Potential Role of Modifier Genes Influencing Transforming Growth Factor-β1 Levels in the Development of Vascular Defects in Endoglin Heterozygous Mice with Hereditary Hemorrhagic Telangiectasia
2001; Elsevier BV; Volume: 158; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64673-1
ISSN1525-2191
AutoresAnnie Bourdeau, Marie E. Faughnan, Merry‐Lynn McDonald, Andrew D. Paterson, Ian R. Wanless, Michelle Letarte,
Tópico(s)Tracheal and airway disorders
ResumoHereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder because of mutations in the genes coding for endoglin (HHT1) or ALK-1 (HHT2). The disease is associated with haploinsufficiency and a murine model was obtained by engineering mice that express a single Endoglin allele. Of a total of 171 mice that were observed for 1 year, 50 developed clinical signs of HHT. Disease prevalence was high in 129/Ola strain (72%), intermediate in the intercrosses (36%), and low in C57BL/6 backcrosses (7%). Most mice first presented with an ear telangiectasia and/or recurrent external hemorrhage. One-third of mice with HHT showed severe vascular abnormalities such as dilated vessels, hemorrhages, liver and lung congestion, and/or brain and heart ischemia. Disease sequelae included stroke, hydrocephalus, fatal hemorrhage, and congestive heart failure. Thus the murine model reproduces the multiorgan manifestions of the human disease. Levels of circulating latent transforming growth factor (TGF)-β1 were significantly lower in the 129/Ola than in the C57BL/6 strain. Intercrosses and 129/Ola mice expressing reduced endoglin also showed lower plasma TGF-β1 levels than control. These data suggest that modifier genes involved in the regulation of TGF-β1 expression act in combination with a single functional copy of endoglin in the development of HHT. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder because of mutations in the genes coding for endoglin (HHT1) or ALK-1 (HHT2). The disease is associated with haploinsufficiency and a murine model was obtained by engineering mice that express a single Endoglin allele. Of a total of 171 mice that were observed for 1 year, 50 developed clinical signs of HHT. Disease prevalence was high in 129/Ola strain (72%), intermediate in the intercrosses (36%), and low in C57BL/6 backcrosses (7%). Most mice first presented with an ear telangiectasia and/or recurrent external hemorrhage. One-third of mice with HHT showed severe vascular abnormalities such as dilated vessels, hemorrhages, liver and lung congestion, and/or brain and heart ischemia. Disease sequelae included stroke, hydrocephalus, fatal hemorrhage, and congestive heart failure. Thus the murine model reproduces the multiorgan manifestions of the human disease. Levels of circulating latent transforming growth factor (TGF)-β1 were significantly lower in the 129/Ola than in the C57BL/6 strain. Intercrosses and 129/Ola mice expressing reduced endoglin also showed lower plasma TGF-β1 levels than control. These data suggest that modifier genes involved in the regulation of TGF-β1 expression act in combination with a single functional copy of endoglin in the development of HHT. Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal-dominant disorder that affects blood vessels and has a prevalence of ∼1:8000.1Plauchu H de Chadarévian JP Bideau A Robert J-M Age-related clinical profile of hereditary hemorrhagic telangiectasia in an epidemiologically recruited population.Am J Med Genet. 1989; 32: 291-297Crossref PubMed Scopus (504) Google Scholar HHT is primarily associated with frequent nosebleeds (epistaxis), telangiectases, and internal vascular lesions.2Shovlin CL Guttmacher AE Buscarini E Faughnan ME Hyland RH Westermann CJ Kjeldsen AD Plauchu H Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome).Am J Med Genet. 2000; 91: 66-67Crossref PubMed Scopus (1362) Google Scholar Patients can develop life-threatening complications such as severe gastrointestinal bleeding and arteriovenous malformations (AVM) (direct connection between a dilated venule and arteriole bypassing the capillary network) of the liver, lung, or brain. Shunting of blood through pulmonary or cerebral AVMs can lead to hypoxemia, stroke, brain abscess, heart failure, and fatal hemorrhage.3Shovlin CL Letarte M Hereditary haemorrhagic telangiectasia and pulmonary arteriovenous malformations: issues in clinical management and review of pathogenic mechanisms.Thorax. 1999; 54: 714-729Crossref PubMed Scopus (354) Google Scholar Clinical manifestations of HHT are highly heterogeneous between families as well as within a given family. Genetic and epigenetic factors have been postulated to account for this diversity. Two genes are responsible for HHT, ENG (ENDOGLIN) mutated in HHT14McAllister KA Grogg KM Johnson DW Gallione CJ Baldwin MA Jackson CE Helmbold EA Markel DS McKinnon WC Murrell J McCormick MK Pericak-Vance MA Heutink P Oostra BA Haitjema T Westerman CJJ Porteous ME Guttmacher AE Letarte M Marchuk DA Endoglin, a TGF-β binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1.Nat Genet. 1994; 8: 345-351Crossref PubMed Scopus (1270) Google Scholar and ACVRL1 (ACTIVIN RECEPTOR-LIKE KINASE 1, also known as ALK-1), mutated in HHT2.5Johnson DW Berg JN Baldwin MA Gallione CJ Marondel I Yoon SJ Stenzel TT Speer M Pericak-Vance MA Diamond A Guttmacher AE Jackson CE Attisano L Kucherlapati R Porteous ME Marchuk DA Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2.Nat Genet. 1996; 13: 189-195Crossref PubMed Scopus (888) Google Scholar HHT1 is associated with a higher incidence of pulmonary AVMs than HHT2, which generally has a later onset.6Berg JN Guttmacher AE Marchuk DA Porteous ME Clinical heterogeneity in hereditary haemorrhagic telangiectasia: are pulmonary arteriovenous malformations more common in families linked to endoglin?.J Med Genet. 1996; 33: 256-257Crossref PubMed Scopus (120) Google Scholar Severity of HHT is not correlated with the type of mutation or its position.7Shovlin CL Hughes JM Scott J Seidman CE Seidman JG Characterization of endoglin and identification of novel mutations in hereditary hemorrhagic telangiectasia.Am J Hum Genet. 1997; 61: 68-79Abstract Full Text PDF PubMed Scopus (144) Google Scholar Mutated ENG is rarely expressed in HHT1 patients and only as an intracellular species.8Pece N Vera S Cymerman U White Jr, RI Wrana JL Letarte M Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative.J Clin Invest. 1997; 100: 2568-2579Crossref PubMed Scopus (132) Google Scholar The current model for HHT1 is haploinsufficiency, because of reduced levels of functional endoglin at the surface of endothelial cells.8Pece N Vera S Cymerman U White Jr, RI Wrana JL Letarte M Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative.J Clin Invest. 1997; 100: 2568-2579Crossref PubMed Scopus (132) Google Scholar, 9Pece-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: 2171-2181Crossref PubMed Scopus (95) Google Scholar, 10Cymerman U Vera S Pece-Barbara N Bourdeau A White Jr, RI Dunn J Letarte M Identification of hereditary hemorrhagic telangiectasia type 1 in newborns by protein expression and mutation analysis of endoglin.Pediatr Res. 2000; 47: 24-35Crossref PubMed Scopus (80) Google Scholar, 11Bourdeau A Cymerman U Paquet ME Meschino W McKinnon WC Guttmacher AE Becker L Letarte M Endoglin expression is reduced in normal vessels but still detectable in arteriovenous malformations of patients with hereditary hemorrhagic telangiectasia type 1.Am J Pathol. 2000; 156: 911-923Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Haploinsufficiency in ALK-1 also seems to be associated with HHT2.12Abdalla SA Pece-Barbara N Vera S Tapia E Paez E Bernabeu C Letarte M Analysis of ALK-1 and endoglin in newborns from families with hereditary hemorrhagic telangiectasia type 2.Hum Mol Genet. 