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Clinical Chemistry 50: 2003-2011, 2004. First published September 16, 2004; 10.1373/clinchem.2004.035287
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(Clinical Chemistry. 2004;50:2003-2011.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Mutation Analysis in Spanish Patients with Hereditary Hemorrhagic Telangiectasia: Deficient Endoglin Up-regulation in Activated Monocytes

Francisco Sanz-Rodriguez1,2,2, Africa Fernandez-L.1,2, Roberto Zarrabeitia3, Alfonso Perez-Molino3, Jose R. Ramírez4, Eliecer Coto5, Carmelo Bernabeu1 and Luisa M. Botella1,a

1 Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
2 Departamento de Biología y Genética, Facultad de Biología, Universidad Autonoma de Madrid, Madrid, Spain.
3 Servicio de Medicina Interna, Hospital Sierrallana, Torrelavega (Cantabria), Spain.
4 Departamento de Anatomia Patológica, Hospital Central de la Defensa, Gómez Ulla, Madrid, Spain.
5 Genética Molecular-Instituto Reina Sofia de Investigación Renal, Hospital Central de Asturias, Oviedo, Spain.

aAddress correspondence to this author at: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, 28040 Madrid, Spain. Fax 34-915360432; e-mail cibluisa{at}cib.csic.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Mutations in the endoglin (ENG) or ALK1 genes are responsible for hereditary hemorrhagic telangiectasia types 1 and 2 (HHT1 and HHT2), respectively, a dominant vascular dysplasia caused by haploinsufficiency. No formal mutation studies of patients with HHT have been conducted in Spain.

Methods: ENG and ALK1 mutation analyses were carried out in 13 Spanish HHT patients diagnosed according to the Curaçao criteria. Because endoglin is up-regulated at the cell surface during the monocyte-macrophage transition, endoglin concentrations in activated monocytes were determined by immunofluorescence flow cytometry in a systematic analysis. As controls, 40 non-HHT volunteers were studied for up-regulation of endoglin in activated monocytes.

Results: The mutation responsible for HHT was identified in eight patients belonging to two unrelated families. One of the families has a nonsense mutation in exon 4 (c.511C>T; R171X) of the ENG gene, and accordingly the disorder was identified as HHT1. The other family has a missense mutation affecting exon 8 (c.1120C>T; R374W) of the ALK1 gene, and hence is a HHT2 family. Interestingly, endoglin up-regulation was deficient in activated monocytes of both HHT1 and HHT2 patients compared with controls. By contrast, endoglin up-regulation was age-independent in control donors across a broad range of ages. The extent of endoglin up-regulation in activated monocytes was most diminished in those patients with the most severe symptoms.

Conclusions: Endoglin up-regulation in activated monocytes is impaired in HHT1 and HHT2 patients and is age-dependent in both HHT types. Endoglin expression may predict the clinical severity of HHT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hereditary hemorrhagic telangiectasia (HHT;1 OMIM 187300), or Rendu–Osler–Weber syndrome, is an inherited autosomal dominant disease affecting 1 in 10 000 individuals as assessed in several human populations(1)(2). HHT exhibits age-dependent onset and variable penetrance(1)(3)(4). The disease is characterized by abnormal vascular structures, which lead to epistaxis, telangiectasia, and anemia as well as visceral arteriovenous malformations (AVMs) in the lung, brain, and liver. These may contribute to serious health outcomes, such as stroke, seizures, brain abscess, and hemorrhage(5).

HHT is a genetically heterogeneous disorder. The first locus, HHT1, was assigned to chromosome 9q33–34 where endoglin (ENG) was identified as the mutated gene(6)(7). Endoglin (CD105) is a transmembrane homodimeric glycoprotein produced mainly in endothelial cells, but is also present at lower concentrations in activated monocytes(8)(9)(10). It may function as an auxiliary transforming growth factor-ß (TGF-ß) co-receptor(11)(12) and as a modulator of cell migratory and adhesive properties(13)(14). The second locus, HHT2, is the ALK1 (activin receptor-like kinase 1) gene and maps to chromosome 12q(15)(16). ALK1 is also expressed on endothelial cells, and ALK1 is a type I receptor for the TGF-ß superfamily. TGF-ß and other members of this superfamily signal through type I and II serine/threonine kinase receptors(17). After ligand binding, the type II receptor recruits the type I receptor, and the latter is transphosphorylated, leading to its activation. Once activated, the type I receptor transduces signals by phosphorylating a family of transcriptional coactivators, the Smads(18), which leads to their translocation to the nucleus and interaction with TGF-ß-responsive genes. Because endoglin and ALK1 are involved in the TGF-ß signaling pathway and mutations in either of the genes coding for these proteins lead to HHT, it is hypothesized that both proteins participate in a common mechanism of vascular development and/or homeostasis.

