Dedifferentiated early postnatal lung myofibroblasts redifferentiate in adult disease.

Alveolarization ensures sufficient lung surface area for gas exchange, and during bulk alveolarization in mice (postnatal day [P] 4.5-14.5), alpha-smooth muscle actin (SMA)+ myofibroblasts accumulate, secrete elastin, and lay down alveolar septum. Herein, we delineate the dynamics of the lineage of early postnatal SMA+ myofibroblasts during and after bulk alveolarization and in response to lung injury. SMA+ lung myofibroblasts first appear at ∼ P2.5 and proliferate robustly. Lineage tracing shows that, at P14.5 and over the next few days, the vast majority of SMA+ myofibroblasts downregulate smooth muscle cell markers and undergo apoptosis. Of note, ∼8% of these dedifferentiated cells and another ∼1% of SMA+ myofibroblasts persist to adulthood. Single cell RNA sequencing analysis of the persistent SMA- cells and SMA+ myofibroblasts in the adult lung reveals distinct gene expression profiles. For instance, dedifferentiated SMA- cells exhibit higher levels of tissue remodeling genes. Most interestingly, these dedifferentiated early postnatal myofibroblasts re-express SMA upon exposure of the adult lung to hypoxia or the pro-fibrotic drug bleomycin. However, unlike during alveolarization, these cells that re-express SMA do not proliferate with hypoxia. In sum, dedifferentiated early postnatal myofibroblasts are a previously undescribed cell type in the adult lung and redifferentiate in response to injury.

The age of bone marrow dictates the clonality of smooth muscle-derived cells in atherosclerotic plaques.

Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to ~70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin ß3 resulting in increased tumor necrosis factor [TNF]-α signaling. TNFα signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin ß3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

Vascular pathobiology of pulmonary hypertension.

Pulmonary hypertension (PH), increased blood pressure in the pulmonary arteries, is a morbid and lethal disease. PH is classified into several groups based on etiology, but pathological remodeling of the pulmonary vasculature is a common feature. Endothelial cell dysfunction and excess smooth muscle cell proliferation and migration are central to the vascular pathogenesis. In addition, other cell types, including fibroblasts, pericytes, inflammatory cells and platelets contribute as well. Herein, we briefly note most of the main cell types active in PH and for each cell type, highlight select signaling pathway(s) highly implicated in that cell type in this disease. Among others, the role of hypoxia-inducible factors, growth factors (e.g., vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor-ß and bone morphogenetic protein), vasoactive molecules, NOTCH3, Kruppel-like factor 4 and forkhead box proteins are discussed. Additionally, deregulated processes of endothelial-to-mesenchymal transition, extracellular matrix remodeling and intercellular crosstalk are noted. This brief review touches upon select critical facets of PH pathobiology and aims to incite further investigation that will result in discoveries with much-needed clinical impact for this devastating disease.

Propranolol: A "Pick and Roll" Team Player in Benign Tumors and Cancer Therapies.

Research on cancer therapies focuses on processes such as angiogenesis, cell signaling, stemness, metastasis, and drug resistance and inflammation, all of which are influenced by the cellular and molecular microenvironment of the tumor. Different strategies, such as antibodies, small chemicals, hormones, cytokines, and, recently, gene editing techniques, have been tested to reduce the malignancy and generate a harmful microenvironment for the tumor. Few therapeutic agents have shown benefits when administered alone, but a few more have demonstrated clear improvement when administered in combination with other therapeutic molecules. In 2008 (and for the first time in the clinic), the therapeutic benefits of the ß-adrenergic receptor antagonist, propranolol, were described in benign tumors, such as infantile hemangioma. Propranolol, initially prescribed for high blood pressure, irregular heart rate, essential tremor, and anxiety, has shown, in the last decade, increasing evidence of its antitumoral properties in more than a dozen different types of cancer. Moreover, the use of propranolol in combination therapies with other drugs has shown synergistic antitumor effects. This review highlights the clinical trials in which propranolol is taking part as adjuvant therapy at single administration or in combinatorial human trials, arising as a good pick and roll partner in anticancer strategies.

The Role of Propranolol as a Repurposed Drug in Rare Vascular Diseases.

