Copyright © 2005, European Society of Cardiology
Reduced angiogenic responses in adult endoglin heterozygous mice
aInstituto "Reina Sofía" de Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain
bCancer Research Program, The Hospital for Sick Children, Toronto, Canada M5G1X8
cDepartmento de Anatomía e Histología Humanas, Universidad de Salamanca, Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
dThe Heart and Stroke Lewar Centre of Excellence, University of Toronto, Toronto, Canada
eCentro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28040 Madrid, Spain
* Corresponding author. Tel.: +34 923 294 472; fax: +34 923 294 669. Email address: jmlnovoa{at}usal.es
Received 5 October 2005; revised 8 November 2005; accepted 14 November 2005
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentsReferences |
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Objective: To determine if angiogenesis is altered in adult Endoglin heterozygous (Eng+/–) mice, the animal model for the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1).
Methods: Primary cultures of endothelial cells were generated from Eng+/– and Eng+/+ mice and analyzed for proliferation, migration, and ability to form capillary-like tubes. Endothelial cells derived from umbilical veins of newborns (HUVEC) with an HHT1 genotype were also tested for capillary formation. Two in vivo models of angiogenesis were tested in the Eng+/– and Eng+/+ mice: Matrigel implant-dependent angiogenesis and reperfusion following hindlimb ischemia.
Results: The Eng+/– endothelial cells displayed significantly reduced proliferation and migration, increased collagen production, and decreased NO synthase expression and vascular endothelial growth factor (VEGF) secretion. They also showed impaired capillary tube formation in vitro, as did the HHT1 HUVEC. These endothelial cell-specific abnormalities were associated with impaired Matrigel-dependent capillary tube formation in vivo and delayed reperfusion following hindlimb ischemia.
Conclusions: Although vascular development is normal in Eng+/– mice, angiogenic abnormalities were observed in the adult mice and their isolated endothelial cells. These results suggest that a normal level of endoglin is required for full angiogenic activity.
KEYWORDS Angiogenesis; Endoglin; HHT1; Nitric oxide; VEGF
This article is referred to in the Editorial by Maio et al. (pages 781–783) in this issue.
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentsReferences |
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Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by focal telangiectases and arteriovenous malformations (AVMs). Mutations in the genes encoding endoglin (ENG) and activin-like kinase (ALK1) are associated with HHT1 and HHT2, respectively. Haploinsufficiency is the disease model for HHT as mutant proteins are generally not expressed or are non-functional (see review by Abdalla and Letarte [1]).
Endoglin (CD105) is a 180-kDa homodimeric transmembrane glycoprotein expressed mainly in endothelial cells and known to modulate cellular responses to ligands of the transforming growth factor-β (TGF-β) superfamily [2,3]. Endoglin is up-regulated in tissues undergoing angiogenesis and in vitro inhibition of its expression on endothelial cells impairs this process [4]. Eng-null mice die at mid-gestation from defective angiogenesis and severe cardiovascular abnormalities, while Endoglin heterozygous (Eng+/–) mice have normal life spans, but are predisposed to develop HHT-like vascular abnormalities [4–7]. We also demonstrated that endothelial nitric oxide (NO) synthase (eNOS) expression was reduced and that NO synthesis was impaired in these mice [8,9]. Endoglin has been found in endothelial caveolae, where it associates with eNOS and modulates its activation by promoting eNOS/Hsp90 association [9]. Endoglin heterozygous cells (human and murine) not only have reduced eNOS levels, but show uncoupled eNOS activity and generate eNOS-derived superoxide during agonist-induced activation [9]. However, the characterization of endoglin-dependent endothelial mechanisms, necessary to support normal angiogenesis and whether or not these are altered in Eng+/– mice, a physiologically and clinically relevant model of HHT1, remain to be elucidated.
We report that isolated murine Eng+/– endothelial cells display reduced proliferation and migration, impaired capillary tube formation and reduced eNOS activity and VEGF secretion. These changes were associated with decreased blood vessel formation in in vivo models of angiogenesis, indicating that endoglin contributes to normal adult angiogenesis.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentsReferences |
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2.1 Mice
All procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Eng+/– and Eng+/+ mice were sex- and age-matched 8- to 12-week-old N3–N5 C57BL/6 backcrosses.