The development of powerful molecular approaches for amplifying and sequencing genomic DNA from HHT patients allows, in most cases, determination of the genetic mutation responsible for the disease. However, the provision of a sequence-based diagnostic service is hampered by the predominance of novel mutations in affected individuals (at least in ENG and ALK1) and difficulties in detecting mutations in the heterozygous state, especially when a single base change underlies the cause of the disease. Thus, the diagnosis of HHT currently remains at the clinical level. Consequently, on behalf of the Scientific Advisory Board of the HHT Foundation, a consensus clinical profile, known as the Curaçao criteria, was developed and is currently used for clinical diagnosis of HHT(19). These criteria include four traits: epistaxis (spontaneous, recurrent nose bleeds); telangiectasias (multiple at characteristic sites, such as the lips, oral cavity, fingers, and nose); visceral lesions (AVMs in the lung, brain, liver, or spinal cord); and a family history. In accordance with these criteria, the HHT diagnosis is definite if three of four of these criteria are present; it is possible or suspected if two criteria are present and unlikely if fewer than two criteria are present.

Haploinsufficiency of the endoglin protein is currently accepted as the molecular basis for HHT1. Mutated endoglin proteins do not appear to be produced at the cell surface, and some nonsense mutations give rise to undetectable mRNA transcripts(3)(20)(21), leading to a loss of protein production. Haploinsufficiency has also been reported for HHT2(22)(23). These observations suggest that direct measurement of the endoglin protein would constitute a useful diagnostic approach. However, the restricted expression of ENG and ALK1, mainly in endothelial cells, hampers direct measurement of the concentrations of the proteins encoded by these genes. In a few cases of HHT pregnancies, endothelial cells derived from the umbilical cord vein (HUVECs) have been used as a tissue source for the direct study of endoglin mRNA and protein concentrations(9)(10)(22).

Interestingly, endoglin is up-regulated during the process of monocyte activation(8). This property has recently been used to quantify the relative endoglin concentrations in monocytes derived from HHT1 patients(9)(10). These authors concluded, however, that the true sensitivity and specificity of the test were difficult to establish. In this work, we present the first study formally conducted in Spain with HHT patients, of mutation analysis accompanied by flow cytometric quantification of endoglin up-regulation in activated monocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
cell culture
Peripheral blood mononuclear cells (PBMCs) were obtained from whole venous blood. Briefly, 5 mL of blood were used to recover mononuclear cells by Ficoll density gradient centrifugation using Lymphoprep Separation Medium (ICN) and centrifugation at 1500g for 30 min at 22 °C. Mononuclear cells were subsequently collected and washed in phosphate-buffered saline (PBS). Partially differentiated monocytes were subsequently obtained by plating of mononuclear cells on plastic in 6-cm wells of P-6 Falcon plates. Plated cells were incubated for 21 h in DMEM (Gibco BRL) supplemented with 100 mL/L fetal calf serum.

blood samples, mutation analysis, and reverse transcription-pcr
Informed consent was obtained from all HHT patients and healthy non-HHT volunteers participating in the study. Positive diagnoses of HHT were based on the Curaçao criteria(19). The mutations in individuals analyzed in this study were confirmed by automated DNA sequence analysis. Genomic DNA was isolated from peripheral blood lymphocytes by use of the QIAamp Mini Kit (Qiagen). The 15 exons of the ENG gene were amplified by PCR using HotMaster Polymerase (Eppendorf) and sequenced by a cycle-sequencing protocol (Applied Biosystems) using previously reported primers(20). The nine exons of the ALK1 gene were amplified and sequenced as described above, using primers reported elsewhere(22)(23). For reverse transcription-PCR (RT-PCR), total monocyte RNA was extracted by use of the RNAeasy Kit (Qiagen). RNA (1 µg) from monocytes plated for 20 min or 21 h was subjected to RT-PCR using the AMV reverse transcriptase (La Roche). One fourth of the total RNA-derived cDNA was subjected to PCR as described above, with primers corresponding to the coding region of exon 7 of ALK1: sense, 5'-CTTCATCGCCTCAGACAT-3'; antisense, 5'-GATGCAACACTGCAGGTT-3'.

