Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

Kaposi sarcoma (KS) is an angioproliferative and invasive tumor caused by Kaposi sarcoma-associated herpesvirus (KSHV). The cellular origin of KS tumor cells remains contentious. Recently, evidence has accrued indicating that KS may arise from KSHV-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT), but the transformation process has been largely unknown. In this study, we investigated the KSHV-mediated MEndT process and found that KSHV infection rendered MSCs incomplete endothelial lineage differentiation and formed hybrid mesenchymal/endothelial (M/E) state cells characterized by simultaneous expression of mesenchymal markers Nestin/PDGFRA/α-SAM and endothelial markers CD31/PDPN/VEGFR2. The hybrid M/E cells have acquired tumorigenic phenotypes in vitro and the potential to form KS-like lesions after being transplanted in mice under renal capsules. These results suggest a homology of KSHV-infected MSCs with Kaposi sarcoma where proliferating KS spindle-shaped cells and the cells that line KS-specific aberrant vessels were also found to exhibit the hybrid M/E state. Furthermore, the genetic analysis identified KSHV-encoded FLICE inhibitory protein (vFLIP) as a crucial regulator controlling KSHV-induced MEndT and generating hybrid M/E state cells for tumorigenesis. Overall, KSHV-mediated MEndT that transforms MSCs to tumorigenic hybrid M/E state cells driven by vFLIP is an essential event in Kaposi sarcomagenesis.

Recent Advances on Cell-Based Co-Culture Strategies for Prevascularization in Tissue Engineering

Currently, the fabrication of a functional vascular network to maintain the viability of engineered tissues is a major bottleneck in the way of developing a more advanced engineered construct. Inspired by vasculogenesis during the embryonic period, the in vitro prevascularization strategies have focused on optimizing communications and interactions of cells, biomaterial and culture conditions to develop a capillary-like network to tackle the aforementioned issue. Many of these studies employ a combination of endothelial lineage cells and supporting cells such as mesenchymal stem cells, fibroblasts, and perivascular cells to create a lumenized endothelial network. These supporting cells are necessary for the stabilization of the newly developed endothelial network. Moreover, to optimize endothelial network development without impairing biomechanical properties of scaffolds or differentiation of target tissue cells, several other factors, including target tissue, endothelial cell origins, the choice of supporting cell, culture condition, incorporated pro-angiogenic factors, and choice of biomaterial must be taken into account. The prevascularization method can also influence the endothelial lineage cell/supporting cell co-culture system to vascularize the bioengineered constructs. This review aims to investigate the recent advances on standard cells used in in vitro prevascularization methods, their co-culture systems, and conditions in which they form an organized and functional vascular network.

Kaposi’s sarcoma-associated herpesvirus promotes mesenchymal-to-endothelial transition by resolving the bivalent chromatin of PROX1 gene

Increasing evidence suggests that Kaposi’s sarcoma (KS) arises from Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT). KSHV infection promotes MSC differentiation of endothelial lineage and acquisition of tumorigeneic phenotypes. To understand how KSHV induces MEndT and transforms MSCs to KS cells, we investigated the mechanism underlying KSHV-mediated MSC endothelial lineage differentiation. Like embryonic stem cells, MSC differentiation and fate determination are under epigenetic control. Prospero homeobox 1 (PROX1) is a master regulator that controls lymphatic vessel development and endothelial differentiation. We found that the PROX1 gene in MSCs harbors a distinctive bivalent epigenetic signature consisting of both active marker H3K4me3 and repressive marker H3K27me3, which poises expression of the genes, allowing timely activation upon differentiation signals or environmental stimuli. KSHV infection effectively resolves the bivalent chromatin by decreasing H3K27me3 and increasing H3K4me3 to activate the PROX1 gene. vIL-6 signaling leads to the recruitment of MLL2 and SET1 complexes to the PROX1 promoter to increase H3K4me3, and the vGPCR-VEGF-A axis is responsible for removing PRC2 from the promoter to reduce H3K27me3. Therefore, through a dual signaling process, KSHV activates PROX1 gene expression and initiates MEndT, which renders MSC tumorigenic features including angiogenesis, invasion and migration.

Transcriptional activity of Npas4l controls endothelial lineage specification and restriction in zebrafish

Die Bildung von Blutgefäßen ist essentiell für die Entwicklung und Homöostase von Wirbeltieren und die Endothelzellspezifikation ist ein wichtiger erster Schritt in diesem Prozess. Das früheste bekannte Ereignis bei der Endothelzellspezifikation im Zebrafisch ist die Expression des bHLH-PAS-Transkriptionsfaktor-Gens npas4l. Ich habe eine transgene V5-Linie zum Nachweis des markierten Npas4l auf Proteinebene und eine Gal4-VP16-Reporterlinie zur Visualisierung und Verfolgung von npas4l exprimierenden Zellen in vivo generiert. Beide Linien können bereits in frühen Entwicklungsstadien nachgewiesen werden und komplementieren auch starke npas4l-Mutanten Allele. Um npas4l Reporter exprimierende Zellen in npas4l Mutanten zu verfolgen, habe ich anschließend eine mutierte Variante der Gal4-Reporterlinie erzeugt. Diese Mutante trägt eine Insertion in der Region, die die DNA-Bindedomäne kodiert. Dadurch stört sie die Npas4l-Funktion, aber nicht die Reporterexpression. Dieses mutierte Reporterallel komplementiert nicht die npas4l-Mutanten und zeigt einen starken Phänotyp, was darauf hindeutet, dass es sich um ein funktionelles Nullallel handelt. Phänotypische Analysen zeigten, dass npas4l-Reporter positive Zellen in npas4l-Mutanten nicht spezifizieren oder zur Mittelachse wandern. Stattdessen tragen sie zu den vom intermediären Mesoderm abgeleiteten pronephrischen Tubuli und dem vom paraxialen Mesoderm abgeleiteten Skelettmuskel bei. Ich habe diese Phänotypen durch Einzelzell-RNAseq an den npas4l-Reporter positiven Zellen in npas4l+/- und npas4l-/- Embryonen bestätigt. Zusammen erklären diese beiden alternativen Zellschicksale den Großteil der beobachteten Veränderungen zwischen den Genotypen. Npas4l ist dafür bekannt die Expression der drei Transkriptionsfaktorgene etsrp, tal1 und lmo2 zu fördern. Ich stellte die Hypothese auf, dass das Fehlen jedes dieser Transkriptionsfaktoren in npas4l-Mutanten verschiedene Aspekte des npas4l-Phänotyps verursacht. Daher habe ich Mutantenlinien für alle drei Gene generiert und sie sowohl in vaskulären Reporterlinien als auch im npas4l-Reporterhintergrund analysiert. Die Daten legen nahe, dass verschiedene Gene unterschiedliche Prozesse während der frühen Endothelentwicklung regulieren. In npas4l-/- und etsrp-/- Embryonen differenzieren npas4l-Reporter exprimierende Zellen nicht zu Endothelzellen und tragen stattdessen zur Skelettmuskelzellpopulation bei. In npas4l-/- und tal1-/- Embryonen können npas4l-Reporter exprimierende Zellen nicht migrieren und tragen stattdessen zu der Bildung der pronephrischen Tubuli bei. Um die Beziehung zwischen diesen Faktoren besser zu verstehen, habe ich getestet, ob die Injektion von etsrp-, tal1- oder lmo2-mRNA verschiedene Aspekte des npas4l-Phänotyps retten würde. npas4l-, etsrp- und tal1-Mutanten zeigen alle schwere vaskuläre Phänotypen. Einige Endothelzellen und vaskuläre Strukturen bleiben jedoch in jeder Mutante erhalten. Der Phänotyp ist am stärksten in npas4l-/- Embryonen, aber selbst in diesen Embryonen können einige fli1a-positive Endothelzellen in der Schwanzregion beobachtet werden. Es war unklar, ob sich diese Population von Endothelzellen unabhängig von der Npas4l-, Tal1- und Etsrp-Funktion entwickelt oder als Folge einer restlichen tal1- oder etsrp-Expression unabhängig von Npas4l. Um diese Frage zu untersuchen, habe ich Doppelmutanten generiert und nach dem Vorhandensein von fli1a-positiven Endothelzellen in diesen Mutanten gesucht. Während fli1a-positive Endothelzellen in npas4l-/- und npas4l-/-;tal1-/- Embryonen deutlich vorhanden sind, können keine solchen Zellen in npas4l-/-;etsrp-/- oder etsrp-/-;tal1-/- Embryonen beobachtet werden. Diese Daten deuten darauf hin, dass sich im Zebrafisch keine Endothelzellen entwickeln können, wenn zugleich npas4l und etsrp oder etsrp und tal1 gestört sind. Während der Verlust von etsrp zu stärkeren Defekten in npas4l-Mutanten führt, gibt es keinen zusätzlichen Phänotyp, der durch den Verlust von tal1verursacht wird, was darauf hindeutet, dass die Expression von etsrp, aber nicht die von tal1, unabhängig von Npas4l auftreten kann. Diese Idee wird durch die Beobachtung unterstützt, dass etsrp, aber nicht tal1-Expression in den meisten fli1a-exprimierenden Zellen in npas4l-/- Embryonen beobachtet wird. Dennoch wird der Großteil -Expression durch Npas4l reguliert. tal1-mRNA-Injektionen reichten aus, um eine Wildtyp-ähnliche vaskuläre Musterbildung im Bauchbereich der npas4l-/- Embryonen wiederherzustellen, einschließlich der Rettung sowohl der Zellmigration als auch der Differenzierung. Da Npas4l mehrere unterschiedliche transkriptionelle Effektoren hat, war eine so starke Rettung durch nur einen dieser Effektoren unerwartet. In den geretteten Mutanten wurde die bilaterale Population von npas4l-Reporter-positiven pronephrischen Tubuluszellen nicht entdeckt, aber die Anzahl der ektopischen npas4l-Reporter exprimierenden Muskelzellen war im Vergleich zu nicht injizierten npas4l-Mutanten gleichbleibend. ...

Abstract 17503: ETV2 Functions as a Pioneer Factor and Regulates the Endothelial Lineage

Background: Genetic mutations perturb the multipotent progenitors, which results in congenital cardiovascular disease. Therefore, it is essential to decipher the pioneer factors and the regulatory pathways that govern the specification and differentiation of mesodermal progenitors and use this information to develop targeted therapies to promote cardiovascular regeneration. Etv2 as an essential transcription factor for the development of cardiac, endothelial and hematopoietic lineages. In the present study, we used ES/EB differentiation and MEF reprogramming systems, to define Etv2 as a novel pioneer factor that relaxes the closed chromatin and drives endothelial development. Results: Using the iHA-Etv2 ES cell line, we engineered a mouse that inducibly overexpresses ETV2. The bulk RNA-seq, single cell RNA-seq data and ATAC-seq experiments showed that inducing Etv2 in MEFs and ES/EBs activated the downstream endothelial marker genes and promoted the development of endothelial lineages, supporting the notion that Etv2 functioned as a master regulator to drive the endothelial lineage development in different cellular contexts. We found that similar to other known pioneer factors, Etv2 was intrinsically able to target and bind the nucleosomes, and this capability appeared to be independent of the cellular context. To further define the mechanism, we performed Etv2, Brg1 and H3K27ac ChIP-seq analyses during MEF reprogramming and ES/EB differentiation. We found that Brg1 maintains and stabilizes the binding of Etv2 on the nucleosome, and Etv2 requires Brg1 to activate downstream genes during reprograming. Conclusion: In these studies, we defined Etv2 as a novel pioneer factor that relaxed the closed chromatin and promoted the endothelial lineage in both ES/EB differentiation and MEF reprogramming. The definition of these mechanisms will enhance our understanding of cardiovascular development and regeneration and serve as a platform for therapeutic applications for patients with congenital or aging related cardiovascular diseases.

Epigenetic regulation of the lineage specificity of primary human dermal lymphatic and blood vascular endothelial cells

Lymphatic and blood vascular endothelial cells (ECs) share several molecular and developmental features. However, these two cell types possess distinct phenotypic signatures, reflecting their different biological functions. Despite significant advances in elucidating how the specification of lymphatic and blood vascular ECs is regulated at the transcriptional level during development, the key molecular mechanisms governing their lineage identity under physiological or pathological conditions remain poorly understood. To explore the epigenomic signatures in the maintenance of EC lineage specificity, we compared the transcriptomic landscapes, histone composition (H3K4me3 and H3K27me3) and DNA methylomes of cultured matched human primary dermal lymphatic and blood vascular ECs. Our findings reveal that blood vascular lineage genes manifest a more ‘repressed’ histone composition in lymphatic ECs, whereas DNA methylation at promoters is less linked to the differential transcriptomes of lymphatic versus blood vascular ECs. Meta-analyses identified two transcriptional regulators, BCL6 and MEF2C, which potentially govern endothelial lineage specificity. Notably, the blood vascular endothelial lineage markers CD34, ESAM and FLT1 and the lymphatic endothelial lineage markers PROX1, PDPN and FLT4 exhibited highly differential epigenetic profiles and responded in distinct manners to epigenetic drug treatments. The perturbation of histone and DNA methylation selectively promoted the expression of blood vascular endothelial markers in lymphatic endothelial cells, but not vice versa. Overall, our study reveals that the fine regulation of lymphatic and blood vascular endothelial transcriptomes is maintained via several epigenetic mechanisms, which are crucial to the maintenance of endothelial cell identity.