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Regular Article |
From the Cancer and Blood Program,*
The Hospital
for Sick Children and Department of Immunology, University of Toronto,
Toronto; the Division of Respiratory Medicine,
Department of Medicine, St. Michael’s Hospital, University of Toronto,
Toronto; The Centre for Applied Genomics,
The
Hospital for Sick Children and Department of Public Health Sciences,
University of Toronto, Toronto; and the Department of Laboratory
Medicine and Pathobiology,
University of
Toronto Health Network, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1:8000.1
HHT is primarily associated with
frequent nosebleeds (epistaxis), telangiectases, and internal vascular
lesions.2
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.3 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 HHT14 and ACVRL1 (ACTIVIN RECEPTOR-LIKE KINASE 1, also known as ALK-1), mutated in HHT2.5 HHT1 is associated with a higher incidence of pulmonary AVMs than HHT2, which generally has a later onset.6 Severity of HHT is not correlated with the type of mutation or its position.7 Mutated ENG is rarely expressed in HHT1 patients and only as an intracellular species.8 The current model for HHT1 is haploinsufficiency, because of reduced levels of functional endoglin at the surface of endothelial cells.8-11 Haploinsufficiency in ALK-1 also seems to be associated with HHT2.12
Both endoglin and ALK-1 are components of the transforming growth factor-ß (TGF-ß) superfamily of receptors that are predominantly expressed on vascular endothelium. Endoglin cannot bind ligands of the TGF-ß superfamily by itself. However it binds TGF-ß1, TGF-ß3, activin-A, and bone morphogenetic protein-7 and -2 via association with their respective ligand-binding receptors.13 Endoglin modulates several cellular responses to TGF-ß1 but its role in regulating effects of other ligands has yet to be demonstrated.14,15 ALK-1 is a type I receptor recently shown to bind TGF-ß1 in endothelial cells.16 Thus the receptor complex for TGF-ß1 on vascular endothelium contains endoglin associated with the ligand binding receptor, TßR-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.17-19 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.18 The yolk sac defects in Endoglin null embryos were similar to those observed in mice lacking ALK-1, TGF-ß1, TßR-II, and Smad5.16,20-22 We also observed that some Endoglin heterozygous mice (referred to as End+/- mice) developed external signs of HHT such as telangiectases and bleeds.18
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-ß1 that is further reduced in End+/- mice. Our data suggest that endoglin haploinsufficiency, combined with the effects of modifier genes that regulate TGF-ß1 expression, are responsible for the heterogeneity and severity of HHT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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End+/- mice were generated by homologous recombination using embryonic stem cells of 129/Ola origin.18 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.
Genotyping of Mice
The genotype of each mouse was first assessed by ß-galactosidase staining made possible by the presence of a LacZ reporter gene driven by the Endoglin promoter in the targeting construct.18 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.18
HHT Phenotype Assessment
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.
Immunohistochemical Staining
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 
-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 -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.11
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.
Measurement of Plasma TGF-ß1 Levels
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-ß 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-ß1 levels were assessed by enzyme-linked immunosorbent assay using TGF-ß1 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.
Statistical Analysis
Results of the plasma TGF-ß 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-ß 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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)
.
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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 Figures 1 and 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.23
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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)
.
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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 (Figures 1 and 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.
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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 -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)
.
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. 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.24,25 Cardiac Changes in End+/- Mice with HHT
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.
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TGF-ß1 Plasma Levels Influenced by 129 Background Genes
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.
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. The nonparametric
Mann-Whitney test revealed that TGF-ß1 plasma levels were
significantly lower in End+/+ 129 than in
both End+/+ B6 (P =
0.025) and End+/+ intercrosses
(P = 0.003). This implies that the 129 mouse
strain has lower circulating levels of TGF-ß1 than the B6 strain.