flow cytometry
For identification of human peripheral blood monocytes, fresh PBMCs and PBMCs cultured for 21 h were aliquoted and stained with a monoclonal antibody (mAb) cocktail containing anti-CD3, anti-CD14, anti-CD16, anti-CD11c, and anti-CD11b. Subsequently, cells were washed twice with cold PBS and incubated with the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Dako). Finally, cells were washed with cold PBS and fixed with a solution of 37 mL/L formaldehyde in cold PBS. A minimum of 10 000 stained cells were analyzed in a Coulter Epics XL flow cytometry system. Cells that were negative for CD3 and CD16 and positive for CD11c, CD11b, and CD14 were subsequently analyzed for endoglin production.

For analysis of endoglin up-regulation in differentiated monocytes, normal and HHT PBMCs were obtained, and monocytes were differentiated to macrophages by culture for 21 h as described above. For single-color flow cytometry, PBMCs from 10 mL of blood were divided equally and used at times 0 and 21 h after adherence. PBMCs or activated monocytes were then incubated with saturating amounts of mAb P4A4 [anti-endoglin(24)], Bear1 (anti-CD11b; kindly provided by Dr. Carl Figdor, University Medical Center, Nijmegen, The Netherlands), or an isotype-matched control for 30 min at 4 °C in cold PBS. Incubations were conducted in the presence of human {gamma}-globulins to block nonspecific binding and were followed by incubation with FITC-conjugated F(ab')2 goat anti-mouse IgG.

Samples were analyzed by flow cytometry as described above. Dead cells and contaminating lymphocytes were excluded by their forward- and side-scatter properties. A gate was set on CD11b+ cells, and these were analyzed for endoglin production. Endoglin values are reported in terms of the expression index, which is the product of the percentage of cells producing endoglin (positive cells) multiplied by the mean fluorescence intensity of the entire cell population (positive and negative).

statistical analysis
To analyze the age dependence of the endoglin expression index in non-HHT patients, we calculated the Pearson correlations among complete variables (age and endoglin expression index at 0 and 21 h) and the pooled correlations, taking into account that the factor-defining data originated from six sets of experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
endoglin up-regulation during activation of monocytes
In the present work, endoglin up-regulation in the monocyte/macrophage transition was studied in controls and in HHT patients. The methodology for the flow cytometric analysis was essentially as described previously(8)(9)(10). Forward- and side-scatter analysis of control PBMCs together with single-color flow cytometry analysis allowed the identification of monocytes as a CD14-positive, CD3-negative, CD16-negative, CD11b-positive population (data not shown). We determined the basal concentrations of endoglin on freshly PBMCs and on cells obtained after 21 h of adherence (Fig. 1 ). At time 0 h, monocytes produced low amounts of CD105 (12.4%, with a mean fluorescence intensity of 0.4). After 21 h of adherence, there was no significant change in CD11b production in the activated monocyte population, but 86% of these cells became CD105+, with a 2.3-fold increase in the mean fluorescence intensity. At all time points, the gate representing lymphocytes was CD3+ and <5% of the CD105+ mean fluorescence (data not shown).



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  		Figure 1. Flow cytometry profiles from PBMCs.

Healthy PBMCs from 10 mL of blood were equally divided into 5-mL aliquots for two analyses. Freshly isolated PBMCs (0 h) and PBMCs that had undergone 21 h of monocyte activation by plastic adherence in culture (21 h) were subjected to single-color flow cytometry using mAb P4A4 (anti-CD105) followed by FITC-conjugated F(ab')2 goat anti-mouse IgG. The background produced by the control irrelevant antibody was subtracted from the final analysis of endoglin (CD105). The percentage of positive cells and the mean fluorescence intensity (in parentheses) are indicated.

analysis of endoglin up-regulation in a panel of non-hht individuals
Cymerman et al.(10) stressed the need for a large number of probands to establish the sensitivity and specificity of the predictive value of endoglin up-regulation in activated monocytes for HHT. Hence, we surveyed endoglin up-regulation in activated monocytes in blood samples from 40 non-HHT volunteers covering a broad age range (20–80 years). This presumptive non-HHT population was based on the absence of Curaçao criteria symptoms for the disease(19).

The specific flow cytometry technique was used in six series of experiments encompassing a total of 40 donors, averaging a minimum of 3 individuals per decade of age. In all cases, the apparent endoglin concentration was measured by flow cytometry using a mAb to human endoglin. The amount of endoglin was recorded in terms of the expression index (Fig. 2A ). Although at 0 time the endoglin expression index in monocytes was almost negligible (0.06–12), endoglin production was markedly up-regulated after 21 h in culture, with endoglin expression index values fluctuating between 120 and 210; the mean for the controls was 175. This variability is likely derived from the blood collection process, fluctuations attributable to variation in hormonal and growth factor (i.e., TGF-ß) concentrations(10), sample processing, and antibody reactivity inherent to each sample set collected and processed at the same time.



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  		Figure 2. Endoglin expression indexes in monocytes at 0 and 21 h of differentiation from non-HHT and HHT patients.

(A), expression index of endoglin in monocytes from non-HHT volunteers after 0 and 21 h of incubation. PBMCs from 10 mL of blood from non-HHT volunteers were equally divided into 5-mL aliquots for two analyses of endoglin (CD105) production by flow cytometry. One analysis was performed with freshly isolated PBMCs (0 h; dotted line), whereas the other was performed after 21 h of incubation in culture medium at 37 °C (21 h; solid line). The endoglin values at 0 and 21 h are reported as the expression index (the result of multiplying the percentage of positive cells and the mean fluorescence intensity). The non-HHT population included volunteers spanning a broad age range. Each sample was analyzed in duplicate, and each age decade is represented by the mean of at least three different donors. No apparent correlation between age and endoglin expression index is apparent in activated monocytes (after 21 h incubation). (B), endoglin up-regulation is decreased in HHT patients and inversely related to their age and the severity of symptoms. Up-regulation of endoglin in partially differentiated monocytes is shown after adherence for 0 or 21 h. The endoglin concentration is reported as the expression index. Flow cytometry measurements were made on duplicate samples from 13 HHT patients diagnosed according to the Curaçao criteria. Endoglin up-regulation shows an inverse age-dependent correlation. * indicate samples from patients with markedly low up-regulation. Two 45-year-old patients are included (45a and 45b). The mutation for patient 45a had not been identified by the time of publication, and patient 45b had a mutation in ALK1, as indicated in Fig. 2Up of the online Data Supplement. The low endoglin up-regulation of the 45a sample is associated with severe symptoms. The dotted line near the center of the graph indicates the theoretical 50% up-regulation in endoglin according to the heterozygous condition of the mutations and the haploinsufficiency hypothesis. The dot-dashed line indicates the mean for the non-HHT population and is shown for comparison.

The existence of a putative age dependence in endoglin up-regulation during the monocyte-macrophage transition in the control population was discarded based on two statistical tests: (a) the Pearson general correlation test using all 40 probands, and (b) a matrix of intragroup correlations taking the data as a pool of a series of six independent experiments. In the first case, the Pearson test correlation coefficients of age and endoglin production (at 0 and 21 h) were 0.030 and –0.219, respectively. Consistent with this finding, the matrix of intragroup correlations gave values of 0.011 and –0.274, both approaches demonstrating the lack of significant correlations. Moreover, as HHT typically displays a late onset, we repeated the Pearson correlation test after excluding the 0–10 age group. The Pearson correlations were then 0.071 and –0.265, whereas the corresponding intragroup matrix correlations were 0.042 and –0.322 at 0 and 21 h, respectively. Again, the correlation between age and endoglin production in the control probands was not significant. Therefore, from this statistical analysis we can conclude that in non-HHT patients, up-regulation of endoglin during the macrophage-monocyte transition is independent of age.

endoglin up-regulation in activated monocytes is age-dependent in the hht population
Endoglin up-regulation in HHT patients is shown in Fig. 2BUp . The clinical symptoms of these patients are summarized according to Curaçao criteria in Table 1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol50/issue11/. We observed a statistically significant decrease in endoglin up-regulation compared with the mean response of the non-HHT population. As a control, the up-regulation in a healthy age-paired donor was measured in each experiment to assess possible experimental fluctuations. This approach permits the up-regulation data to be reported in terms of the healthy control for each experiment and family. As can be seen in Fig. 2BUp , except in the youngest patients (range, 12–22 years), endoglin up-regulation was markedly lower than the mean for the control population. Moreover, in individuals 45 years and older, at least in the case of endoglin haploinsufficiency, the up-regulation decreased to below the theoretical 50% threshold represented by the control population. These data suggest that endoglin up-regulation is inversely correlated with age and, surprisingly, independent of the locus (ENG or ALK1) affected; the initial screening was done with a panel of patients being classified as HHT based on the clinical Curaçao criteria before genetic analysis was used to determine which locus was mutated (see below). Two different 45-year-old patients (patients 45a and 45b) showed distinctly different endoglin up-regulation patterns. Interestingly, the endoglin up-regulation shown by these two patients was correlated to the severity of the clinical symptoms. Thus, patient 45a, with almost null up-regulation, matched four Curaçao criteria, presenting with nine lung AVMs larger than 3 mm and two cerebral AVMs. These results suggest that the degree of endoglin up-regulation in activated monocytes may be a molecular marker related to the severity of the HHT disease.

mutation and endoglin up-regulation analyses in a hht1 family
We studied endoglin production in two affected members of a HHT family and compared the production with that of an unaffected relative. This family was diagnosed with HHT according to the Curaçao criteria; the eldest patient (81 years of age) presented with all four Curaçao criteria, whereas his affected son (40 years of age) presented with three (see Table 1 in the online Data Supplement). DNA sequencing of all endoglin exons showed that there was a nonsense mutation in c.511C>T (exon 4) leading to the change of an arginine to a stop codon (R171X; Fig. 1Up in the online Data Supplement). From the clinical point of view, it is noteworthy mentioning that the 81-year-old patient suffered from an epidural abscess and that he is the first Spanish HHT patient with an identified mutation showing infectious brain complications. The pedigree of this family is shown in Fig. 3 . The sequencing chromatogram is presented in Fig. 1Up in the online Data Supplement.



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  		Figure 3. Pedigree of the Spanish HHT1 family.

The familial pedigree, as far as is known, is presented. {blacksquare}, affected individuals, according to the Curaçao criteria. Arrows indicate the two patients studied.

Histograms representing the endoglin expression index values of monocytes at time 0 and after 21 h in culture are shown in Fig. 4 . In both affected members, endoglin up-regulation was significantly decreased in comparison with the healthy relative. However, whereas endoglin up-regulation in the eldest patient was <20% of the control, his son had 70% of the expected endoglin up-regulation compared with an unaffected relative. Cymerman et al.(10) reported decreased endoglin up-regulation (<70%) in activated monocytes from 67 clinically diagnosed HHT family members. This study assumed a HHT1 phenotype for all 67, although the endoglin mutation was confirmed in fewer than one half (27 patients) of them. Unfortunately, no relationship between this up-regulation and age was studied.



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  		Figure 4. Endoglin up-regulation in activated monocytes from a HHT1 family.

Endoglin expression index in monocytes after 0 and 21 h of culture are shown in comparison with a non-HHT control belonging to the same family. Up-regulation in the oldest member of the family (81 years of age; left) and his 40-year-old son (right) is compared with that in healthy controls ({square}). Numbers above the columns indicate the percentage of up-regulation, with respect to controls (represented arbitrarily as 100%).