2.2 Isolation and characterization of endothelial cells
Mouse aortic endothelial cells (MAECs) from Eng+/+ and Eng+/– mice were isolated and cultured up to 4 passages as previously described [8]. The identity and purity of the cells were determined by indirect immunofluorescence using monoclonal antibodies (mAb) to CD31 (Santa Cruz), von Willebrand factor (Sigma) and endoglin (MJ178), followed by Cy3 or Alexa Fluor 488-conjugated secondary antibodies (Jackson Immunoresearch) [10]. Cells were visualized under a Zeiss fluorescence microscope equipped with a digital camera and analyzed using Openlab (Improvision, Inc.). Human umbilical vein endothelial cells (HUVECs) were derived from newborns with an ENG mutation, or from control babies. These cells were characterized and maintained in complete M199 medium supplemented with 10% FBS and 30 µg/ml endothelial mitogen. Samples from HHT1 families were tested by protein and mutation analysis as described.
2.3 Proliferation and migration assays
MAECs were plated at 5 x 103 cells/well and proliferation assessed by counting the number of viable cells, determined by crystal violet nuclei staining, over 8 days. Cell cycle analysis was evaluated by flow cytometry.
Confluent monolayers of Eng+/+ and Eng+/– MAECs were wounded using a sterile pipette and the extent of wound closure was determined over 24 h by calculating migrated distance/total wound distance using digital microscopy. Endothelial cell migration was also assessed by the Boyden Chamber assay, used in the bottom chamber medium with 2% (negative control) or 10% serum. After 12 h, inserts were washed with PBS and migrated cells fixed with 10% glutaraldehyde, stained with 2% crystal violet, treated with 10% acetic acid and quantified at 595 nm.
2.4 Collagen synthesis and VEGF secretion
MAECs were grown to 70–80% confluence and serum-starved for 24 h. Cells were grown in the presence and absence of TGF-β1 (1 ng/ml) or in the presence of anti-TGF-β1 antibody (100 ng/mL; R&D Systems, Minneapolis, MN, USA). The collagen content in the medium was quantified by measuring [3H]-proline incorporation over a period of 24 h [10]. Media VEGF accumulation over a 24-h period was determined by ELISA (QuantikineTM, R&D Systems) and normalized for DNA content.
2.5 Western blot
Tissue or cell protein extracts were prepared in 10 mM Tris buffer containing 1% Triton X-100 supplemented with protease inhibitors (Roche). Protein extracts were quantified by the Bradford method and 20 µg were fractionated by SDS–PAGE, transferred onto nitrocellulose and probed with a rabbit polyclonal antibody to eNOS (1:1000, Santa Cruz Biotechnology) or rat monoclonal anti-endoglin (MJ7/18). Protein levels were normalized to 
-tubulin levels.
2.6 In vitro and in vivo Matrigel assays
Eng+/+ and Eng+/– MAECs or HUVECs derived from newborns with different ENG mutations or from control newborns (n=6/group) were plated at 5 x 104 cells/well in 6-well plates coated with 0.5 mL of growth factor free MatrigelTM (Becton Dickinson). After 6 h, images were taken at 20 x magnification using an inverted microscope and a digital camera; capillary tube number, area and wall width were quantified using Openlab software (Improvision Inc.).
Separately, Eng+/– mice and Eng+/+ littermates (n=4/group) were anaesthetized with ketamine/xylazine (100/10 mg/kg i.p.) and 0.4 mL of MatrigelTM (with growth factors) was injected into the subcutaneous space along the dorsal midline. Mice were sacrificed 12 days later and Matrigel plugs were carefully removed. Samples were fixed in 4% paraformaldehyde, paraffin embedded, cross-sectioned and stained with haematoxylin/eosin. Sections were analyzed using Openlab software and quantified for the number of structures having circular or ellipsoid shapes with a visible lumen with or without blood.