Rare Diseases (RD) are defined by their prevalence in less than 5 in 10,000 of the general population. Considered individually, each RD may seem insignificant, but together they add up to more than 7000 different diseases. Research in RD is not attractive for pharmaceutical companies since it is unlikely to recover development costs for medicines aimed to small numbers of patients. Since most of these diseases are life threatening, this fact underscores the urgent need for treatments. Drug repurposing consists of identifying new uses for approved drugs outside the scope of the original medical indication. It is an alternative option in drug development and represents a viable and risk-managed strategy to develop for RDs. In 2008, the "off label" therapeutic benefits of propranolol were described in the benign tumor Infantile Hemangioma. Propranolol, initially prescribed for high blood pressure, irregular heart rate, essential tremor, and anxiety, has, in the last decade, shown increasing evidence of its antiangiogenic, pro-apoptotic, vasoconstrictor and anti-inflammatory properties in different RDs, including vascular or oncological pathologies. This review highlights the finished and ongoing trials in which propranolol has arisen as a good repurposing drug for improving the health condition in RDs.

Distinct roles of KLF4 in mesenchymal cell subtypes during lung fibrogenesis.

During lung fibrosis, the epithelium induces signaling to underlying mesenchyme to generate excess myofibroblasts and extracellular matrix; herein, we focus on signaling in the mesenchyme. Our studies indicate that platelet-derived growth factor receptor (PDGFR)-ß+ cells are the predominant source of myofibroblasts and Kruppel-like factor (KLF) 4 is upregulated in PDGFR-ß+ cells, inducing TGFß pathway signaling and fibrosis. In fibrotic lung patches, KLF4 is down-regulated, suggesting KLF4 levels decrease as PDGFR-ß+ cells transition into myofibroblasts. In contrast to PDGFR-ß+ cells, KLF4 reduction in α-smooth muscle actin (SMA)+ cells non-cell autonomously exacerbates lung fibrosis by inducing macrophage accumulation and pro-fibrotic effects of PDGFR-ß+ cells via a Forkhead box M1 to C-C chemokine ligand 2-receptor 2 pathway. Taken together, in the context of lung fibrosis, our results indicate that KLF4 plays opposing roles in PDGFR-ß+ cells and SMA+ cells and highlight the importance of further studies of interactions between distinct mesenchymal cell types.

Targeting ß2-Adrenergic Receptors Shows Therapeutical Benefits in Clear Cell Renal Cell Carcinoma from Von Hippel-Lindau Disease.

Von Hippel-Lindau (VHL), is a rare autosomal dominant inherited cancer in which the lack of VHL protein triggers the development of multisystemic tumors such us retinal hemangioblastomas (HB), CNS-HB, and clear cell renal cell carcinoma (ccRCC). ccRCC ranks third in terms of incidence and first in cause of death. Standard systemic therapies for VHL-ccRCC have shown limited response, with recurrent surgeries being the only effective treatment. Targeting of ß2-adrenergic receptor (ADRB) has shown therapeutic antitumor benefits on VHL-retinal HB (clinical trial) and VHL-CNS HB (in vitro). Therefore, the in vitro and in vivo antitumor benefits of propranolol (ADRB-1,2 antagonist) and ICI-118,551 (ADRB-2 antagonist) on VHL-/- ccRCC primary cultures and 786-O tumor cell lines have been addressed. Propranolol and ICI-118,551 activated apoptosis inhibited gene and protein expression of HIF-2α, CAIX, and VEGF, and impaired partially the nuclear internalization of HIF-2α and NFĸB/p65. Moreover, propranolol and ICI-118,551 reduced tumor growth on two in vivo xenografts. Finally, ccRCC patients receiving propranolol as off-label treatment have shown a positive therapeutic response for two years on average. In summary, propranolol and ICI-118,551 have shown antitumor benefits in VHL-derived ccRCC, and since ccRCCs comprise 63% of the total RCCs, targeting ADRB2 becomes a promising drug for VHL and other non-VHL tumors.

Review of Pharmacological Strategies with Repurposed Drugs for Hereditary Hemorrhagic Telangiectasia Related Bleeding.

The diagnosis of hereditary hemorrhagic telangiectasia (HHT) is based on the Curaçao criteria: epistaxis, telangiectases, arteriovenous malformations in internal organs, and family history. Genetically speaking, more than 90% of HHT patients show mutations in ENG or ACVRL1/ALK1 genes, both belonging to the TGF-ß/BMP9 signaling pathway. Despite clear knowledge of the symptoms and genes of the disease, we still lack a definite cure for HHT, having just palliative measures and pharmacological trials. Among the former, two strategies are: intervention at "ground zero" to minimize by iron and blood transfusions in order to counteract anemia. Among the later, along the last 15 years, three different strategies have been tested: (1) To favor coagulation with antifibrinolytic agents (tranexamic acid); (2) to increase transcription of ENG and ALK1 with specific estrogen-receptor modulators (bazedoxifene or raloxifene), antioxidants (N-acetylcysteine, resveratrol), or immunosuppressants (tacrolimus); and (3) to impair the abnormal angiogenic process with antibodies (bevacizumab) or blocking drugs like etamsylate, and propranolol. This manuscript reviews the main strategies and sums up the clinical trials developed with drugs alleviating HHT.

