Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-β (TGF-β) signaling receptors to bind TGF-β family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-β, TGF-β signaling receptors, and TGF-β–stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglinpromoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated theendoglin promoter and those of collagen α1(I), urokinase-type plasminogen activator, TGF-β1, and TGF-β receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the C-terminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-β signaling complex in vascular repair.

Hereditary hemorrhagic telangiectasia (HHT) is a multisystemic inherited vascular dysplasia that leads to nosebleeds and visceral arteriovenous malformations (AVMs). Anti-angiogenic drugs thalidomide and bevacizumab have been increasingly used off-label with variable results. The HHT working group within the ERN for Rare Multisystemic Vascular Diseases (VASCERN), developed a questionnaire-based retrospective capture of adverse events (AEs) classified using the Common Terminology Criteria for Adverse Events. Sixty-nine HHT patients received bevacizumab, 37 (50.6%) for high output cardiac failure/hepatic AVMs, and 32 (49.4%) for bleeding; the 69 patients received bevacizumab for a mean of 11 months for a total of 63.8 person/years treatment. 67 received thalidomide, all for epistaxis and/or gastrointestinal bleeding; they received thalidomide for a mean of 13.4 months/patient for a total of 75 person/years treatment. AEs were reported in 58 patients, 33 with bevacizumab, 37 with thalidomide. 32 grade 1–3 AEs related to bevacizumab were reported with an average incidence rate of 50 per 100 person-years. 34 grade 1–3 AEs related to thalidomide were reported with an average incidence rate of 45.3 per 100 person-years. Bevacizumab AEs were more common in females (27 AEs in 46 women) than males (6 in 23, p < 0.001). Thalidomide AEs occurred at more similar rates in males (25 AEs in 41 men, 60.9%) and females (12 in 26 (46.2%), but were more common in ENG patients (17 in 17) than in ACVRL1 (14 in 34, p < 0.0001). For bevacizumab, the most common reports were of joint pains (7/69, 10%), headache (3/69, 4.4%) and proteinuria (2/69, 3%), and for thalidomide, peripheral neuropathy (12/67, 18%); drowsiness (8/67, 12%); and dizziness (6/67, 9%). Fatal adverse events were more common in males (p = 0.009), and in patients with ENG pathogenic variants (p = 0.012). One fatal AE was possibly related to bevacizumab (average incidence rate: 1.5 per 100 person-years); 3 fatal AEs were possibly related to thalidomide (average incidence rate: 4 per 100 person-years). With potential increase in use of Bevacizumab and Thalidomide in HHT patients, data presented support appropriate weighing of the toxicities which can arise in HHT settings and the practice recommendations for their prevention and management.

Upon inflammation, monocyte-derived macrophages (MΦ) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and efficient initial response (GM-MΦ) and a good resolution (M-MΦ) of the inflammatory process. The functional activity of polarized MΦ is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MΦ that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MΦ secretome involved in the shedding of soluble endoglin. We find that the GM-MΦ secretome contains metalloprotease 12 (MMP-12), a GM-MΦ specific marker that may account for the anti-angiogenic activity of the GM-MΦ secretome. Cell surface endoglin is present in both GM-MΦ and M-MΦ, but soluble endoglin is only detected in GM-MΦ culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MΦ and endothelial cells. These data demonstrate a direct correlation between GM-MΦ polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.