2.7 Hindlimb ischemia and reperfusion
Eng+/+ and Eng+/– mice were anaesthetized with isoflurane and the left femoral artery was ligated 2–3 mm distal to the inguinal ligament. In some animals, NO synthesis was inhibited with 1 mM NG-nitro-L-arginine methyl ester (L-NAME; Sigma) in the drinking water. Hindlimb perfusion was measured every other day up to 28 days by laser Doppler (Periflux 2B, Perimed) using a 0.45-mm probe, and the results are expressed as the ratio of perfusion in the ischemic versus non-ischemic limb. Perfusion studies have been completed in 10 untreated animals per group and in 6 animals per group in mice treated with L-NAME. Four days after femoral ligation, groups of animals (6 per group) were anaesthetized with isoflurane and the adductor muscle rapidly excised from both ischemic and non-ischemic limbs, rinsed in PBS and snap-frozen in liquid nitrogen for total protein and RNA extractions.
2.8 Northern blot
Total RNA was isolated from tissues by phenol/chloroform extraction. About 20 µg was fractionated on a 2% formaldehyde agarose gel, transferred onto a nylon membrane, probed with random 32P-labeled fragments to murine endoglin (938 bp, GenBank Accession No.: X77952
[GenBank]
). The 28S ribosomal RNA (700 bp) served as loading control and specific bands were visualized by radioautography.
2.9 Statistical analysis
Between-group comparisons were performed by ANOVA and corrected for repeated measures when appropriate. If ANOVA revealed overall significant differences, individual means were evaluated post hoc using Bonferroni's procedure. Results are expressed as the mean ± S.E.M.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentsReferences |
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3.1 Characterization of Eng+/+ and Eng+/– MAECs
Endothelial sprouts from 4 to 6 aortic rings per mouse were first visible after 9 days and grew into a homogeneous population as observed by flow cytometry analyses of size and granularity (data not shown). In culture, Eng+/+ MAECs formed typical cobblestone monolayers (Fig. 1A), whereas Eng+/– MAEC showed a more elongated morphology (Fig. 1B). Immunofluorescence staining of Eng+/+ MAEC revealed von Willebrand factor, CD31 and endoglin expression, confirming their endothelial phenotype (Fig. 1C, D and E).
-smooth muscle actin was absent in MAEC cultures (Fig. 1F) while readily detectable in rat smooth muscle cells (Fig. 1G). MAECs from Eng+/– mice showed the same positive CD31 and von Willebrand factor staining patterns than those from Eng+/+ mice and no -smooth muscle actin staining (data not shown). However, Western blot analysis showed reduced endoglin levels corresponding to 50% of those found in Eng+/+ cells (Fig. 1H).
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3.2 Eng+/– MAECs display reduced proliferation and migration and increased collagen synthesis
Endothelial cell proliferation and migration are key early events in angiogenesis. The effects of endoglin heterozygosity on cellular proliferation were tested by determining the increase in cell number during 8 days of growth in complete medium. Eng+/– MAECs proliferated significantly slower than Eng+/+ MAECs reaching a 9-fold increase in total cell count by day 6, compared to a 16-fold increase for Eng+/+ cells (Fig. 2A). Flow cytometry analysis revealed a greater percentage of Eng+/– cells arrested in G0/G1 (76 ± 2%) compared to Eng+/+(68 ± 2%) cells. The proportion of cells in S+G2M phases was significantly lower in Eng+/– (20%) than in Eng+/+ cells (29%) (Fig. 2B).
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) and TGF-β1 (50 pM for 24 h) – stimulated collagen synthesis (#), quantified by [3H]-proline incorporation, were increased in Eng+/– MAECs (n=8/group; P<0.01 vs. Eng+/+). In both groups, collagen synthesis was stimulated by TGF-β1 (*P<0.01 vs. untreated), but basal levels were not affected by incubation of the cells for 24 h with an anti-TGF-β antibody.Endothelial cell migration was analysed by two independent techniques, which yielded similar results. Eng+/– MAEC confluent monolayers when damaged by a scratch wound assay migrated a significantly shorter distance, covering 21.9 ± 4.1% of the total wound versus 38.2 ± 5.1% for Eng+/+ cells (Fig. 2C). Similarly, the number of migrated cells assayed using a Boyden chamber was significantly lower in the Eng+/– than in Eng+/+ MAECs (Fig. 2D).