The difference in endoglin up-regulation according to the age of the patient is shown in Fig. 4Up ; as can be seen, the older the individual, the less the extent of up-regulation. This age-related up-regulation is coincident with the severity of the symptoms such that the patient with the most depressed up-regulation (81 years of age) had more severe clinical manifestations of HHT (see Table 1 in the online Data Supplement). This result, diminished endoglin induction, may provide the molecular mechanism for the reported higher penetrance and increasing severity of HHT symptoms with increased age(4).

mutation and endoglin up-regulation analyses in a hht2 family
Tests for endoglin up-regulation in activated monocytes were performed in blood samples from six HHT2 patients in a single affected family. The pedigree of this family is shown in Fig. 5 . These patients were diagnosed as certain HHT carriers according to the Curaçao criteria. Although DNA sequencing of the ENG locus showed no mutations, sequencing of the ALK1 locus revealed a missense mutation c.1120C>T (exon 8) leading to a change from arginine to tryptophan (R374W). The sequencing chromatograms for two affected members of this family and one healthy control relative are provided in Fig. 2Up in the online Data Supplement. This ALK1 mutation has already been described as pathogenic in HHT2(23).



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  		Figure 5. Pedigree of the Spanish HHT2 family.

Known members of this family are shown. Filled symbols represent affected individuals, according to the Curaçao criteria. Arrows indicate the six patients studied.

Table 1 in the online Data Supplement lists the clinical symptoms of each patient in this family. The ALK1 mutation is present in six affected members but not in controls (unaffected members) of the same family. Fig. 6 shows the comparison of endoglin up-regulation with genotype. In particular, the results shown in Fig. 6B emphasize that the expression index for endoglin up-regulation in those HHT2 patients is age-dependent, as compared with controls. This up-regulation decreases from almost 90% in the youngest patients (12 and 18 years) to 5% in the eldest (69 years). The 47-year-old patient (Fig. 6B , indicated with asterisk) suffers from gastrointestinal bleeding (see Table 1 in the online Data Supplement). Thus, not only in HHT1, but also in HHT2 patients, the percentage of endoglin increase in activated monocytes is age-dependent and is related to the severity of clinical symptoms (Fig. 6 ; also see Table 1 in the online Data Supplement). This result leads us to speculate about a role of ALK1 in endoglin up-regulation during monocyte differentiation. Because endoglin shows deficient up-regulation in HHT2 patients (Fig. 6 ), we postulate that normal concentrations of functional ALK1 are necessary for endoglin up-regulation during the process of monocyte activation. ALK1 has been shown to be expressed mainly in endothelial cells(25), but nothing was known about its possible expression in human monocytes. Although Panchenko et al.(26) showed that ALK1 was also present in rat macrophages, to our knowledge, nothing has been reported to date about ALK1 production in human monocytes. We were able to amplify ALK1-specific cDNA by use of total RNA from activated monocytes. The results of specific RT-PCR amplification of the ALK1 mRNA in human monocytes after 20 min or 21 h of adhesion to culture plates are shown in Fig. 3AUp of the online Data Supplement. A specific DNA fragment corresponding to ALK1 mRNA could be amplified in both samples. Furthermore, DNA sequencing of this fragment confirmed that it was actually cDNA of ALK1 exon 7 (Fig. 3BUp in the online Data Supplement). This result demonstrates that activated monocytes produce ALK1.



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  		Figure 6. Endoglin up-regulation in activated monocytes from patients in a HHT2 family.

(A), endoglin up-regulation in activated monocytes from patients in the same HHT2 family and healthy controls indicated in Fig. 2Up . The endoglin concentration is reported as the expression index. Flow cytometry measurements were made in duplicates. (B), percentage of expression index for endoglin in monocytes from six HHT2 patients in relation to the healthy control. These data indicate a clear age dependence of endoglin up-regulation in the HHT2 family. * indicates a patient whose percentage of endoglin up-regulation is lower than expected according to age. The dotted line indicates the theoretical 50% up-regulation in endoglin for the mutation in the heterozygous state.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Haploinsufficiency is the molecular basis currently accepted for the clinical manifestations in HHT1 and HHT2 patients(3)(20)(22). Because endothelial cells from adult HHT patients are currently very difficult to obtain, the use of activated monocytes from peripheral blood represents an alternative method to measure endoglin concentrations in patients clinically diagnosed as having HHT. The role of endoglin in the monocytic lineage remains to be elucidated, but it can be postulated that endoglin gene expression might be necessary for proper macrophage performance. Supporting this view, there are several reports on deficiencies in the immune response of HHT(27)(28)(29). Thus, HHT patients show higher occurrences of purulent meningococcal encephalitis(27). In addition, brain abscesses(28), recurrent Staphylococcus aureus extracerebral infections(29), and epidural abscess (this work) have been observed in these patients. Nevertheless, more systematic studies are required to assess this immunologic correlation.