2000; 9: 1227-1237Crossref PubMed Scopus (105) Google Scholar Both endoglin and ALK-1 are components of the transforming growth factor-β (TGF-β) superfamily of receptors that are predominantly expressed on vascular endothelium. Endoglin cannot bind ligands of the TGF-β superfamily by itself. However it binds TGF-β1, TGF-β3, activin-A, and bone morphogenetic protein-7 and -2 via association with their respective ligand-binding receptors.13Barbara 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-β superfamily.J Biol Chem. 1999; 274: 584-594Crossref PubMed Scopus (501) Google Scholar Endoglin modulates several cellular responses to TGF-β1 but its role in regulating effects of other ligands has yet to be demonstrated.14Lastres P Letamendía A Zhang H Rius C Almendro N Raab U López LA Langa C Fabra A Letarte M Bernabéu C Endoglin modulates cellular responses to TGF-β1.J Cell Biol. 1996; 133: 1109-1121Crossref PubMed Scopus (283) Google Scholar, 15Guerrero-Esteo M Lastres P Letamendia A Perez-Alvarez MJ Langa C Lopez LA Fabra A Garcia-Pardo A Vera S Letarte M Bernabeu C Endoglin overexpression modulates cellular morphology, migration, and adhesion of mouse fibroblasts.Eur J Cell Biol. 1999; 78: 614-623Crossref PubMed Scopus (84) Google Scholar ALK-1 is a type I receptor recently shown to bind TGF-β1 in endothelial cells.16Oh SP Seki T Goss KA Imamura T Yi Y Donahoe PK Li L Miyazono K ten Dijke P Kim S Li E Activin receptor-like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis.Proc Natl Acad Sci USA. 2000; 97: 2626-2631Crossref PubMed Scopus (737) Google Scholar Thus the receptor complex for TGF-β1 on vascular endothelium contains endoglin associated with the ligand binding receptor, TβR-II, and signaling via the type I receptors ALK-1 or ALK-5. Recently a crucial role for endoglin in angiogenesis was demonstrated in mice deficient in the Endoglin (Eng) gene, which showed multiple vascular and cardiac defects leading to death in early embryos.17Li DY Sorensen LK Brooke BS Urness LD Davis EC Taylor DG Boak BB Wendel DP Defective angiogenesis in mice lacking endoglin.Science. 1999; 284: 1534-1537Crossref PubMed Scopus (727) Google Scholar, 18Bourdeau A Dumont DJ Letarte M A murine model of hereditary hemorrhagic telangiectasia.J Clin Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (392) Google Scholar, 19Arthur 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 TGFβ receptor, is required for extraembryonic angiogenesis and plays a key role in heart development.Dev Biol. 2000; 217: 42-53Crossref PubMed Scopus (384) Google Scholar From embryonic day 9.0, the primitive vascular plexus of yolk sac failed to remodel into mature vessels causing vascular channel dilation, rupture, and hemorrhage. Internal bleeding was also observed in the embryo implying vessel fragility. Endocardial cushion formation, essential for valve development and heart septation did not occur. Pericardial edema was also observed.18Bourdeau A Dumont DJ Letarte M A murine model of hereditary hemorrhagic telangiectasia.J Clin Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (392) Google Scholar The yolk sac defects in Endoglin null embryos were similar to those observed in mice lacking ALK-1, TGF-β1, TβR-II, and Smad5.16Oh SP Seki T Goss KA Imamura T Yi Y Donahoe PK Li L Miyazono K ten Dijke P Kim S Li E Activin receptor-like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis.Proc Natl Acad Sci USA. 2000; 97: 2626-2631Crossref PubMed Scopus (737) Google Scholar, 20Dickson MC Martin JS Cousins FM Kulkarni AB Karlsson S Akhurst RJ Defective haematopoiesis and vasculogenesis in transforming growth factor-β1 knock out mice.Development. 1995; 121: 1845-1854Crossref PubMed Google Scholar, 21Oshima M Oshima H Taketo MM TGF-β receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis.Dev Biol. 1996; 179: 297-302Crossref PubMed Scopus (563) Google Scholar, 22Chang H Huylebroeck D Verschueren K Guo Q Matzuk MM Zwijsen A Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects.Development. 1999; 126: 1631-1642Crossref PubMed Google Scholar We also observed that some Endoglin heterozygous mice (referred to as End+/− mice) developed external signs of HHT such as telangiectases and bleeds.18Bourdeau A Dumont DJ Letarte M A murine model of hereditary hemorrhagic telangiectasia.J Clin Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (392) Google Scholar We now report the full characterization and validation of the murine model of HHT. Phenotypic heterogeneity, including severe visceral involvement, is described for 50 End+/−mice with external signs of disease. We also demonstrate that the 129/Ola strain is more susceptible to HHT and has a lower level of plasma TGF-β1 that is further reduced in End+/− mice. Our data suggest that endoglin haploinsufficiency, combined with the effects of modifier