Several in vitro studies have demonstrated that endoglin expression regulates extracellular matrix (ECM) production. Increased collagen secretion is an important step in the resolution phase of angiogenesis, but may have inhibitory effects on endothelial migratory function during the activation phase. Basal [3H]-proline incorporation was significantly greater in Eng+/– compared to Eng+/+ MAECs (Fig. 2E). TGF-β1 treatment caused a 1.7-fold increase in collagen production in both groups, resulting in significantly higher total levels in Eng+/– cells. Incubation with a neutralizing antibody to TGF-β1 had no effect on collagen accumulation in either cell type, suggesting that the elevated basal collagen synthesis in the Eng+/– cells was not caused by higher endogenous levels of TGF-β1.
3.3 Decreased VEGF secretion by Eng+/– MAECs
Given that VEGF is a central mediator of endothelial cell proliferation and migration, its basal levels were quantified in the culture medium. They were significantly reduced in Eng+/– MAECs versus control Eng+/+ cells (Fig. 3A). TGF-β1 significantly increased VEGF levels in both groups, resulting in higher total levels in Eng+/+ cells. A neutralizing antibody to TGFβ1 did not affect baseline VEGF secretion, suggesting that the lower levels observed in Eng+/– cells are not due to a lower concentration of endogenous TGF-β1.
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3.4 Reduced eNOS expression in Eng+/– MAECs
The mitogenic and chemotactic effects of VEGF on endothelial cells are NO-dependent and necessitate normal eNOS expression and activity. Consistent with previous observations [8,9], Eng+/– MAECs displayed significantly reduced eNOS levels relative to Eng+/+ cells (Fig. 3B).
3.5 Abnormal capillary-tube formation in vitro and in vivo
When plated on Matrigel, endothelial cells rapidly organize and form capillary-like structures. Eng+/– MAEC generated a less extensive capillary network than the Eng+/+ cells (Fig. 4A). HUVECs derived from six newborns with a distinct ENG mutation, and therefore an HHT1 genotype, expressed reduced levels of endoglin (ranging from 37% to 64%) relative to the control group, as assessed by metabolic labelling [11]. A median of 45% was estimated for 30 newborns with HHT1 while unaffected newborns had a median of 98% of control HUVEC, as reviewed recently and in keeping with the haploinsufficiency model [1]. The HHT1 HUVECs generated an altered capillary network when plated on Matrigel (Fig. 4B). A lower number of capillary tubes (101 ± 4.5 vs. 118 ± 5, P<0.05) was observed (Fig. 4C), with greater luminal areas reaching 11,765 ± 611 µm2 vs. 10,252 ± 434 µm2 in the control group (P<0.05) (Fig. 4D). Moreover, the walls of the capillary tubes formed by HUVECs with an ENG mutation were significantly thicker than those of control HUVEC (16.4 ± 1.7 µm versus 10.0 ± 0.6 µm, P<0.01) (Fig. 4E).
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Matrigel plugs were implanted dorsally in Eng+/– and Eng+/+ mice as a way to monitor endothelial cell outgrowth and invasion of extracellular matrix in vivo. Representative sections of the implanted Matrigel plugs show much reduced numbers of invading cells in the Eng+/– mice (Fig. 4F). Quantitative analysis revealed a significantly lower number of blood vessels (11 ± 0.9 vessels/mm2) in Eng+/– mice, relative to Eng+/+ mice (25 ± 3.3 vessels/mm2, P<0.01) (Fig. 4G).
3.6 Lower post-ischemic capillary generation and reperfusion in Eng+/– mice
The importance of NO and the capacity for ischemia-induced angiogenesis in Eng+/– mice were evaluated by measuring reperfusion recovery in femoral artery-ligated hindlimb following chronic treatment with or without L-NAME. Eng+/– mice had significantly lower overall hindlimb reperfusion compared to Eng+/+ mice and L-NAME significantly reduced this parameter to similar levels in both groups of mice (Fig. 5A).