Hereditary haemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome, is an autosomal dominant vascular disease. The clinical manifestations are epistaxis, mucocutaneous and gastrointestinal telangiectases, and arteriovenous malformations. There are two predominant types of HHT caused by mutations in Endoglin (ENG) and activin receptor-like kinase 1 (ALK1) (ACVRL1) genes, HHT1 and HHT2, respectively. No cure for HHT has been found and there is a current need to find new effective drug treatments for the disease. Some patients show severe epistaxis which interferes with their quality of life. We report preliminary results obtained with Raloxifene to treat epistaxis in postmenopausal HHT women diagnosed with osteoporosis. We tried to unravel the molecular mechanisms involved in the therapeutic effects of raloxifene. ENG and ACVRL1 genes code for proteins involved in the transforming growth factor β pathway and it is widely accepted that haploinsufficiency is the origin for the pathogenicity of HHT. Therefore, identification of drugs able to increase the expression of those genes is essential to propose new therapies for HHT. In vitro results show that raloxifene increases the protein and mRNA expression of ENG and ALK1 in cultured endothelial cells. Raloxifene also stimulates the promoter activity of these genes, suggesting a transcriptional regulation of ENG and ALK1. Furthermore, Raloxifene improved endothelial cell functions like tubulogenesis and migration in agreement with the reported functional roles of Endoglin and ALK1. Our pilot study provides a further hint that oral administration of raloxifene may be beneficial for epistaxis treatment in HHT menopausal women. The molecular mechanisms of raloxifene involve counteracting the haploin-sufficiency of ENG and ALK1.

Hereditary hemorrhagic telangiectasia (HHT) or Osler–Weber–Rendu syndrome is an autosomal dominant vascular disorder characterized by telangiectases and internal arteriovenous malformations. It is caused by mutations in elements of the transforming growth factor-β (TGF-β) receptor complex: endoglin , a co-receptor, responsible for HHT1, or ALK1 (activin receptor-like kinase 1), a type I receptor leading to HHT2. Recently, we have established cultures of HHT endothelial cells, primary targets of the disease. These cells showed deficient TGF-β signaling and angiogenesis, representing a useful human model to study the molecular mechanism of this disease. To understand the pathogenic mechanism underlying HHT, we have used total RNA probes to compare HHT versus non-HHT cells by expression microarrays. This work represents a systematic study to identify target genes affected in HHT cells. Given the similarity of symptoms in HHT1 and HHT2, special interest has been put on the identification of common targets for both HHT types. As a result, 277 downregulated and 63 upregulated genes were identified in HHT versus control cells. These genes are involved in biological processes relevant to the HHT pathology, such as angiogenesis, cytoskeleton, cell migration, proliferation and NO synthesis. The type of misregulated genes found in HHT endothelial cells lead us to propose a model of HHT pathogenesis, opening new perspectives to understand this disorder. Moreover, as the disease is originated by mutations in proteins of the TGF-β receptor complex, these results may be useful to find out targets of the TGF-β pathway in endothelium.

Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Eng^fl/flLysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Eng^fl/flLysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients. Endoglin is a transmembrane protein and an auxiliary receptor for TGF-β with an important role in the homeostasis of the vessel wall. However, endoglin was originally identified as a human cell surface antigen expressed in a pre-B leukemic cell line. Mutations in ENG are responsible for the Hereditary Hemorrhagic Telangiectasia type 1 (HHT1) or Rendu-Osler-Weber syndrome. HHT is a rare disease, with a prevalence of 1/5,000 to 1/8,000. It is an autosomal dominant disorder characterized by a multisystemic vascular dysplasia, recurrent hemorrhages and arteriovenous malformations in internal organs. Interestingly, endoglin expression is also triggered during the monocyte-macrophage differentiation process. In our laboratory, we described that up-regulation of endoglin during in vitro differentiation of blood monocytes is age-dependent and impaired in monocytes from HHT patients, suggesting a role of endoglin in macrophages. In the present work, we first analyzed endoglin expression during differentiation of peripheral blood monocytes to macrophages under in vitro and in vivo conditions. Next, to investigate endoglin’s role in macrophage function in vivo, a myeloid-lineage specific endoglin knock-out mouse line was generated (Eng^fl/flLysMCre). Endoglin deficiency in macrophages predisposed animals to spontaneous infections and led to delayed endotoxin-induced mortality. Phagocytic activity by peritoneal macrophages was reduced in the absence of endoglin and altered expression of TGF-β target genes was consistent with an altered balance of TGF-β signaling. The results show a novel role for endoglin in mouse macrophages, which if analogous in human macrophages, may explain, at least in part, the increased infection rates seen in HHT patients.