genes that regulate TGF-β1 expression, are responsible for the heterogeneity and severity of HHT. End+/− mice were generated by homologous recombination using embryonic stem cells of 129/Ola origin.18Bourdeau A Dumont DJ Letarte M A murine model of hereditary hemorrhagic telangiectasia.J Clin Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (392) Google Scholar Male chimeras were mated with wild-type 129/Ola (129) (Harlan UK, Bicester, UK) giving rise to 129 inbred progeny, homozygous at all alleles but Endoglin. Male chimeras were also mated with C57BL/6 (B6) females (Taconic Farms, Germantown, NY) yielding an F1 progeny, with a heterozygous B6/129 genome. The F1 were backcrossed to wild-type B6 mice, giving an N2 generation, with a 50% probability at any locus of being heterozygous (B6/129) or homozygous for B6 alleles. Intercrosses of End+/− F1 mice yielded the F2 generation with at any particular locus a ratio of genotypes of 1:2:1 for 129 homozygous, B6/129 heterozygous, and B6 homozygous, respectively. Mice were kept in ventilated racks in a germ-free facility and all protocols were approved by the Ethics Committee of the Hospital for Sick Children Laboratory Animal Services. The genotype of each mouse was first assessed by β-galactosidase staining made possible by the presence of a LacZ reporter gene driven by the Endoglin promoter in the targeting construct.18Bourdeau A Dumont DJ Letarte M A murine model of hereditary hemorrhagic telangiectasia.J Clin Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (392) Google Scholar At weaning time (3 weeks), an ear punch from each pup was washed in phosphate-buffered saline, fixed in 0.25% glutaraldehyde for 15 minutes, washed three times for 5 minutes, stained overnight at 30°C in X-gal solution, and observed under a microscope. Blue vessels are associated with End+/− mice because all End−/− embryos die in utero at day 10 to 10.5. The Endoglin genotype was often confirmed by multiplex polymerase chain reaction using tail DNA.18Bourdeau A Dumont DJ Letarte M A murine model of hereditary hemorrhagic telangiectasia.J Clin Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (392) Google Scholar End+/+ and End+/− mice were observed daily and all signs of HHT were recorded in a FileMaker Pro data base. The age of onset of external signs was recorded: telangiectases (location, frequency/week), interstitial bleeding (location, severity, frequency/week), breathing capacity, mobility, weight loss, moribund state, and other manifestations such as ear or tail loss. Internal signs were also carefully examined during autopsy. Each mouse was dissected to evaluate dilation of inner skin vessels, interstitial bleeding and its origin. Organs including spleen, liver, intestine, kidneys, lungs, heart, and brain were analyzed for parameters such as size, color, edema, effusion, prominence of vessels, and presence of telangiectases and hemorrhages (focal or diffuse). Other severe consequences from hemorrhage such as hydrocephalus and stroke were also noted. Histological examination allowed to determine more precisely the site and extent of hemorrhage, number and size of vessels, organization of vessel wall components, ischemia, congestion, and infiltration. Organs from End+/− and control mice were immediately embedded in OCT and frozen on isopentane/dry ice. Cryosections (7 μm) were fixed for 10 minutes in acetone, washed briefly in TBST (0.01 mol/L Tris, pH 7.4, 0.16 mol/L NaCl, 0.2% Tween 20), dipped 5 seconds in 0.1 N HCl to remove endogenous alkaline phosphatase and washed thoroughly in TBST. Sections were then blocked with 5% normal rabbit serum (DAKO, Mississauga, Ontario, Canada) for 20 minutes, blocked sequentially with avidin and biotin solution (Vector Laboratories, Burlington, Ontario, Canada) for 20 minutes, and washed. Sections were incubated at 4°C for 2 hours with optimal concentrations of primary antibodies. These were mAb JC7/18 to endoglin (CD105, purified IgG, 2 μg/ml; Pharmingen, Mississauga, Ontario, Canada), mAb MEC13.3 to PECAM-1 (CD31, purified IgG, 5 μg/ml; Pharmingen), mAb 1A4 to α-smooth muscle cell actin (ascites, diluted 8000-fold; Sigma, Oakville, Ontario, Canada), and nonimmune rat