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Non-ischemic and ischemic adductor muscle vascularity was quantified in tissue sections by counting the number of CD31 positive vessels. Non-ischemic muscles showed similar vessel density in Eng+/+ and Eng+/– mice (Fig. 5B; basal). Following ischemia, Eng+/– mice had a significantly lower overall adductor muscle vessel density; most noticeable were the differences at 4 and 14 days (60% of control values). By day 28, capillary density was similar in both groups. When the rate of perfusion was calculated per vessel, a trend at 4 days and a significant increase at 14 days were observed in the Eng+/– muscles, suggesting that increased flow was compensating for reduced vascularity (Fig. 5C).
3.7 Endoglin and eNOS expression in ischemic adductor muscles
Endoglin mRNA was significantly reduced in Eng+/– non-ischemic muscles, due to expression of a single Eng allele. Following ischemia, muscle endoglin mRNA levels were increased in both groups, maintaining a higher level in Eng+/+ mice (Fig. 5D). eNOS protein levels were significantly lower in non-ischemic Eng+/– than in Eng+/+ muscles and remained so despite a significant ischemia-induced increase in both groups (Fig. 5E).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentsReferences |
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Our findings demonstrate that endothelial cell proliferation, migration and capillary tube formation are impaired in an animal model of HHT1, the Eng+/– mouse. In addition to in vitro findings, two different in vivo angiogenic assays showed reduced vessel formation. However, a higher rate of perfusion per vessel was observed in the hind limb ischemia model. Increases in local blood flow, associated with HHT, may compensate for the angiogenic defects and account for normal perfusion 28 days after injury-induced ischemia.
VEGF regulates both vasculogenesis and angiogenesis. It was demonstrated that VEGF induced proliferation of HUVECs and their ability to form vessel-like structures in vitro was NO-dependent [12]. Long-term VEGF exposure stimulates eNOS protein levels while short-term stimulation of NO production was mediated by PI-3 Kinase signaling. It was subsequently shown that the AKT serine threonine kinase, known to phosphorylate eNOS and cause its activation and NO production, was the downstream effector of VEGF stimulated actin reorganization and endothelial cell migration [13,14]. NO promotes endothelial cell proliferation and migration, while NOS inhibitors suppress these responses. Endothelial cell growth and angiogenic sprouting are impaired in eNOS–/– mice. Association with heat shock protein 90 (Hsp90) is required for eNOS activation and NO production [15]. Impaired eNOS/Hsp90 association causes uncoupling of the enzyme and production of eNOS-derived superoxide (O2–) [16]. Agents that perturb eNOS/Hsp90 association or uncouple eNOS activity were shown to attenuate endothelial cell proliferation, migration and capillary tube formation [17,18]. Endoglin was shown to associate with eNOS and modulate its activation by promoting eNOS/Hsp90 association. Eng+/– endothelial cells have uncoupled eNOS activity, evidenced by less NO and more eNOS-derived superoxide (O2–) production during agonist-induced activation [9]. Therefore, the abnormalities in proliferation, migration and capillary formation observed in Eng+/– cells may be due to uncoupled eNOS activity and reduced NO production.
Our data also revealed a decrease in basal VEGF production in Eng+/– versus Eng+/+ endothelial cells, which may contribute to the observed reduction in proliferation, migration and capillary tube formation. Although NO was shown to stimulate VEGF expression in human and rat vascular smooth muscle cells [19], our findings suggest a potential endothelial autocrine loop whereby eNOS activity and NO levels may regulate VEGF production. TGF-β-stimulated increase in VEGF secretion was unchanged in both groups of cells and treatment with a neutralizing antibody to TGF-β did not alter basal VEGF secretion suggesting that the reduced basal VEGF production in Eng+/– cells is independent of TGF-β.
Basal collagen synthesis was elevated in Eng+/– cells, consistent with a reduced migratory activity. Fibronectin synthesis was also increased (data not shown), indicating that extracellular matrix deposition is higher in Eng+/– cells. This likely leads to increased cell adhesion and hindered locomotion. The stimulation of collagen synthesis by TGF-β1 still occurred in Eng+/– endothelial cells, indicating that reduced endoglin levels did not affect this response. Furthermore, treatment with a neutralization antibody to TGF-β did not affect basal collagen production suggesting that the increased collagen synthesis in Eng+/– endothelial cells is independent of TGF-β.