This observation is further supported by comparing TGF-ß1 levels of
End+/- 129 mice with those of
End+/- B6 backcrosses
(P = 0.001). Because the intercrosses have a
mixed genome, a trend but no significant difference was achieved by
comparing End+/- 129 mice and
End+/- intercrosses
(P = 0.077). Furthermore, expression of a single
allele of Endoglin seems to lead to a further reduction in
the level of circulating TGF-ß1 because
End+/- intercrosses had lower levels than
End+/+ littermate controls
(P = 0.001). These results indicate that the
lower level of plasma TGF-ß1 seen in 129 mice combined with the
effect of reduced endoglin in End+/- could
contribute to the development of HHT.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine model of HHT reproduces the human disease. For example, cutaneous telangiectases are found in 90% of End+/- mice with HHT (ears mostly) and present in 80 to 90% of people (nasal and labial mucosae, skin of face and hands).1 It is more appropriate to compare visceral involvement in murine HHT to that of human HHT1, because pulmonary and cerebral AVMs are much more frequent in HHT1 than HHT2.6,10,26 Although AVMs could not be directly visualized, multiple abnormally dilated vessels along with focal and diffuse hemorrhage were found in the lungs of 50% of End+/- mice with HHT, which is similar to that observed in HHT1 patients. Intracranial hemorrhage was seen in 30% of mice with HHT and occurred at 2 to 4 weeks of age in 3 of 22 cases, before any external sign of disease. Similarly, fatal hemorrhage because of rupture of a cerebral AVM has been reported in infants and newborns with HHT24,27 and confirmed in a newborn with HHT1.11 Hydrocephalus was observed in 8% of mice with HHT but has only been reported in one human HHT case.28 It likely occurred subsequent to the massive subarachnoid hemorrhage, as documented for several human cases of hydrocephalus.29,30 Liver abnormalities such as dilated vessels, congestion, and/or hemorrhage were seen in 80% of HHT mice, based on histological examination. Case reports of HHT have also described disseminated intrahepatic telangiectases, which might be present but remain asymptomatic in many patients. Serious liver complications, seen in 20% of HHT patients include heart failure, portal hypertension, biliary disease, ascites, and nodular transformation.31-36 The murine model of HHT will help elucidating the mechanisms that lead to the initiation and progression of vascular abnormalities. A telangiectasia arises from the dilation of a postcapillary venule and direct fusion with an arteriole, bypassing the capillary network.37 This implies that regulation of the normal angiogenic process of vessel branching is altered in HHT. The 50% reduction in endoglin observed in endothelial cells of all vessels of individuals with an Endoglin mutation8,10,11 and in mice engineered to express a single allele of the gene must predispose vessels to dilation. However, additional genetic and environmental factors seem necessary to trigger the development of vascular abnormalities as suggested by their heterogeneity in human and mice with a single functional copy of endoglin.
Our observations that End+/- mice on a 129 background develop disease whereas those on the B6 background do not, suggest that allelic variations between strains are responsible for the marked differences in vascular phenotype. To confirm the presence of a genetic modifier and exclude the influence of an unrecognized environmental factor, intercrosses of End+/- heterozygous F1 offspring were examined. The F2 mice showed intermediate disease prevalence, consistent with the inheritance of modifier alleles from both strains. Thus a single Endoglin allele is necessary but not sufficient to cause disease, because a large proportion of End+/- mice do not display clinical signs, when observed for 1 year. The 129 mice are high angiogenesis responders compared to B6 and other inbred strains of mice,38 supporting the presence of genetic factors that control angiogenic potential and blood vessel homeostasis. In particular, the majority of wild-type 129/Ola mice were shown to have reduced numbers of peripheral vessels in liver and lungs and to exhibit natural large intrahepatic connections between portal and hepatic veins, when compared to B6 mice.39,40 This might in part also explain the high susceptibility of this strain to hepatic manifestations of HHT.
Lower levels of plasma TGF-ß1 observed in 129/Ola mice might be indicative of a vascular system susceptible to dilation. Circulating TGF-ß1 serves as a prognostic marker for several diseases. Elevated levels have been associated with the pathogenesis of chronic fibrotic and autoimmune diseases, atherosclerosis, and carcinogenesis, whereas deficient levels have been reported in stenosis of major coronary vessels.41 Our observations that End+/- mice have reduced levels of plasma TGF-ß1 suggest that HHT is not only associated with propensity to vasodilation but also with less circulating TGF-ß1. Our preliminary data show that umbilical vein endothelial cells derived from newborns with an Endoglin mutation, produce significantly less TGF-ß1 than do normal ones (M Letarte, ML McDonald, S Vera, Hospital for Sick Children, Toronto, unpublished 2000). This suggests that endothelial cells contribute to the production of plasma TGF-ß1.
TGF-ß1 also acts by autocrine and paracrine mechanisms that are likely to play a role in the pathology of HHT. TGF-ß1 can stimulate the activity of its own promoter, and this autoregulation might explain the prolongation of secretion and autocrine action of TGF-ß1 after an initial stimulus.42 If endothelial cells from End+/- mice secrete less TGF-ß1 than normal cells, we can propose that the level of endoglin controls this autoregulatory pathway. Less endoglin would then lead to reduced autocrine effects of TGF-ß1. This factor plays a crucial role in vascular homeostasis by regulating the synthesis of extracellular matrix proteins that stabilize interactions between endothelial, mesenchymal, and smooth muscle cells of the vessel wall.20,43 A decrease in both local and circulating TGF-ß1 levels, will lead to unstable cellular interactions in the vessel wall, dilated vessels, and vascular abnormalities. Such alterations could impair other angiogenic regulatory mechanisms and lead to deterioration of the vascular network associated with the progression of HHT. Reduced TGF-ß1 levels must play a role in vascular remodeling of cerebral and pulmonary human AVMs, that leads to extremely dilated and tortuous vessels with variable thickness of smooth muscle cells, disorganized adventitia, and active angiogenesis.11 Such changes were also observed in mice at advanced stages of the disease. A dysregulation in the mechanisms responsible for maintaining interactions between intimal, medial, and adventitial layers of vessels is thus the likely cause of the progression and expansion of vascular lesions.