IgG (5 μg/ml, Sigma). Slides were washed and incubated for 1 hour at 4°C with biotinylated rabbit anti-rat IgG (diluted 400-fold, Vector Laboratories). For α-smooth-muscle cell actin detection, biotinylated polyclonal antibody from the LSAB kit was used (DAKO). The streptavidin-alkaline-phosphatase amplification system (StreptABC/AP, DAKO) was used and the enzymatic reaction was performed as described.11Bourdeau A Cymerman U Paquet ME Meschino W McKinnon WC Guttmacher AE Becker L Letarte M Endoglin expression is reduced in normal vessels but still detectable in arteriovenous malformations of patients with hereditary hemorrhagic telangiectasia type 1.Am J Pathol. 2000; 156: 911-923Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Some sections were counterstained with 5% neutral red (Sigma). Tissue morphology was assessed with both frozen and paraffin-embedded sections stained with hematoxylin and eosin and/or Masson's trichrome. Blood was collected from both End+/−and littermate controls End+/+ using heparinized hematocrit tubes by tail bleeds from live mice or at the time of autopsy. A total of 173 mice were analyzed: 129 backcrosses (n = 45), B6 backcrosses (n = 42), and intercrosses (n = 84). The plasma was recovered by centrifugation and kept at −70°C. Plasma was then diluted 1:200 and latent TGF-β was activated by acid treatment with 1 N HCl and lowering to pH 2 for 15 minutes. The samples were then neutralized to pH 7.6 with 1 N NaOH. The TGF-β1 levels were assessed by enzyme-linked immunosorbent assay using TGF-β1 Emax ImmunoAssay system with the internal standard provided, following the manufacturer's instructions (Promega, Madison, WI). Two to four dilutions were done per sample, and all measurements were done in triplicate. Results of the plasma TGF-β levels were analyzed using the statistical software package, SPSS V.5. As the data did not show a normal distribution, the nonparametric Mann-Whitney test was used to compare TGF-β levels between groups. The data are reported as median plus the 25th and 75th percentile values. Group differences with P values < 0.05 were considered significant. Mice with a single allele of the Endoglin gene were found to spontaneously develop clinical signs of HHT. To characterize onset, progression, and mechanism of disease, we studied 171 End+/− mice for a minimum of 25 weeks and a maximum of 52 weeks. Mice who developed early onset of disease and died before 25 weeks were also included. A diagnosis of HHT was made in 50 mice based on their End+/− genotype and at least one of these criteria: the presence of telangiectases on the ears, skin, tail, or genitals; external bleeds from nose/mouth, ears, tail, genitals, or intestine; and/or vascular abnormalities in viscera such as lungs, brain, liver, and intestine. In 90% of cases, telangiectases were the first signs of disease whereas 52% of HHT mice experienced external bleeds. Telangiectases on the ears, and bleeding from nose/mouth and ears were the most frequent external HHT signs in the End+/− mice (Figure 1). Disease severity was highly variable; some mice had a mild phenotype whereas others (32%) reached an agonal phase because of rupture of major vessels that caused fatal internal hemorrhage (Figure 1). To examine visceral involvement, 22 of 50 End+/− mice with life-threatening signs of HHT and/or at an advancing age were sacrificed. Direct microscopic examination of the mesenteric surface of the small intestine revealed a telangiectasia, seen as a network of dilated vessels, in an End+/− HHT mouse but not in the End+/+ littermate control (Figure 2, a and b). Antibodies to endoglin and PECAM-1, specifically stained endothelial cells of arteries, veins, and capillaries in the submucosa in control and End+/− HHT mice (Figure 2, c–f). Abnormally dilated arteries and veins were noted within the submucosa and serosa of the HHT mouse (Figure 2, d and f). Levels of expression of endoglin and PECAM-1 were similar in vessels of the control intestine (Figure 2, c and e). However, in the HHT mouse, endoglin-staining intensity was much reduced, compared to PECAM-1 (Figure 2, d and f). This lower level of endoglin on endothelial cells of End+/− mice reflects the