KSHV infection of endothelial precursor cells with lymphatic characteristics as a novel model for translational Kaposi's sarcoma studies

Author summaryKaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS). The main proliferative component of KS, spindle cells, express markers of lymphatic and blood endothelium. Endothelial precursor cells, which are circulating endothelial colony forming cells (ECFCs), have been proposed as the source of spindle cells. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are readily infected by KSHV, maintain the viral episomes and show modest transformation of the cells, which the infected blood ECFCs and all uninfected ECFCs failed to show. The lymphatic ECFCs express SOX18, which supported the maintenance of high copy numbers of KSHV genomes. The KSHV-infected lymphatic ECFCs persisted in vivo and recapitulated the phenotype of KS tumor cells such as high number of viral genome copies and spindling morphology. These KS tumor cell hallmarks were significantly reduced by SOX18 chemical inhibition using a small molecule SM4 treatment. These data suggest that KSHV-infected lymphatic ECFCs could be the progenitors of KS spindle cells and are a promising model for the translational studies to develop new therapies for KS.Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS.Peer reviewe

Angiopoietin–Tie signalling in the cardiovascular and lymphatic systems

Abstract Endothelial cells that form the inner layer of blood and lymphatic vessels are important regulators of vascular functions and centrally involved in the pathogenesis of vascular diseases. In addition to the vascular endothelial growth factor (VEGF) receptor pathway, the angiopoietin (Ang)–Tie system is a second endothelial cell specific ligand–receptor signalling system necessary for embryonic cardiovascular and lymphatic development. The Ang–Tie system also regulates postnatal angiogenesis, vessel remodelling, vascular permeability and inflammation to maintain vascular homoeostasis in adult physiology. This system is implicated in numerous diseases where the vasculature has an important contribution, such as cancer, sepsis, diabetes, atherosclerosis and ocular diseases. Furthermore, mutations in the TIE2 signalling pathway cause defects in vascular morphogenesis, resulting in venous malformations and primary congenital glaucoma. Here, we review recent advances in the understanding of the Ang–Tie signalling system, including cross-talk with the vascular endothelial protein tyrosine phosphatase (VE-PTP) and the integrin cell adhesion receptors, focusing on the Ang–Tie system in vascular development and pathogenesis of vascular diseases

KSHV infection of endothelial precursor cells with lymphatic characteristics as a novel model for translational Kaposi's sarcoma studies

Author summaryKaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS). The main proliferative component of KS, spindle cells, express markers of lymphatic and blood endothelium. Endothelial precursor cells, which are circulating endothelial colony forming cells (ECFCs), have been proposed as the source of spindle cells. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are readily infected by KSHV, maintain the viral episomes and show modest transformation of the cells, which the infected blood ECFCs and all uninfected ECFCs failed to show. The lymphatic ECFCs express SOX18, which supported the maintenance of high copy numbers of KSHV genomes. The KSHV-infected lymphatic ECFCs persisted in vivo and recapitulated the phenotype of KS tumor cells such as high number of viral genome copies and spindling morphology. These KS tumor cell hallmarks were significantly reduced by SOX18 chemical inhibition using a small molecule SM4 treatment. These data suggest that KSHV-infected lymphatic ECFCs could be the progenitors of KS spindle cells and are a promising model for the translational studies to develop new therapies for KS.Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS.Peer reviewe

Autoinhibition of Jak2 Tyrosine Kinase Is Dependent on Specific Regions in Its Pseudokinase Domain

Jak tyrosine kinases have a unique domain structure containing a kinase domain (JH1) adjacent to a catalytically inactive pseudokinase domain (JH2). JH2 is crucial for inhibition of basal Jak activity, but the mechanism of this regulation has remained elusive. We show that JH2 negatively regulated Jak2 in bacterial cells, indicating that regulation is an intrinsic property of Jak2. JH2 suppressed basal Jak2 activity by lowering the V(max) of Jak2, whereas JH2 did not affect the K(m) of Jak2 for a peptide substrate. Three inhibitory regions (IR1–3) within JH2 were identified. IR3 (residues 758–807), at the C terminus of JH2, directly inhibited JH1, suggesting an inhibitory interaction between IR3 and JH1. Molecular modeling of JH2 showed that IR3 could form a stable α-helical fold, supporting that IR3 could independently inhibit JH1. IR2 (725–757) in the C-terminal lobe of JH2, and IR1 (619–670), extending from the N-terminal to the C-terminal lobe, enhanced IR3-mediated inhibition of JH1. Disruption of IR3 either by mutations or a small deletion increased basal Jak2 activity, but abolished interferon-γ–inducible signaling. Together, the results provide evidence for autoinhibition of a Jak family kinase and identify JH2 regions important for autoregulation of Jak2

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Abstract Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling