Blood vascular networks in vertebrates are essential to tissue survival. Establishment of a fully functional vasculature is complex and requires a number of steps including vasculogenesis and ...angiogenesis that are followed by differentiation into specialized vascular tissues (i.e., arteries, veins, and lymphatics) and organ-specific differentiation. However, an equally essential step in this process is the pruning of excessive blood vessels. Recent studies have shown that pruning is critical for the effective perfusion of blood into tissues. Despite its significance, vessel pruning is the least understood process in vascular differentiation and development. Two recently published PLOS Biology papers provide important new information about cellular dynamics of vascular regression.
Inflammation and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). CD74 is a receptor for the proinflammatory cytokine macrophage migration ...inhibitory factor (MIF). This ligand/receptor complex initiates survival pathways and cell proliferation, and it triggers the synthesis and secretion of major proinflammatory factors and cell adhesion molecules.
We hypothesized that the MIF/CD74 signaling pathway is overexpressed in idiopathic PAH (iPAH) and contributes to a proinflammatory endothelial cell (EC) phenotype.
Primary early passage cultures of human ECs isolated from lung tissues obtained from patients with iPAH and controls were examined for their ability to secrete proinflammatory mediators and bind inflammatory cells with or without modulation of the functional activities of the MIF/CD74 complex. In addition, we tested the efficacies of curative treatments with either the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies on the aberrant proinflammatory EC phenotype in vitro and in vivo and on the progression of monocrotaline-induced pulmonary hypertension.
In human lung tissues, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions are markedly up-regulated in the endothelium of distal iPAH pulmonary arteries. Circulating MIF levels are increased in the serum of patients with PAH compared with control subjects, and T-cell lymphocytes represent a source of this overabundance. In addition, CD74 is highly expressed in the endothelium of muscularized pulmonary arterioles and in cultured pulmonary ECs from iPAH, contributing to an exaggerated recruitment of peripheral blood mononuclear cells to pulmonary iPAH ECs. Finally, we found that curative treatments with the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies partially reversed development of pulmonary hypertension in rats and substantially reduced inflammatory cell infiltration.
We report here that CD74 and MIF are markedly increased and activated in patients with iPAH, contributing to the abnormal proinflammatory phenotype of pulmonary ECs in iPAH.
Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor‐like ...kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15 weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC‐specifying transcription factor GATA‐binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9‐KO) mice as compared with wild‐type mice. LSECs from Bmp9‐KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9‐KO mice had a significantly reduced number of fenestrae. This was already observable in 2‐week‐old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis.
Pericytes and their crosstalk with endothelial cells are critical for the development of a functional microvasculature and vascular remodeling. It is also known that pulmonary endothelial dysfunction ...is intertwined with the initiation and progression of pulmonary arterial hypertension (PAH). We hypothesized that pulmonary endothelial dysfunction, characterized by abnormal fibroblast growth factor-2 and interleukin-6 signaling, leads to abnormal microvascular pericyte coverage causing pulmonary arterial medial thickening.
In human lung tissues, numbers of pericytes are substantially increased (up to 2-fold) in distal PAH pulmonary arteries compared with controls. Interestingly, human pulmonary pericytes exhibit, in vitro, an accentuated proliferative and migratory response to conditioned media from human idiopathic PAH endothelial cells compared with conditioned media from control cells. Importantly, by using an anti-fibroblast growth factor-2 neutralizing antibody, we attenuated these proliferative and migratory responses, whereas by using an anti-interleukin-6 neutralizing antibody, we decreased the migratory response without affecting the proliferative response. Furthermore, in our murine retinal angiogenesis model, both fibroblast growth factor-2 and interleukin-6 administration increased pericyte coverage. Finally, using idiopathic PAH human and NG2DsRedBAC mouse lung tissues, we demonstrated that this increased pericyte coverage contributes to pulmonary vascular remodeling as a source of smooth muscle-like cells. Furthermore, we found that transforming growth factor-β, in contrast to fibroblast growth factor-2 and interleukin-6, promotes human pulmonary pericyte differentiation into contractile smooth muscle-like cells.
To the best of our knowledge, this is the first report of excessive pericyte coverage in distal pulmonary arteries in human PAH. We also show that this phenomenon is directly linked with pulmonary endothelial dysfunction.
ALK1 is a type I receptor of the TGF-β family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that ...blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.