expression of a single allele. We randomly tested 20 of 50 HHT mice for fecal occult blood in their stools and found 11 positive ones including mouse 44.1 described in Figure 1, Figure 2. Although we could not exclude that positive tests were caused by ingesting blood, there were no external bleeds observed in mouse 93.3 and three others with HHT (Figure 1 and data not shown). These findings demonstrate the presence of fecal blood as early as 9 weeks and correlate with the presence of microscopic telangiectases on the intestinal surface. Hematocrit values were normal even in older mice with HHT, unlike in elderly human patients where intestinal bleeds lead to chronic anemia.23Vase P Grove O Gastrointestinal lesions in hereditary hemorrhagic telangiectasia.Gastroenterology. 1986; 91: 1079-1083Abstract Full Text PDF PubMed Scopus (115) Google Scholar On autopsy, gross morphology revealed that 48% of End+/− HHT mice had liver abnormalities, including prominent vessels, telangiectases in one or several segments, focal or severe hemorrhage, and hepatomegaly (Figure 3a). However, histological examination revealed 80% of dissected mice had vessel enlargement, hepatic congestion, and/or hemorrhage (Figure 1). Endoglin was detected on endothelial cells of all types of vessels including central veins and sinusoidal endothelium in End+/+ control mice as illustrated in Figure 3b. Liver sections from an End+/− HHT mouse showed dilated central veins when compared to control, and signs of hepatic congestion in a case of mild disease (Figure 3c). At lower magnification, a marked increase in the number of vessels was noticeable, especially in the subcapsular region in mild cases of disease (Figure 3e) compared to littermate controls (Figure 3d). As disease progressed, sinusoidal dilation and hepatocellular atrophy were seen near the central veins in severe cases of HHT (Figure 3f). Several End+/− HHT mice had difficulty breathing, manifested by tachypnea and marked inspiratory effort. Pulmonary involvement was seen, mostly on the ventral aspect of the upper lobes, by gross morphological examination in 33% of sacrificed HHT mice whereas microscopic analysis revealed abnormalities in 50% of cases (Figure 1, Figure 4). Gross changes included dilated vessels, visible telangiectases, and hemorrhages ranging in severity from focal to diffuse (Figure 4; a, b, and c). Histological sections demonstrated abnormally large vessels with increased thickness of the adventitial layer in lungs of End+/− HHT mice compared to littermate controls (Figure 4, d and e). At higher magnification, congestion of the alveolar capillaries was apparent (Figure 4, f and g). These findings suggest that pulmonary lesions tend to develop more often in the dependent areas of affected lungs, upper lobes in the case of mice and lower lobes in humans. Most of the End+/− HHT mice first showed external signs of disease between 7 and 43 weeks, as described in Figure 1. Animals 77.5, 111.3, and 111.140 were exceptions as they seemed unwell and developed cephalic changes at 2 to 4 weeks of age, before appearance of telangiectasia or external bleeds. These affected mice developed dome-shaped head, limb weakness, kyphosis, lethargy, drowsiness, and emaciation (Figure 5a). On cranial exposure, severe subarachnoid hemorrhage was found, accompanied by an expanded calvarium and underlying brain with hydrocephalus (Figure 5b). Bilateral enlargement of the ventricles with thinning of the cerebral cortex is shown on a cross-section stained with α-smooth muscle cell actin (Figure 5c). Cortical atrophy was likely because of necrosis or apoptosis resulting from elevated intracranial pressure. A higher magnification shows normal smooth muscle cell distribution and endoglin expression on small cerebral vessels (Figure 5, d and e). A fourth case, among the 22 dissected End+/− HHT mice, experienced a subdural hemorrhage followed by hydrocephalus (Figure 1). Other brain manifestations were seen in 45% of End+/− HHT mice after histological examination (Figure 1). These included focal subdural, subgalial, or subarachnoid hemorrhage and brain infarction. Two HHT mice (95.1 and 75.71) had a stroke manifested by hemiplegia, facial