TGF-beta-mediated endothelial to mesenchymal transition (EndMT) and the functional assessment of EndMT effectors using CRISPR/Cas9 gene editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-beta), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-beta-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-beta-induced EndMT. Using these techniques, we provide evidence that TGF-beta 2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.Cancer Signaling networks and Molecular Therapeutic

Inhibiting endothelial cell function in normal and tumor angiogenesis using BMP type I receptor macrocyclic kinase inhibitors

Simple Summary Anti-angiogenesis agents have shown anti-cancer activity by preventing blood vessel ingrowth, thereby limiting tumour growth and metastasis. Although these molecules lead to prolonged overall survival of cancer patients, therapy resistance is easily acquired. Therefore, novel inhibitors against other signaling pathways mediating angiogenesis are needed to achieve more efficient and sustainable targeting of the angiogenesis process. Here, we synthesized and identified two compounds belonging to a new class of small molecules termed macrocyclics that selectively inhibit bone morphogenetic protein receptor kinase activity. One compound also inhibits vascular endothelial growth factor-induced signalling. Treatment studies using in vitro cultured cells and zebrafish embryos revealed that both compounds impaired endothelial cell function and decreased normal and tumour-induced angiogenesis. Both compounds might provide a steppingstone for the development of novel-angiogenesis therapeutic agents. Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.Cancer Signaling networks and Molecular Therapeutic

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR(+) cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.Cancer Signaling networks and Molecular Therapeutic

Fibrodysplasia ossificans progressiva: what have we achieved and where are we now?: Follow-up to the 2015 Lorentz workshop

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics.Diabetes mellitus: pathophysiological changes and therap

Signal Transduction: Gain of Activin Turns Muscle into Bone

Cancer Signaling networks and Molecular Therapeutic

Endothelial-to-mesenchymal transition in cardiovascular diseases: Developmental signaling pathways gone awry

Therapeutic cell differentiatio

TGF-beta-induced endothelial to mesenchymal transition in disease and tissue engineering

Endothelial to mesenchymal transition (EndMT) is a complex biological process that gives rise to cells with multipotent potential. EndMT is essential for the formation of the cardiovascular system during embryonic development. Emerging results link EndMT to the postnatal onset and progression of fibrotic diseases and cancer. Moreover, recent reports have emphasized the potential for EndMT in tissue engineering and regenerative applications by regulating the differentiation status of cells. Transforming growth factor beta (TGF-beta) engages in many important physiological processes and is a potent inducer of EndMT. In this review, we first summarize the mechanisms of the TGF-beta signaling pathway as it relates to EndMT. Thereafter, we discuss the pivotal role of TGF-beta-induced EndMT in the development of cardiovascular diseases, fibrosis, and cancer, as well as the potential application of TGF-beta-induced EndMT in tissue engineering