Hereditary haemorrhagic telangiectasia, HHT, is an autosomal dominant disorder that affects approximately 1 in 8000 people. HHT1 is associated with mutations in the ENG (Endoglin) gene and with haploinsufficiency. The disorder is characterized by focally dilated vessels, which can lead to arteriovenous malformations and serious complications even in young children. In the current study, umbilical cord and placenta samples from newborns with ENG mutations were analyzed to estimate the level of corresponding protein and look for potential vascular dysplasia. We confirmed, using metabolic labelling and flow cytometry, that endoglin levels were significantly reduced to median values of 47 per cent (range 32-56 per cent) and 58 per cent (46-90 per cent), respectively, in human umbilical vein endothelial cells derived from newborns with ENG mutations (HHT1 group; n=18) relative to samples from newborns shown not to have the familial mutation (non-HHT group). We also quantified the relative expression of endoglin by estimating the endoglin/PECAM-1 staining ratio in tissue sections. We observed significantly lower values in the HHT1 group, compared to the non-HHT group for the umbilical vein (n=9; median 0.6 vs 0.9; ranges 0.2-1.0 and 0.5-1.5) and for placental stem villus vessels (n=9 and 10; median 0.42 vs 0.93; ranges 0.24-0.58 and 0.56-1.18). No differences in the estimated umbilical vein cross-sectional area and in the proportion of vessels present in placental villi were observed in sections from the HHT1 group relative to the non-HHT group. Thus, blood vessels from HHT1 individuals are maintained intact in the umbilical vein and placenta during pregnancy and delivery, despite a significant reduction in endoglin expression.

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 mite 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)-beta1 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-beta1 levels than control These data suggest that modifier genes involved in the regulation of TGF-beta1 expression act in combination with a single functional copy of endoglin in the development of HHT.

Background and Purpose-A high incidence of arteriovenous malformations (AVMs) is associated with hereditary hemorrhagic telangiectasia type 1. Endoglin, the gene mutated in this disorder, is expressed at reduced levels on blood vessels of these patients. Since endoglin is a component of the transforming growth factor-beta receptor complex critical for vascular development and homeostasis, we determined its expression in sporadic cerebral AVMs and in normal brain vessels.Methods-Twenty cerebral AVMs and 10 normal brain samples were analyzed for endoglin, platelet endothelial cell adhesion molecule 1 (PECAM-1), alpha -smooth muscle cell actin, vimentin, and desmin by immunohistochemistry.Results-in normal brain, endoglin was found not only on the endothelium of all vessels but also on the adventitial layer of arteries and arterioles. In cerebral AVMs, the numerous vessels present expressed endoglin on both endothelium and adventitia. Arterialized veins, identified by lack of elastin and uneven thickness of smooth muscle cells, revealed endoglin-positive mesenchymal cells in the adventitia and perivascular connective tissue. These cells were fibroblasts since they expressed vimentin but not actin and/or desmin.Conclusions-This is the first report of endoglin expression on adventitia of normal brain arteries and on arterialized veins in cerebral AVMs. Increasing numbers of endoglin-positive endothelial and adventitial cells were seen in sporadic cerebral AVMs, but endoglin density was normal. Thus, it is not involved in the generation of these lesions. However, the presence of endoglin on Fibroblasts in the perivascular stroma suggests an active role for this protein in vascular remodeling in response to increased blood flow and shear stress.

Endoglin is predominantly expressed on endothelium and is mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1). We report the analysis of endoglin in tissues of a newborn (family 2), who died of a cerebral arteriovenous malformation (CAVM), and in a lung specimen surgically resected from a 78-year-old patient (family 5), with a pulmonary AVM (PAVM). The clinically affected father of the newborn revealed a novel mutation that was absent in his parents and was identified as a duplication of exons 3 to 8, by quantitative multiplex polymerase chain reaction. The corresponding mutant protein (116-kd monomer) and the missense mutant protein (80-kd monomer) present in family 5 were detected only as transient intracellular species and were unreactive by Western blot analysis and immunostaining. Normal endoglin (90-kd monomer) was reduced by 50% on peripheral blood-activated monocytes of the HHT1 patients. When analyzed by immunostaining and densitometry, presumed normal blood vessels of the newborn lung and brain and vessels adjacent to the adult PAVM showed a 50% reduction in the endoglin/PECAM-1 ratio. A similar ratio was observed in the CAVM and PAVM, suggesting that all blood vessels of HHT1 patients express reduced endoglin in situ and that AVMs are not attributed to a focal loss of endoglin.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterized by dilated vessels and arteriovenous malformations. Phenotypic heterogeneity, such as age of onset, severity of disease and organ involvement, is explained in part by two genes being mutated, endoglin (HHT1) and ALK-1 (HHT2). Haploinsufficiency is the mechanism responsible for HHT. This implies that position and type of mutations cannot explain heterogeneity, because mutant proteins are not expressed at the cell surface and consequently cannot interfere with normal function. Based on this model, we generated mice expressing only one allele of endoglin, but in two different inbred strains, 129/Ola and C57BL/6. Phenotypic heterogeneity was also observed among the HHT mice and was very dependent on the genetic background. Our data strongly suggest that additional genes, contributed by the 129/Ola strain, are responsible for the vascular anomalies associated with HHT. The murine model is faithful to the human disease and should allow us to identify the modifier genes of HHT as well as to test potential therapeutic interventions.