flaccidity, and ptosis. Brain histology revealed a localized subarachnoid hemorrhage with blood infiltration into the cortex, not seen in an unaffected adjacent region (Figure 5, f and g). At higher magnification, lymphocytic infiltration and inflamed vessels with fibrinoid necrosis of the media are seen in the hemorrhagic area. A threefold thickening of adventitia and loosening of muscle bundles were also observed. The neuropile was vacuolated and early traces of infarction were seen (Figure 5h). These hemorrhagic strokes are reminiscent of neurological defects observed in human HHT with cerebral involvement.24Kikuchi K Kowada M Sasajima H Vascular malformations of the brain in hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber disease).Surg Neurol. 1994; 41: 374-380Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 25Kadoya C Momota Y Ikegami Y Urasaki E Wada S Yokota A Central nervous system arteriovenous malformations with hereditary hemorrhagic telangiectasia: report of a family with three cases.Surg Neurol. 1994; 42: 234-239Abstract Full Text PDF PubMed Scopus (38) Google Scholar Pulmonary and hepatic congestion were observed concurrently in 9 of 22 End+/− HHT mice along with hypertrophy of the myocardium (50 to 500%) suggesting congestive heart failure (Figure 1). Heart sections demonstrated biventricular hypertrophy with dilatation especially of the left atria, and of the coronary arteries (Figure 6). Organizing thrombi such as shown in Figure 6b were seen in the atrium of four mice with HHT and could have caused embolic events to brain and coronary arteries. Ischemic regions associated with muscle necrosis were noted in three animals (Figure 6, a and b). Immunostaining of endoglin confirmed the vascular hypertrophy seen in some animals and revealed large dilated coronary vessels (Figure 6, c and d). Cardiac failure was likely secondary to the high output from the dilated hepatic and possibly pulmonary arterial circuits. Our findings suggest two major causes of death in End+/− HHT mice: massive hemorrhage occurring after rupture of a major vessel, such as abdominal aorta, renal artery, and cerebral arteries, or chronic congestive heart failure, occurring as a result of high output failure because of pulmonary, hepatic, or coronary vascular abnormalities. For analysis of phenotype/genotype correlations, the age of onset and the various HHT manifestations were recorded. Seventy-two percent of End+/− mice on the 129 background observed for 41.8 ± 12.6 weeks, and 36% of End+/− F2 intercrosses observed for 43.6 ± 8.9 weeks, developed HHT whereas only 7% of B6 backcrosses (N2) did when observed for an even significantly longer time of 48.7 ± 14.3 weeks (P = 0.002). The age of onset was highly variable. It ranged from 1 to 37 weeks in 129 mice, 14 to 43 weeks in F2 intercrosses, and 37 to 51 weeks in B6 backcrosses (Figure 7). These data indicate an earlier onset and higher susceptibility to HHT in 129 than B6 strain and intermediate age of onset and disease prevalence in F2 intercrosses, suggesting that the 129 background contributes some disease modifier alleles. TGF-β1 has multiple effects that include the regulation of its own production. Endoglin as a component of the receptor complex, is capable of modulating several responses to TGF-β1.25Kadoya C Momota Y Ikegami Y Urasaki E Wada S Yokota A Central nervous system arteriovenous malformations with hereditary hemorrhagic telangiectasia: report of a family with three cases.Surg Neurol. 1994; 42: 234-239Abstract Full Text PDF PubMed Scopus (38) Google Scholar We ascertained whether plasma TGF-β1 levels were different in the two strains studied and whether endoglin reduction could further alter these levels. Initial studies revealed no detectable active TGF-β1 in the plasma. We thus measured levels of total TGF-β1 (latent plus active, after acid treatment) in plasma from 173 End+/+ and End+/− mice of 129 strain and B6 background (backcrosses and intercrosses). As the distribution of TGF-β1 in the various groups did not follow a normal distribution, the median and the 25th and 75th percentiles are shown for each group in Table 1. The no
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