The finding that Eng+/– endothelial cells display impaired proliferation and migration is consistent with reports demonstrating that endoglin: i) promotes cell proliferation and TGF-β/ALK-1 signaling [20]; ii) is up-regulated in angiogenesis [21]; iii) down-regulation impairs proliferation and increases cell apoptosis [4]; and iv) normal cellular levels are required for the formation of new blood vessels [22].
In addition to the impaired in vitro angiogenic activity of isolated Eng+/– endothelial cells, Eng+/– mice showed impaired angiogenesis in vivo. The migration of adjacent cells into Matrigel implants and their ability to form vessels was markedly inhibited in the Eng+/– mice. The lesser migration of Eng+/– cells may be due to their lower production of NO, and the consequent reduction in metalloproteinase activity as reported in our recent study [23]. We demonstrated that NO elicits migration in murine and bovine aortic endothelial cells, by increasing extracellular MMP-13 expression, which leads to higher collagen breakdown.
Hindlimb revascularization following femoral artery ligation, was significantly lower in Eng+/– mice during the first 2-week recovery period, but reached normal levels after 28 days suggesting a delay in neo-vascularization. Interestingly, there were relatively large differences in hindlimb vascularity between Eng+/+ and Eng+/– mice, which did not correlate with the much smaller differences in perfusion. This suggests that despite a reduction in local vascularity in Eng+/– mice, there is increased collateral blood flow as evidenced by an elevated perfusion per vessel. These observations are consistent with the impaired myogenic reactivity and enhanced endothelium-dependent dilatation observed in Eng+/– resistance or feeding vessels, due to uncoupled eNOS activity [9]. Indeed, chronic treatment of mice with L-NAME reduced hindlimb perfusion to a similar level in both groups of mice suggesting that the increased perfusion per vessel in Eng+/– mice is entirely NOS-dependent. This endothelial abnormality in Eng+/– resistance vessels leads to increased local blood flow and maintains adequate oxygen delivery in response to tissue metabolic demand, despite reduced vascularity during the early phase of recovery. As recently proposed [9], consequent excessive increases in hemodynamic stress may abnormally dilate or damage downstream fragile venular and capillary structures and represent an early event in the pathogenesis of HHT-type vascular lesions.
Angiogenesis is crucial for tumor growth and progression. Without an increased blood supply from newly formed vessels, neoplastic cell aggregates remain dependent on diffusion for nutrients, oxygenation and the removal of waste metabolites. Under these conditions, their growth is restricted to a maximum of 1–2 mm3 and most remain clinically quiescent. The increased expression of endoglin in proliferating endothelial cells and in tumor vasculature suggests that it may be highly relevant in the diagnostic, prognostic and potential treatment of solid malignancies [21]. We have found that endothelial cell proliferation, migration and capillary tube formation are impaired in cultured Eng+/– endothelial cells and that these abnormalities may be related to reduced NO bioavailability and VEGF production, and to an increase in collagen deposition. Reduced vessel formation was observed in two distinct in vivo angiogenic assays. Interestingly, hindlimb reperfusion per vessel following ischemia was in fact elevated in Eng+/– mice as would be expected from impaired arterial myogenic reactivity and enhanced endothelium-dependent dilation in collateral Eng+/– resistance vessels [9]. Therefore, such inherent tendencies for increased local blood flow in Eng+/– mice can serve to maintain adequate tissue oxygenation during early reduction in neo-vascularization. However, such a seemingly beneficial vascular response would cause excessive hemodynamic stress leading to abnormal venular dilatation or capillary damage, and subsequent HHT-type vascular lesions.
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Acknowledgments |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentsReferences |
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This study was supported by grants from Comisión Interministerial de Ciencia y Tecnología, (SAF2001/1701 and BFU2004-00285/BFI to JMLN and SAF2004-01390 to CB), Fondo de Investigación Sanitaria (PI020200 to CB), HHT Foundation International (CB) and The Heart and Stroke Foundation of Canada (ML). Dr. M. Jerkic was supported by a Fellowship from Instituto Reina Sofía de Investigación Nefrológica. Marta Prieto and Miguel Pericacho are fellows of Junta de Castilla y Leon.
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Notes |
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1 These authors contributed equally to the paper.
Time for primary review 21 days
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