Endoglin (CD105), an accessory protein of the TGF-beta receptor superfamily, is highly expressed on endothelial cells. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the Endoglin gene, leading to haploinsufficiency. To generate a disease model and ascertain the role of endoglin in development, we generated mice lacking 1 or both copies of the gene. Endoglin null embryos die at gestational day 10.0-10.5 due to defects in vessel and heart development. Vessel formation appears normal until hemorrhage occurs in yolk sacs and embryos. The primitive vascular plexus of the yolk sac fails to mature into defined vessels, and vascular channels dilate and rupture. Internal bleeding is seen in the peritoneal cavity, implying fragile vessels. Heart development is arrested at day 9.0, and the atrioventricular canal endocardium fails to undergo mesenchymal transformation and cushion-tissue formation. These data suggest that endoglin is critical for both angiogenesis and heart valve formation. Some heterozygotes, either with an inbred 129/Ola or mixed C57BL/6-129/Ola background, show signs of HHT, such as telangiectases or recurrent nosebleeds. In this murine model of HHT, it appears that epigenetic factors and modifier genes, some of which are present in 129/Ola, contribute to disease heterogeneity.

Endoglin (CD105) is a homodimeric cell surface component of the TGF-beta 1 receptor complex, which is expressed at high levels on vascular endo-thelium and at lower levels on activated monocytes. It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1. To date, each family has a distinct endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein. We generated stable transfectants of full-length human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain. Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants. Ten of these mono-clonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin: Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N-terminal region of 204 amino acids encoded by exons 1 to 5. Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 all bound to a region of 54 amino acids encoded mostly by exon 7. Monoclonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.

For the last 5 years, attention has focused on the nongenomic effects of 1,25-(OH)2D3, but considerably less is known about the mechanisms of the nonnuclear actions of 24,25-(OH)2D3. The present study examines and compares the rapid (5-90 s) effects of 100 pM to 10 nM 24,25-(OH)2D3, 10 pM to 1 nM 1,25-(OH)2D3, and 1-100 nM 25-OHD3 on the formation of inositol phosphates and lipids in confluent mouse osteoblasts. 24,25-(OH)2D3 and 25-OHD3 effects were dose dependent; those of 1,25-(OH)2D3 were dose dependent in a bell-shaped manner. The two dihydroxylated metabolites induced a multiphasic response in inositol 1,4,5-trisphosphate (IP3) formation with three stimulation peaks; the IP3 response to 25-OHD3 was monophasic. The amplitude of the IP3 response to 24,25-(OH)2D3 was greater and its oscillation period was slower than that induced by 1,25-(OH)2D3. The diacylglycerol (DAG) responses to secosteroids showed two stimulation peaks that appeared at different times depending on the secosteroid used. Pretreatment with neomycin totally inhibited the first DAG response; neomycin had no effect on the second peak of DAG induced by 25-OHD3, whereas it partially blocked the second response of DAG to 24,25-(OH)2D3 and 1,25-(OH)2D3. These data show for the first time that 24,25-(OH)2D3 can modulate phospholipid metabolism in confluent mouse osteoblasts as early as 5-10 s. The first pathway used by all three secosteroids is that of the hydrolysis of phosphatidylinositol 4,5-bisphosphate via phospholipase C activation, leading to the formation of the two second messengers, IP3 and DAG, since neomycin totally blocked the response. Thus, the action of these secosteroids on the osteoblast membrane may also implicate several steps of the phosphatidylcholine cycle, according to the metabolite tested. Finally, these data point to a direct interaction of vitamin D metabolites with specific membrane recognition moieties.

The involvement of arachidonic acid (AA) and its metabolites in the control of PTH secretion by porcine parathyroid cells was investigated. Increasing the extracellular calcium concentration from 0.5 to 2 mM increased free [3H]AA release and decreased PTH secretion from labeled parathyroid cells as a function of time (1-30 min). Free [3H]AA in the medium was significantly increased (+153 +/- 6%) after 5 min, while PTH secretion was significantly decreased (-75 +/- 7%) only after 15 min, suggesting a link between the two. [3H]AA release was associated with a decrease in [3H]AA incorporated into phosphatidylinositol, phosphatidic acid, and phosphatidylcholine, suggesting that these phospholipids are the major source of AA. Exogenous phospholipase-A2 (PL-A2; 1-500 mU/ml) and AA (5-40 microM) inhibited PTH secretion in a dose-dependent manner. PTH secretion inhibited by 2 mM Ca2+ was restored by two PL-A2 inhibitors, indomethacin (30 microM) and mepacrine (50 microM). The cyclooxygenase pathway inhibitor ibuprofen (20 microM) did not restore PTH secretion of affect high Ca(2+)-, AA-, or PL-A2-inhibited PTH secretion. Two inhibitors of the lipoxygenase pathway (LO), phenidone (1 microM) and baicalein (0.1 microM), a relatively selective 12-LO inhibitor, blunted high Ca(2+)-induced inhibition of PTH secretion (+101 +/- 10% and +105 +/- 6%, respectively), but nordihydroguaiaretic acid, which inhibits the 5-LO pathway, did not restore PTH secretion inhibited by high Ca2+, AA, or PL-A2. These results suggested that AA and agents that cause its liberation inhibit PTH secretion. AA may act via the 12-LO, but not via the 5-LO or cyclooxygenase, pathway. Thus, 12-LO products may be second messengers in parathyroid cells.

We examined the rapid effects (less than 120 sec) of 1,25(OH)2D3 on membrane phospholipid hydrolysis in porcine parathyroid cells and compared these effects to those produced by extracellular Ca2+. Cells were labeled with [3H]myo-inositol or [14C]arachidonic acid for 3 h, then exposed to various 1,25(OH)2D3 concentrations in 0.5 mM Ca2+ for different time periods or to 2 mM [Ca2+]. Parathyroid cells showed a biphasic increase in diacylglycerol (DAG), monoacylglycerol (MG), phosphatidic acid (PA), and inositol trisphosphate (IP3) in response to 1,25(OH)2D3 (10(-12)-10(-8) M) or to 2 mM [Ca2+]. This effect was rapid (within 5 sec) and dose-dependent, with a maximal stimulation with 10 pM of 1,25(OH)2D3. At this concentration, the first peak of DAG, MG, and IP3 was at 5 sec and reached 176 +/- 9%, 134 +/- 4%, and 154 +/- 13%, respectively vs. basal levels. For PA, the first maximum increase was at 20 sec (130 +/- 6%). At 30 sec MG, PA, and IP3 returned to basal levels, whereas the decrease in DAG was under the basal level (-25 +/- 5%). The second peak reached a maximum at 60 sec for the four products (145 +/- 8%, 119 +/- 5%, 125 +/- 6%, and 175 +/- 20%, respectively) then decreased to basal level at 120 sec. High extracellular Ca2+ (2 mM) and fluoride (5 mM) also produced similar increase in phosphatidylinositol metabolites, except that DAG levels returned to basal level at 30 sec. In conclusion, the present data shows the existence of rapid effects of 1,25(OH)2D3 in porcine parathyroid cells. The short time sequence suggests that they are mediated by a direct interaction with the membrane, possibly through a receptor-mediated process linked to phospholipase C by a G-protein.

Ten-week-old pigs were treated with 4 different treatment schedules of porcine calcitonin for 2 months. Groups C1 and C4 received continuous treatment: C1 had daily IM injections (4 IU/kg/BW (body weight) each injection), and C4 was infused with a minipump implanted subcutaneously delivering 4 IU/kg/BW/day. Groups C2 and C3 received intermittent calcitonin treatment (each injection 4 IU/kg/BW): C2 was given 1 out of every four days, C3 was injected 5 consecutive days out of 20 days. The total dosage received in C1 versus C4 and C2 versus C3 were the same. Results were evaluated by histomorphometry after double tetracycline labeling on iliac trabecular bone. Resorption surfaces were decreased in groups C2, C3 and C4, but bone volume, osteoclast surfaces, and interstitial bone thickness were not modified in any group receiving calcitonin. Osteoblast and mineralizing surfaces were increased in group C2, C3 and C4. Plasma 1,25-dihydroxyvitamin D concentration and bone formation rate were increased in groups C2 and C4. Plasma immunoreactive parathyroid hormone levels and parathyroid weights were not increased in any treated groups. In conclusion, 2-month calcitonin treatment did not decrease the amount of bone resorbed in growing pigs. Continuous calcitonin infusion and intermittent calcitonin administration induced an increase in the extent of active bone formation which might be in part dependent on an increased production of 1,25 dihydroxyvitamin D.