15. Roberts AB, Sporn MB. Physiological actions and clinical applications of transforming growth factor-ß (TGF-ß). Growth Factors. 1993; 8: 1–9.[Medline] [Order article via Infotrieve]
  16. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-ß family signalling. Nature. 2003; 425: 577–584.[CrossRef][Medline] [Order article via Infotrieve]
  17. Ten Dijke P, Hill CS. New insights into TGF-ß-Smad signalling. Trends Biochem Sci. 2004; 29: 265–273.[CrossRef][Medline] [Order article via Infotrieve]
  18. Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, ten Dijke P. Balancing the activation state of the endothelium via two distinct TGF-ß type I receptors. EMBO J. 2002; 21: 1743–1753.[CrossRef][Medline] [Order article via Infotrieve]
  19. Blanco FJ, Santibanez JF, Guerrero-Esteo M, Langa C, Vary CP, Bernabeu C. Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factor-ß receptor complex. J Cell Physiol. 2005; 204: 574–584.[CrossRef][Medline] [Order article via Infotrieve]
  20. Lebrin F, Goumans MJ, Jonker L, Carvalho RL, Valdimarsdottir G, Thorikay M, Mummery C, Arthur HM, ten Dijke P. Endoglin promotes endothelial cell proliferation and TGF-ß/ALK1 signal transduction. EMBO J. 2004; 23: 4018–4028.[CrossRef][Medline] [Order article via Infotrieve]
  21. Paquet ME, Pece-Barbara N, Vera S, Cymerman U, Karabegovic A, Shovlin C, Letarte M. Analysis of several endoglin mutants reveals no endogenous mature or secreted protein capable of interfering with normal endoglin function. Hum Mol Genet. 2001; 10: 1347–1357.[Abstract/Free Full Text]
  22. Pece-Barbara N, Cymerman U, Vera S, Marchuk DA, Letarte M. Expression analysis of four endoglin missense mutations suggests that haploinsufficiency is the predominant mechanism for hereditary hemorrhagic telangiectasia type 1. Hum Mol Genet. 1999; 8: 2171–2181.[Abstract/Free Full Text]
  23. Lebrin F, Deckers M, Bertolino P, Ten Dijke P. TGF-ß receptor function in the endothelium. Cardiovasc Res. 2005; 65: 599–608.[Abstract/Free Full Text]
  24. Shovlin CL, Letarte M. Hereditary haemorrhagic telangiectasia and pulmonary arteriovenous malformations: issues in clinical management and review of pathogenic mechanisms. Thorax. 1999; 54: 714–729.[Free Full Text]
  25. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 2001; 7: 430–436.[CrossRef][Medline] [Order article via Infotrieve]
  26. Ma N, Stamm C, Kaminski A, Li W, Kleine HD, Muller-Hilke B, Zhang L, Ladilov Y, Egger D, Steinhoff G. Human cord blood cells induce angiogenesis following myocardial infarction in NOD/scid-mice. Cardiovasc Res. 2005; 66: 45–54.[Abstract/Free Full Text]
  27. Nakajima H, Nakajima HO, Tsai SC, Field LJ. Expression of mutant p193 and p53 permits cardiomyocyte cell cycle reentry after myocardial infarction in transgenic mice. Circ Res. 2004; 94: 1606–1614.[Abstract/Free Full Text]
  28. Bajanca F, Luz M, Duxson MJ, Thorsteinsdottir S. Integrins in the mouse myotome: developmental changes and differences between the epaxial and hypaxial lineage. Dev Dyn. 2004; 231: 402–415.[CrossRef][Medline] [Order article via Infotrieve]
  29. Chuva de Sousa Lopes SM, Hassink RJ, Feijen A, van Rooijen MA, Doevendans PA, Tertoolen L, Brutel de la Riviere A, Mummery CL. Patterning the heart, a template for human cardiomyocyte development. Dev Dyn. 2006; 253: 1994–2002.
  30. Chen K, Mehta JL, Li D, Joseph L, Joseph J. Transforming growth factor-ß receptor endoglin is expressed in cardiac fibroblasts and modulates profibrogenic actions of angiotensin II. Circ Res. 2004; 95: 1167–1173.[Abstract/Free Full Text]
  31. Kalkman EA, Bilgin YM, van Haren P, van Suylen RJ, Saxena PR, Schoemaker RG. Determinants of coronary reserve in rats subjected to coronary artery ligation or aortic banding. Cardiovasc Res. 1996; 32: 1088–1095.[Abstract/Free Full Text]
  32. Nelissen-Vrancken HJ, Debets JJ, Snoeckx LH, Daemen MJ, Smits JF. Time-related normalization of maximal coronary flow in isolated perfused hearts of rats with myocardial infarction. Circulation. 1996; 93: 349–355.[Abstract/Free Full Text]
  33. Bourdeau A, Faughnan ME, McDonald ML, Paterson AD, Wanless IR, Letarte M. Potential role of modifier genes influencing transforming growth factor-ß1 levels in the development of vascular defects in endoglin heterozygous mice with hereditary hemorrhagic telangiectasia. Am J Pathol. 2001; 158: 2011–2020.[Abstract/Free Full Text]