To define the role of ERK1/2 signaling in the quiescent endothelium, we induced endothelial
knockout in adult
mice. This resulted in a rapid onset of hypertension, a decrease in eNOS expression, and ...an increase in endothelin-1 plasma levels, with all mice dying within 5 wk. Immunostaining and endothelial fate mapping showed a robust increase in TGFβ signaling leading to widespread endothelial-to-mesenchymal transition (EndMT). Fibrosis affecting the cardiac conduction system was responsible for the universal lethality in these mice. Other findings included renal endotheliosis, loss of fenestrated endothelia in endocrine organs, and hemorrhages. An ensemble computational intelligence strategy, comprising deep learning and probabilistic programing of RNA-seq data, causally linked the loss of ERK1/2 in HUVECs in vitro to activation of TGFβ signaling, EndMT, suppression of eNOS, and induction of endothelin-1 expression. All in silico predictions were verified in vitro and in vivo. In summary, these data establish the key role played by ERK1/2 signaling in the maintenance of vascular normalcy.
Although the causal pathomechanisms contributing to remodelling of the pulmonary vascular bed in pulmonary arterial hypertension (PAH) are still unclear, several analogous features with ...carcinogenesis have led to the emergence of the cancer-like concept. The major similarities concern the altered crosstalk between cells from different tissue types, unexplained proliferation and survival of pulmonary smooth muscle and endothelial cells, the metabolic (glycolytic) shifts, and the association with the immune system. However, major differences between PAH and cancer exist, including the absence of invasion and metastasis, as well as the pathogenic genes involved and the degrees of angiogenesis impairment and genetic instability. It is clear that PAH is not a cancer, but this cancer-like concept has opened a new field of investigation and raises the possibility that antiproliferative and/or oncological drugs may exert therapeutic effects not only in cancer, but also in PAH. Such analogies and differences are discussed here.
Significance At birth, newborns must switch from the fetal aquatic life to the aerial one, by closure of a vessel named the ductus arteriosus. During fetal life, it allows blood to bypass the lungs, ...and a failure of its closure at birth is a major cause of mortality, particularly in preterm neonates. This pathological condition is known as patent ductus arteriosus and occurs in approximately 60% of preterm infants born before 28 wk of gestation. Herein, we show, for the first time to our knowledge, the involvement of two circulating growth factors, bone morphogenetic proteins BMP9 and BMP10, in the anatomical closure of this vessel. This finding will have potential clinical applications in the management of this pathology.
The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFβ family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetically inheritable vascular dysplasia caused by mutations in genes encoding receptors of the transforming growth factor-β ...(TGF-β) family: ENG, encoding endoglin (HHT1), and ACVRL1, encoding activin receptor-like kinase-1 (ALK1; HHT2). Our recent discovery of bone morphogenetic protein 9 (BMP9) as the specific ligand for ALK1 allowed us to reevaluate the functional significance of ACVRL1 mutations. We generated 19 ALK1 mutants reproducing HHT2 mutations (4 were novel mutations) found throughout the protein. We show that all ALK1 mutant proteins were expressed by transfected cells; most of them were present at the cell surface and retained their ability to bind BMP9 (except for the extracellular mutants). However, most were defective in BMP9 signaling. None of the ALK1 mutants had a dominant negative effect on wild-type ALK1 activity. These data demonstrate that mutations of ACVRL1 fit with a functional haploinsufficiency model affecting BMP9 signaling. Our study also identified 4 ACVRL1 mutations (D179A, R386C, R454W, and A482V) that did not alter the BMP9 responses that are polymorphisms and 2 novel mutations that are pathogenic (L381P and I485F). This demonstrates that the analysis of BMP9 responses can be used as a diagnostic tool by geneticists confronted with novel or conflicting ACVRL1 mutations.
The aim of the present work was to address the role of BMP9 in different genetic backgrounds (C57BL/6, BALB/c, and 129/Ola) of mice deleted for
. We found that
deletion led to premature mortality ...only in the 129/Ola strain. We have previously shown that
deletion led to liver sinusoidal endothelial cells (LSEC) capillarization and liver fibrosis in the 129/Ola background. Here, we showed that this is not the case in the C57BL/6 background. Analysis of LSEC from Wild-type (WT) versus
-KO mice in the C57BL/6 background showed no difference in LSEC fenestration and in the expression of differentiation markers. Comparison of the mRNA expression of LSEC differentiation markers between WT C57BL/6 and 129/Ola mice showed a significant decrease in Stabilin2, Plvap, and CD209b, suggesting a more capillary-like phenotype in WT C57BL/6 LSECs. C57BL/6 mice also had lower BMP9 circulating concentrations and hepatic Vegfr2 mRNA levels, compared to the 129/Ola mice. Taken together, our observations support a role for BMP9 in liver endothelial cell fenestration and prevention of fibrosis that is dependent on genetic background. It also suggests that 129/Ola mice are a more suitable model than C57BL/6 for the study of liver fibrosis subsequent to LSEC capillarization.
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