Previous reports(10) analyzing endoglin concentrations in activated monocytes of HHT patients concluded that this approach is more difficult than the use of umbilical cord-derived HUVECs because the endoglin concentrations are 5- to 20-fold lower and are likely modulated by hormones and growth factors present in the serum.

The relative deficiency of endoglin in activated monocytes from HHT1 patients is expected and has been used as complementary to endoglin analysis in HUVECs (newborns) and in mutation studies to differentiate between HHT1 and HHT2 families(10). In the present work, we have determined endoglin up-regulation in activated monocytes from a large non-HHT sample of 40 donors spanning a broad range of ages to assess the diagnostic sensitivity of this method. Analysis of HHT patients demonstrated significantly lower endoglin up-regulation in monocytes compared with non-HHT samples. In the HHT1 family studied, the decrease in endoglin up-regulation in monocytes was more pronounced in the oldest patient, who also had more severe symptoms. The situation concerning ALK1, the other locus affected in HHT2 patients, would seem in principle different. ALK1 is produced predominantly by endothelial cells(25)(30)(31), and here we have shown that ALK1 is also produced by human monocytes. Unexpectedly, family 2, whose members harbor an ALK1 mutation (HHT2), also showed deficient endoglin up-regulation, as in the case of the ENG mutation (HHT1). The age-dependent failure in endoglin up-regulation was also evident in this HHT2 family: we found an almost perfect inverse relationship between age and endoglin up-regulation in the six affected members studied. On the other hand, the inverse relationship between endoglin up-regulation and age was complete when the severity of symptoms was taken into account. The younger patients (<40 years; see Fig. 6Up ) seemed to compensate for the haploinsufficiency quite well; in those individuals, endoglin up-regulation was >50% of the value in healthy controls (the theoretically expected value). In patients 40–60 years of age, up-regulation was ~50% of the value for healthy controls. Finally, in individuals older than 60, the increase of endoglin in activated monocytes was much less than 50% of the increase in healthy individuals. Endoglin up-regulation was almost totally absent in the most severely affected patients.

The down-regulation of endoglin in activated monocytes derived from HHT2 patients contrasts with a previous report(22) in which endoglin concentrations remained unaffected in HHT2 patients. This apparent discrepancy is likely explained, in part, by the different experimental methods used to determine the endoglin concentration. In the present study, we have used a straightforward quantitative method, without major manipulation of samples, i.e., flow cytometric analysis of endoglin present on the surface of activated monocytes in living cells. By contrast, Abdalla et al.(22) carried out metabolic labeling, immunoprecipitation, and Western blot analyses of endoglin from cellular extracts of HUVECs and activated monocytes, which likely provided only a semiquantitative measurement. They also carried out flow cytometry experiments using HUVECs but did not examine activated monocytes. Furthermore, the age of the patient was not included in their analysis and may also have contributed to the differences observed. For example, we have shown that in HHT2 family 2 (Fig. 6Up ), the endoglin concentrations in the younger affected members (12, 18, and 22 years) were not significantly different from the concentrations in age-matched controls and that endoglin deficiencies became evident only in the older family members (>45 years). Therefore, it would be predicted that in HHT2 HUVECs (newborns, age 0), the amount of endoglin should be close to the concentrations in HUVECs from healthy newborns, in agreement with the HUVEC data reported by Abdalla et al.(22). Unfortunately, these authors provide no information regarding the age of the six HHT2 patients from whom the monocytes were derived and analyzed(22).