  34. Jerkic M, Rodriguez-Barbero A, Prieto M, Toporsian M, Pericacho M, Rivas-Elena JV, Obreo J, Wang A, Perez-Barriocanal F, Arevalo M, Bernabeu C, Letarte M, Lopez-Novoa JM. Reduced angiogenic responses in adult endoglin heterozygous mice. Cardiovasc Res. 2006; 69: 845–854.[Abstract/Free Full Text]
  35. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003; 9: 702–712.[CrossRef][Medline] [Order article via Infotrieve]
  36. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease, part II: cell-based therapies. Circulation. 2004; 109: 2692–2697.[Free Full Text]
  37. Rafii S, Meeus S, Dias S, Hattori K, Heissig B, Shmelkov S, Rafii D, Lyden D. Contribution of marrow-derived progenitors to vascular and cardiac regeneration. Semin Cell Dev Biol. 2002; 13: 61–67.[CrossRef][Medline] [Order article via Infotrieve]
  38. Chuva de Sousa Lopes SM, Feijen A, Korving J, Korchynskyi O, Larsson J, Karlsson S, ten Dijke P, Lyons KM, Goldschmeding R, Doevendans P, Mummery CL. Connective tissue growth factor expression and Smad signaling during mouse heart development and myocardial infarction. Dev Dyn. 2004; 231: 542–550.[CrossRef][Medline] [Order article via Infotrieve]
  39. Sanchez-Elsner T, Botella LM, Velasco B, Langa C, Bernabeu C. Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth factor-ß pathways. J Biol Chem. 2002; 277: 43799–43808.[Abstract/Free Full Text]
  40. Duff SE, Li C, Garland JM, Kumar S. CD105 is important for angiogenesis: evidence and potential applications. Faseb J. 2003; 17: 984–992.[Abstract/Free Full Text]
  41. She X, Matsuno F, Harada N, Tsai H, Seon BK. Synergy between anti-endoglin (CD105) monoclonal antibodies and TGF-ß in suppression of growth of human endothelial cells. Int J Cancer. 2004; 108: 251–257.[CrossRef][Medline] [Order article via Infotrieve]
  42. Kopp HG, Ramos CA, Rafii S. Contribution of endothelial progenitors and proangiogenic hematopoietic cells to vascularization of tumor and ischemic tissue. Curr Opin Hematol. 2006; 13: 175–181.[Medline] [Order article via Infotrieve]
  43. Fernandez LA, Sanz-Rodriguez F, Zarrabeitia R, Perez-Molino A, Hebbel RP, Nguyen J, Bernabeu C, Botella LM. Blood outgrowth endothelial cells from hereditary haemorrhagic telangiectasia patients reveal abnormalities compatible with vascular lesions. Cardiovasc Res. 2005; 68: 235–248.[Abstract/Free Full Text]
  44. Cho SK, Bourdeau A, Letarte M, Zuniga-Pflucker JC. Expression and function of CD105 during the onset of hematopoiesis from Flk1(+) precursors. Blood. 2001; 98: 3635–3642.[Abstract/Free Full Text]
  45. Chen CZ, Li L, Li M, Lodish HF. The endoglin(positive) sca-1(positive) rhodamine(low) phenotype defines a near-homogeneous population of long-term repopulating hematopoietic stem cells. Immunity. 2003; 19: 525–533.[CrossRef][Medline] [Order article via Infotrieve]
  46. Chen CZ, Li M, de Graaf D, Monti S, Gottgens B, Sanchez MJ, Lander ES, Golub TR, Green AR, Lodish HF. Identification of endoglin as a functional marker that defines long-term repopulating hematopoietic stem cells. Proc Natl Acad Sci U S A. 2002; 99: 15468–15473.[Abstract/Free Full Text]
  47. Fernandez Pujol B, Lucibello FC, Gehling UM, Lindemann K, Weidner N, Zuzarte ML, Adamkiewicz J, Elsasser HP, Muller R, Havemann K. Endothelial-like cells derived from human CD14-positive monocytes. Differentiation. 2000; 65: 287–300.[CrossRef][Medline] [Order article via Infotrieve]
  48. Fujiyama S, Amano K, Uehira K, Yoshida M, Nishiwaki Y, Nozawa Y, Jin D, Takai S, Miyazaki M, Egashira K, Imada T, Iwasaka T, Matsubara H. Bone marrow monocyte lineage cells adhere on injured endothelium in a monocyte chemoattractant protein-1-dependent manner and accelerate reendothelialization as endothelial progenitor cells. Circ Res. 2003; 93: 980–989.[Abstract/Free Full Text]
  49. Carvalho RL, Jonker L, Goumans MJ, Larsson J, Bouwman P, Karlsson S, Dijke PT, Arthur HM, Mummery CL. Defective paracrine signalling by TGF-ß in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary haemorrhagic telangiectasia. Development. 2004; 131: 6237–6247.[Abstract/Free Full Text]
 

