DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells.

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Whole-mount immunofluorescence staining of blood and lymphatic vessels in murine small intestine.

The small intestine is an excellent model for studying changes in vasculature in response to different diseases or gene deletions. Here, we present a protocol for whole-mount immunofluorescence staining of blood and lymphatic vessels in the adult mouse small intestine. We describe the steps for perfusion fixation, tissue sample preparation, immunofluorescence staining, and whole-mount preparation of stained samples. Our protocol will enable researchers to visualize and analyze the intricate network of vessels in the small intestine. For complete details on the use and execution of this protocol, please refer to Karaman et al. (2022).1.

A closer look at adipose tissue lymphatics and their markers.

PURPOSE OF REVIEW: Lymphatic vessels are found in most tissues, with the exception of the cornea and the central nervous system. Tissues that have high exposure to antigens, such as the skin and the intestine, have especially extensive lymphatic vascular networks. Despite being densely vascularized with blood vessels, adipose tissue is poorly permeated with lymphatic vasculature. Here, we focus on the recent advances in the research on adipose tissue lymphatics and present a lymphatic-focused analysis of published single-cell and single-nucleus RNA sequencing datasets of adipose tissues. RECENT FINDINGS: Although lymphatic expansion in obesity may limit inflammation and promote glycerol efflux from adipose tissue, lymphatic endothelial cells (LECs) secrete factors that reduce brown adipocyte thermogenesis. Transcriptomic analyses of these cells show that they express common lymphatic markers such as Proxl, but datasets from different studies show great variation in gene expression values due to the low number of captured LECs, depot differences, and species-specific gene expression patterns. SUMMARY: As the importance of LECs in the homeostasis of adipose tissue has become evident, investigators want to shed light on the specific interactions of lymphatics with other cell types in adipose tissues. Extracting LECs from readily available transcriptomics datasets provides a standpoint for investigators for future research. However, systematic studies are needed to reveal unique identities according to depot and species-specific LEC signatures.

Interplay of vascular endothelial growth factor receptors in organ-specific vessel maintenance.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are quintessential for the development and maintenance of blood and lymphatic vessels. However, genetic interactions between the VEGFRs are poorly understood. VEGFR2 is the dominant receptor that is required for the growth and survival of the endothelium, whereas deletion of VEGFR1 or VEGFR3 was reported to induce vasculature overgrowth. Here we show that vascular regression induced by VEGFR2 deletion in postnatal and adult mice is aggravated by additional deletion of VEGFR1 or VEGFR3 in the intestine, kidney, and pancreas, but not in the liver or kidney glomeruli. In the adult mice, hepatic and intestinal vessels regressed within a few days after gene deletion, whereas vessels in skin and retina remained stable for at least four weeks. Our results show changes in endothelial transcriptomes and organ-specific vessel maintenance mechanisms that are dependent on VEGFR signaling pathways and reveal previously unknown functions of VEGFR1 and VEGFR3 in endothelial cells.

Capillary Rarefaction in Obesity and Metabolic Diseases-Organ-Specificity and Possible Mechanisms.

Obesity and its comorbidities like diabetes, hypertension and other cardiovascular disorders are the leading causes of death and disability worldwide. Metabolic diseases cause vascular dysfunction and loss of capillaries termed capillary rarefaction. Interestingly, obesity seems to affect capillary beds in an organ-specific manner, causing morphological and functional changes in some tissues but not in others. Accordingly, treatment strategies targeting capillary rarefaction result in distinct outcomes depending on the organ. In recent years, organ-specific vasculature and endothelial heterogeneity have been in the spotlight in the field of vascular biology since specialized vascular systems have been shown to contribute to organ function by secreting varying autocrine and paracrine factors and by providing niches for stem cells. This review summarizes the recent literature covering studies on organ-specific capillary rarefaction observed in obesity and metabolic diseases and explores the underlying mechanisms, with multiple modes of action proposed. It also provides a glimpse of the reported therapeutic perspectives targeting capillary rarefaction. Further studies should address the reasons for such organ-specificity of capillary rarefaction, investigate strategies for its prevention and reversibility and examine potential signaling pathways that can be exploited to target it.