The reason for the association between the ALK1 mutation and deficient endoglin up-regulation remains to be elucidated, but it could be related to the TGF-ß-dependent regulation of endoglin. Thus, because endoglin is positively regulated by TGF-ß(11)(32)(33) and is a co-receptor of TGF-ß(11)(12)(34), endoglin is probably subject to a positive feedback loop of regulation. HHT1 patients have, in theory, one half the endoglin concentrations of healthy individuals, and this haploinsufficiency may impair the feedback regulation, leading, in the long term, to impaired endoglin production as is observed in older patients. On the other hand, ALK1 is a type I receptor of the TGF-ß superfamily, which is also involved in TGF-ß signaling. Interestingly, Ota et al.(35) reported that endoglin is up-regulated by ALK1 in proliferating endothelial cells. Hence, decreased concentrations of functional ALK1 receptor in HHT2 patients may also lead to improper endoglin up-regulation in monocytes. It can be speculated that during monocyte differentiation, TGF-ß signaling is necessary. According to reports in this field(25)(30)(31)(36), TGF-ß can signal through two type I receptors, ALK1 and ALK5, which display different affinities for TGF-ß1. On stimulation with high concentrations of TGF-ß, both type I receptors are activated, but the signaling through ALK1 is dominant(31). Because endoglin shows deficient up-regulation in HHT2 patients with mutated ALK1, our hypothesis is that normal concentrations of functional ALK1 are necessary for the proper induction of endoglin synthesis during monocyte differentiation. This is in agreement with the results obtained in the present study demonstrating the production of ALK1 in activated monocytes.

Under certain physiologic conditions, such as hypoxia and wound healing(37)(38), the resulting up-regulation of endoglin may be particularly important. Endoglin production may be critical in these situations, in which a significant increase in endoglin production is required. Under such conditions, the amount of endoglin in HHT patients may be insufficient for endothelial cells to meet physiologic requirements, leading to apoptosis(39) and, consequently, to impairment of the vasculature, which is characteristic of HHT(1)(40). From a pathogenic point of view, deficient up-regulation of endoglin in HHT2 patients suggests important consequences, i.e., that endoglin haploinsufficiency is the ultimate trigger mechanism underlying not only HHT1, but also HHT2. Although this is an interesting hypothesis, further experiments are required to assess whether endoglin production in vascular cells in vivo is also affected in HHT2 patients.

HHT is genetically heterogenous; the HHT1 and HHT2 genes are complex (with 15 and 9 exons, respectively), and no prevalent mutation has been found. As seen in this study, the impact of patient age on endoglin up-regulation is a crucial variable, implying that age should be considered during diagnostic assessment. The inverse relationship between age and endoglin up-regulation may explain the general increase in penetrance of the disease during a patient’s life. Finally, the up-regulation of endoglin in activated monocytes appears to be correlated to the severity of the disease, which highlights the physiologic relevance of endoglin production in relation to the symptoms of the HHT.


   Acknowledgments
 
We are indebted to Drs. Michelle Letarte and Ursula Cymerman for excellent suggestions and advice concerning the methods for HHT patient sequencing, Dr. Calvin Vary for helpful editing of the manuscript, Carmen Langa for exceptional technical assistance, Dr. Carmen Gimenez for collaboration in collecting blood samples from the control population, Dr. Laura Barrios for excellent help with the statistical analysis, Dr. Carmelo Morales for allowing us access to his data on Spanish HHT families, MS Victoria Gomez-España and María Jesús Borquez for their help in blood extractions, the HHT Foundation International for support, and to all of the volunteers and HHT patients for their collaboration, without which this study would not have been possible. This work was supported by grants from Ministerio de Educacion y Ciencia, Comunidad Autónoma de Madrid, and Fondo de Investigación Sanitaria (PI020200) to C. Bernabeu. A. Fernandez-Lopez is a predoctoral fellow of Fondo de Investigación Sanitaria (Grant PI020200).


   Footnotes
 
2 These authors should be considered joint first authors.

1 Nonstandard abbreviations: HHT, hereditary hemorrhagic telangiectasia; AVM, arteriovenous malformation; ALK1, activin receptor-like kinase 1; TGF-ß, transforming growth factor-ß; HUVEC, human umbilical cord vein endothelial cell; PBMC, peripheral blood mononuclear cell; RT-PCR, reverse transcription-PCR; mAb, monoclonal antibody; and FITC, fluorescein isothiocyanate.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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