Endoglin (CD105) and VEGF as potential angiogenic and dissemination markers for colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is an important current problem concerning public health due to its high incidence and mortality. Advances in molecular and cellular knowledge and the detection of new disease biomarkers are very important to improve prognosis, prediction, and early diagnosis. In this study, we aimed to analyze the gene and protein expression levels of two angiogenic markers, VEGF and soluble Endoglin, during different tumor stages as well as at different stages of cancer treatment, to predict the diagnosis and evolution of colon and rectal cancer. MATERIAL AND METHODS: This study includes 133 CRC patients (93 with colon cancer and 40 with rectal cancer) on which the gene and protein expression of Endoglin (membrane and soluble form) and VEGF were analyzed by molecular and immunohistochemical techniques on different tumor stage samples and plasma obtained preoperatively as well as 3, 6, and 9 months after resection of the tumor. RESULTS: VEGF and Endoglin gene expressions were higher in tumor tissue than in surrounding non-tumoral tissue for both types of cancer. The VEGF levels in plasma were found to decrease in less aggressive tumors, whereas soluble Endoglin was increased in preoperative samples of patients with metastasis. Membrane Endoglin expression was higher on the vascular endothelium of more aggressive tumors. In contrast, Endoglin expression was mainly in the colon epithelium in less aggressive stage tumors. CONCLUSION: Endoglin and VEGF are proteins with a major role in the tumor angiogenesis process. This study performed with a wide cohort of human samples shows that both proteins seem to be valuable biomarkers in the diagnosis and prognosis of CRC.

Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4.

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction. Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels (sEng+) showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male sEng+ mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4. Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in sEng+ mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners.

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-ß receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-ß receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-ß family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.

MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells.

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.

Soluble endoglin regulates expression of angiogenesis-related proteins and induction of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia.

Endoglin is a transmembrane glycoprotein expressed in vascular endothelium that plays a key role in angiogenesis. Mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1 (HHT1), characterized by arteriovenous malformations (AVMs) in different organs. These vascular lesions derive from abnormal processes of angiogenesis, whereby aberrant vascular remodeling leads to focal loss of capillaries. Current treatments for HHT1 include antiangiogenic therapies. Interestingly, a circulating form of endoglin (also known as soluble endoglin, sEng), proteolytically released from the membrane-bound protein and displaying antiangiogenic activity, has been described in several endothelial-related pathological conditions. Using human and mouse endothelial cells, we find that sEng downregulates several pro-angiogenic and pro-migratory proteins involved in angiogenesis. However, this effect is much reduced in endothelial cells that lack endogenous transmembrane endoglin, suggesting that the antiangiogenic activity of sEng is dependent on the presence of endogenous transmembrane endoglin protein. In fact, sEng partially restores the phenotype of endoglin-silenced endothelial cells to that of normal endothelial cells. Moreover, using an established neonatal retinal model of HHT1 with depleted endoglin in the vascular endothelium, sEng treatment decreases the number of AVMs and has a normalizing effect on the vascular phenotype with respect to vessel branching, vascular density and migration of the vascular plexus towards the retinal periphery. Taken together, these data show that circulating sEng can influence vascular development and AVMs by modulating angiogenesis, and that its effect on endothelial cells depends on the expression of endogenous endoglin.This article has an associated First Person interview with the first author of the paper.

Endoglin and alk1 as therapeutic targets for hereditary hemorrhagic telangiectasia.