CLINICAL PERSPECTIVE

Hereditary hemorrhagic telangiectasia is a rare disease characterized by weak-walled vessels and caused by mutation in cell surface receptors for the cytokine transforming growth factor-ß. Here, we show that mononuclear cells from peripheral blood of patients with 1 type of mutation are defective in their ability to home to the infarct zone in the heart of mice that had undergone experimental myocardial infarction. In contrast to healthy individuals, the mononuclear cells of hereditary hemorrhagic telangiectasia patients are also unable to restore cardiac function in the experimental mice. This indicates for the first time that defective vascular repair may contribute to the origin of hereditary hemorrhagic telangiectasia. More generally, in the context of using autologous bone marrow in cardiovascular repair, patients with intrinsic defects in the ability of their mononuclear cells to form endothelial progenitors may benefit less than expected from bone marrow infusion into the heart.


*    Footnotes
 
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.639161/DC1.

*Drs van Laake and van den Driesche contributed equally to this work. Back

{dagger}Drs Lebrin and Mummery contributed equally to this work. Back




This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
S. Roy, S. Khanna, C. Rink, S. Biswas, and C. K. Sen
Characterization of the acute temporal changes in excisional murine cutaneous wound inflammation by screening of the wound-edge transcriptome
Physiol Genomics, July 9, 2008; 34(2): 162 - 184.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
D. F. Alvarez, L. Huang, J. A. King, M. K. ElZarrad, M. C. Yoder, and T. Stevens
Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity
Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L419 - L430.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
D. Liu, J. Wang, B. Kinzel, M. Mueller, X. Mao, R. Valdez, Y. Liu, and E. Li
Dosage-dependent requirement of BMP type II receptor for maintenance of vascular integrity
Blood, September 1, 2007; 110(5): 1502 - 1510.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
A. Fernandez-Lopez, E. M. Garrido-Martin, F. Sanz-Rodriguez, M. Pericacho, A. Rodriguez-Barbero, N. Eleno, J. M. Lopez-Novoa, A. Duwell, M. A. Vega, C. Bernabeu, et al.
Gene expression fingerprinting for human hereditary hemorrhagic telangiectasia
Hum. Mol. Genet., July 1, 2007; 16(13): 1515 - 1533.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
H. Stepan, T. Kramer, and R. Faber
Maternal Plasma Concentrations of Soluble Endoglin in Pregnancies with Intrauterine Growth Restriction
J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2831 - 2834.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
114/