Our data demonstrate that one or more variable genetic loci in the mouse exert a profound modifying influence on the HHT phenotype. The variable expression within families also suggests an important role for genetic modifiers. Consistent with this hypothesis, we propose that the co-inheritance of mutated Endoglin and specific modifier alleles, predisposes to severe manifestations including the formation of cerebral, pulmonary, and hepatic AVMs. Factors such as increased blood volume and cardiac output combined with hormonal changes such as those observed during pregnancy could then precipitate the growth of AVMs.26 In the absence of modifier genes, mild disease can still occur, and factors such as environment, blood pressure, oxygenation, and shear forces could influence the location of telangiectases.
Modifier alleles that modulate phenotypic outcome in a strain-dependent manner have been reported for several genes including those that control embryonic lethality in the absence of TGF-ß1.44 We can speculate that distinct modifier alleles might contribute to liver, lung, and brain involvement in HHT. Molecules implicated in vascular development and more specifically in the TGF-ß pathway such as TßR-II, ALK-1, ALK-5, and downstream signaling Smads are candidates for genetic mutations or polymorphisms that could affect HHT1. ALK-1, the product of the gene mutated in HHT2, was shown recently to bind TGF-ß1 in endogenous endothelial cells and to be present in a receptor complex in association with TßR-II and endoglin.16 A model was proposed whereby normal angiogenesis would require a balance between activation by TGF-ß1 of ALK-1- and ALK-5-signaling pathways with their respective Smads.16 Endoglin, which can be present in either the ALK-1 or ALK-5 receptor complex, could thus modulate endothelial cell responses to TGF-ß1 via both pathways. Thus modifier genes contributing to the pathophysiology of HHT may code for molecules that participate in the TGF-ß receptor complex or downstream molecules activated on signaling through this complex.
Murine HHT mimics the human disease and provides an excellent model for studying the mechanisms responsible for initiation and progression of this complex vascular disorder. We have demonstrated that endoglin is essential for maintenance of vascular homeostasis as expression of a single allele can lead to abnormal vessels. Our results clearly show genetic variation in susceptibility to disease and suggest that characterization of the murine modifier genes could lead to the identification of susceptibility alleles influencing severity of HHT in human.
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Acknowledgements |
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Footnotes |
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Supported by grant no. NA3434 from the Heart and Stroke Foundation of Ontario, grant no. MT-6247 from the Canadian Institute of Health Research, and an industrial grant from Syn X Pharma Inc.
A. B. was a recipient of a Studentship from the Medical Research Council of Canada; M. E. F. is supported by the Nelson Arthur Hyland Foundation and the Squires Club of Toronto; M. L. is a Terry Fox Research Scientist of the National Cancer Institute of Canada.
Accepted for publication March 7, 2001.
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 Y. Tang, M. L. McKinnon, L. M. Leong, S. A. B. Rusholme, S. Wang, and R. J. Akhurst Genetic modifiers interact with maternal determinants in vascular development of Tgfb1-/- mice Hum. Mol. Genet., July 1, 2003; 12(13): 1579 - 1589. [Abstract] [Full Text] [PDF]
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 S. E. DUFF, C. LI, J. M. GARLAND, and S. KUMAR CD105 is important for angiogenesis: evidence and potential applications FASEB J, June 1, 2003; 17(9): 984 - 992. [Abstract] [Full Text] [PDF]
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D. A. Marchuk, S. Srinivasan, T. L. Squire, and J. S. Zawistowski Vascular morphogenesis: tales of two syndromes Hum. Mol. Genet., April 2, 2003; 12(90001): R97 - 112. [Abstract] [Full Text] [PDF]
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S. van den Driesche, C. L. Mummery, and C. J.J. Westermann Hereditary hemorrhagic telangiectasia: an update on transforming growth factor {beta} signaling in vasculogenesis and angiogenesis Cardiovasc Res, April 1, 2003; 58(1): 20 - 31. [Abstract] [Full Text] [PDF]
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E. Torsney, R. Charlton, A. G. Diamond, J. Burn, J. V. Soames, and H. M. Arthur Mouse Model for Hereditary Hemorrhagic Telangiectasia Has a Generalized Vascular Abnormality Circulation, April 1, 2003; 107(12): 1653 - 1657. [Abstract] [Full Text] [PDF]
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 J. Satomi, R. J. Mount;, M. Toporsian, A. D. Paterson, M. C. Wallace, R. V. Harrison, and M. Letarte Cerebral Vascular Abnormalities in a Murine Model of Hereditary Hemorrhagic Telangiectasia Stroke, March 1, 2003; 34(3): 783 - 789. [Abstract] [Full Text] [PDF]
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 A. Rodriguez-Pena, N. Eleno, A. Duwell, M. Arevalo, F. Perez-Barriocanal, O. Flores, N. Docherty, C. Bernabeu, M. Letarte, and J. M. Lopez-Novoa Endoglin Upregulation During Experimental Renal Interstitial Fibrosis in Mice Hypertension, November 1, 2002; 40(5): 713 - 720. [Abstract] [Full Text] [PDF]
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