INTRODUCTION: Hereditary Haemorrhagic Telangiectasia (HHT) is as an autosomal dominant trait characterized by frequent nose bleeds, mucocutaneous telangiectases, arteriovenous malformations (AVMs) of the lung, liver and brain, and gastrointestinal bleedings due to telangiectases. HHT is originated by mutations in genes whose encoded proteins are involved in the transforming growth factor ß (TGF-ß) family signalling of vascular endothelial cells. In spite of the great advances in the diagnosis as well as in the molecular, cellular and animal models of HHT, the current treatments remain just at the palliative level. Areas covered: Pathogenic mutations in genes coding for the TGF-ß receptors endoglin (ENG) (HHT1) or the activin receptor-like kinase-1 (ACVRL1 or ALK1) (HHT2), are responsible for more than 80% of patients with HHT. Therefore, ENG and ALK1 are the main potential therapeutic targets for HHT and the focus of this review. The current status of the preclinical and clinical studies, including the anti-angiogenic strategy, have been addressed. Expert opinion: Endoglin and ALK1 are attractive therapeutic targets in HHT. Because haploinsufficiency is the pathogenic mechanism in HHT, several therapeutic approaches able to enhance protein expression and/or function of endoglin and ALK1 are keys to find novel and efficient treatments for the disease.

High soluble endoglin levels do not induce changes in structural parameters of mouse heart.

A soluble form of endoglin (sEng) released into the circulation was suggested to be a direct inducer of endothelial dysfunction, inflammation and contributed to the development of hypertension by interfering with TGF-ß signaling in cardiovascular pathologies. In the present study, we assessed the hypothesis that high sEng level-induced hypertension via a possible sEng interference with TGF-ß signaling pathways may result in inflammatory, structural or fibrotic changes in hearts of Sol-Eng+ mice (mice with high levels of soluble endoglin) fed either chow or high-fat diet. Female Sol-Eng+ mice and their age matched littermates with low plasma levels of sEng were fed either chow or high-fat diet (HFD). Heart samples were subsequently analyzed by histology, qRT-PCR and Western blot analysis. In this study, no differences in myocardial morphology/hypertrophy and possible fibrotic changes between Sol-Eng+ mice and control mice were detected on both chow and HFD. The presence of sEng did not significantly affect the expression of selected members of TGF-ß signaling (membrane endoglin, TGFßRII, ALK-5, ALK-1, Id-1, PAI-1 and activated Smad proteins-pSmad 1,5 and pSmad 2,3), inflammation, heart remodeling (PDGFb, Col1A1) and endothelial dysfunction (VCAM-1, ICAM-1) in the hearts of Sol-Eng+ mice compared to control mice on both chow and high-fat diet. High levels of soluble endoglin did not affect microscopic structure (profibrotic and degenerative cardiomyocyte changes), and specific parts of TGF-ß signaling, endothelial function and inflammation in the heart of Sol-Eng+ mice fed both chow diet or HFD. However, we cannot rule out a possibility that a long-term chronic exposure (9 months and more) to soluble endoglin alone or combined with other cardiovascular risk factors may contribute to alterations of heart function and structure in Sol-Eng+ mice, which is the topic in our lab in ongoing experiments.

Soluble endoglin modulates the pro-inflammatory mediators NF-κB and IL-6 in cultured human endothelial cells.

AIMS: Endoglin is a transmembrane glycoprotein, that plays an important role in regulating endothelium. Proteolytic cleavage of membrane endoglin releases soluble endoglin (sEng), whose increased plasma levels have been detected in diseases related to the cardiovascular system. It was proposed that sEng might damage vascular endothelium, but detailed information about the potential mechanisms involved is not available. Thus, we hypothesized that sEng contributes to endothelial dysfunction, leading to a pro-inflammatory phenotype by a possible modulation of the TGF-ß and/or inflammatory pathways. MAIN METHODS: Human umbilical vein endothelial cells (HUVECs) and Human embryonic kidney cell line (HEK293T) were treated with different sEng concentration and time in order to reveal possible effect on biomarkers of inflammation and TGF-ß signaling. IL6 and NFκB reporter luciferase assays, quantitative real-time PCR analysis, Western blot analysis and immunofluorescence flow cytometry were used. KEY FINDINGS: sEng treatment results in activation of NF-κB/IL-6 expression, increased expression of membrane endoglin and reduced expression of Id-1. On the other hand, no significant effects on other markers of endothelial dysfunction and inflammation, including eNOS, peNOSS1177, VCAM-1, COX-1, COX-2 and ICAM-1 were detected. SIGNIFICANCE: As a conclusion, sEng treatment resulted in an activation of NF-κB, IL-6, suggesting activation of pro-inflammatory phenotype in endothelial cells. The precise mechanism of this activation and its consequence remains to be elucidated. A combined treatment of sEng with other cardiovascular risk factors will be necessary in order to reveal whether sEng is not only a biomarker of cardiovascular diseases, but also a protagonist of endothelial dysfunction.

Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response.

Endoglin is an auxiliary receptor for members of the TGF-ß superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-ß 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/fl)LysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Eng(fl/fl)LysMCre 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-ß1 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.

Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury.

After endothelial injury, the transcription factor Krüppel-like factor 6 (KLF6) translocates into the cell nucleus to regulate a variety of target genes involved in angiogenesis, vascular repair and remodeling, including components of the membrane transforming growth factor beta (TGF-ß) receptor complex such as endoglin and activin receptor-like kinase 1. The membrane metalloproteinase 14 (MMP14 or MT1-MMP) targets endoglin to release soluble endoglin and is involved in vascular inflammation and endothelial tubulogenesis. However, little is known about the regulation of MMP14 expression during vascular wounding. In vitro denudation of monolayers of human endothelial cell monolayers leads to an increase in the KLF6 gene transcriptional rate, followed by an upregulation of MMP14 and release of soluble endoglin. Concomitant with this process, MMP14 co-localizes with endoglin in the sprouting endothelial cells surrounding the wound border. MMP14 expression at mRNA and protein levels is increased by ectopic KLF6 and downregulated by KLF6 suppression in cultured endothelial cells. Moreover, after wire-induced endothelial denudation, Klf6 (+/-) mice show lower levels of MMP14 in their vasculature compared with their wild-type siblings. Ectopic cellular expression of KLF6 results in an increased transcription rate of MMP14, and chromatin immunoprecipitation assays show that KLF6 interacts with MMP14 promoter in ECs, this interaction being enhanced during wound healing. Furthermore, KLF6 markedly increases the transcriptional activity of different reporter constructs of MMP14 gene promoter. These results suggest that KLF6 regulates MMP14 transcription and is a critical player of the gene expression network triggered during endothelial repair.

Genome-wide transcriptional and functional analysis of endoglin isoforms in the human promonocytic cell line U937.

Endoglin is an auxiliary cell surface receptor for TGF-ß family members. Two different alternatively spliced isoforms, long (L)-endoglin and short (S)-endoglin, have been reported. S-endoglin and L-endoglin proteins vary from each other in their cytoplasmic tails that contain 14 and 47 amino acids, respectively. A critical role for endoglin in vascular development has primarily been studied in endothelial cells. In addition, endoglin expression is upregulated during monocyte-to-macrophage differentiation; however, little is known about its role in this myeloid context. To investigate the function of endoglin in monocytes, stable transfectants expressing the two endoglin isoforms in the promonocytic human cell line U937 were generated. The differential gene expression fingerprinting of these endoglin transfectants using DNA microarrays and further bioinformatics analysis showed a clear alteration in essential biological functions, mainly those related to "Cellular Movement", including cell adhesion and transmigration. Interestingly, these cellular functions are highly dependent on adhesion molecules, including integrins α1 (CD49a, ITGA1 gene), αL (CD11a, ITGAL gene), αM (CD11b, ITGAM gene) and ß2 (CD18, ITGB2 gene) and the chemokine receptor CCR2 (CD192, CCR2 gene), which are downregulated in endoglin transfectants. Moreover, activin A (INHBA gene), a TGF-ß superfamily member involved in macrophage polarization, was distinctly affected in each endoglin transfectant, and may contribute to the regulated expression of integrins. These data were confirmed by quantitative PCR, flow cytometry and functional tests. Taken together, these results provide new insight into endoglin function in monocytes.

Expression of endoglin isoforms in the myeloid lineage and their role during aging and macrophage polarization.

Endoglin plays a crucial role in pathophysiological processes such as hereditary hemorrhagic telangiectasia (HHT), preeclampsia and cancer. Endoglin expression is upregulated during the monocyte-to-macrophage transition, but little is known about its regulation and function in these immune cells. Two different alternatively spliced isoforms of endoglin have been reported, L-endoglin and S-endoglin. Although L-endoglin is the predominant variant, here, we found that there was an increased expression of the S-endoglin isoform during senescence of the myeloid lineage in human and murine models. We performed a stable isotope labelling of amino acids in cell culture (SILAC) analysis of both L-endoglin and S-endoglin transfectants in the human promonocytic cell line U937. Analysis of differentially expressed protein clusters allowed the identification of cellular activities affected during aging. S-endoglin expression led to decreased cellular proliferation and a decreased survival response to granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced apoptosis, as well as increased oxidative stress. Gene expression and functional studies suggested that there was a non-redundant role for each endoglin isoform in monocyte biology. In addition, we found that S-endoglin impairs the monocytic differentiation into the pro-inflammatory M1 phenotype and contributes to the compromised status of macrophage functions during aging.