Lymphatic-to-blood vessel transition in adult microvascular networks: A discovery made possible by a top-down approach to biomimetic model development

Abstract

Objective: Emerging areas of vascular biology focus on lymphatic/blood vessel mispatterning and the regulation of endothelial cell identity. However, a fundamental question remains unanswered: Can lymphatic vessels become blood vessels in adult tissues? Leveraging a novel tissue culture model, the objective of this study was to track lymphatic endothelial cell fate over the time course of adult microvascular network remodeling.

Methods: Cultured adult Wistar rat mesenteric tissues were labeled with BSI-lectin and time-lapse images were captured over five days of serum-stimulated remodeling. Additionally, rat mesenteric tissues on day 0 and day 3 and 5 post-culture were labeled for PECAM + LYVE-1 or PECAM + podoplanin.

Results: Cultured networks were characterized by increases in blood capillary sprouting, lymphatic sprouting, and the number of lymphatic/blood vessel connections. Comparison of images from the same network regions identified incorporation of lymphatic vessels into blood vessels. Mosaic lymphatic/blood vessels contained lymphatic marker positive and negative endothelial cells.

Conclusions: Our results reveal the ability for lymphatic vessels to transition into blood vessels in adult microvascular networks and discover a new paradigm for investigating lymphatic/blood endothelial cell dynamics during microvascular remodeling.

Keywords: angiogenesis; endothelial cell; lymphangiogenesis; lymphatic; microcirculation.

FIGURE 1.

The Rat Mesentery Culture Model enables real-time investigation of intact microvascular networks. Mesenteric tissue is transferred to a culture plate, quickly spread onto the bottom of a well, secured in place with a membrane insert, and covered with media. During culture microvascular networks, including blood and lymphatic vessels, remain intact and functional. This top-down tissue culture approach offers a multi-cellular/multi-system view of angiogenesis and lymphangiogenesis at the same time.

FIGURE 2.

Time-lapse imaging of blood and lymphatic vessel remodeling following stimulation by serum. Comparison of the same microvascular network labeled with BSI–lectin labeling at t = 0 hr (A) and t = 120 hr (B) after 10% serum stimulation identifies new connections between blood and lymphatic vessels. (C–L) Higher magnification images of the outlined region in (A) and (B) over time. The lymphatic vessel segment forms connections with blood vessels at t = 88 hr and t = 120 hr, marked by arrowheads. Increased blood vessel density is evident by t = 120 hr in culture. Scale bars = 100 μm.

FIGURE 3.

Quantification of angiogenesis, lymphangiogenesis, and lymphatic/blood vessel connections. The effect of serum treatment on capillary sprouting (A), lymphatic sprouting (B), lymphatic blunt ends (C) and lymphatic/blood vessel connections (D) was evaluated by comparing values for the control group (Day 0; uncultured) and at 72 hr (Day 3) and 120 hr (Day 5). * represents a significant difference between groups (p < 0.05).

FIGURE 4.

Time-lapse imaging of blood endothelial cell co-option of a lymphatic vessel. Comparison of the same region in a cultured tissue harvested from a GFP-positive rat time t = 0 hr (A) and t = 64 hr (B) after 10% serum stimulation identifies a new connection between blood and lymphatic vessels. (C–J) Images during the interaction time-course. GFP-positive cell extensions originate from the blood capillary and spread to wrap and co-opt the blunt-ended lymphatic vessel. Scale bars = 100 μm.

FIGURE 5.

Before and after images of lymphatic/blood endothelial cell mispatterning. (A, C) BSI-lectin labeling at time t = 0 hr (before culture) identifies blood and lymphatic vessels. (B) BSI-lectin labeling of the same region at time t = 144 hr (6 days of serum stimulation) identifies new connections to nearby blood vessels. Arrowheads identify connection locations. (D) PECAM and LYVE–1 lymphatic marker labeling at t = 144 hr suggests the original lymphatic vessel segment only partially maintained its lymphatic identity. Scale bars = 100 μm.

FIGURE 6.

Loss of LYVE–1 coverage along lymphatic vessels after 120 hours of serum stimulation. (A–C) LYVE–1-positive lymphatic vessels and PECAM-positive blood vessels at time t = 0 hr. There is almost no gap in lymphatic LYVE–1 coverage. (D–F) LYVE–1-positive lymphatic vessels and PECAM-positive blood vessels after 120 hours of serum stimulation. Arrowheads point to examples of gaps in lymphatic LYVE–1 coverage. Scale bars = 100 μm.

FIGURE 7.

Quantification of lymphatic-to-blood endothelial cell phenotype switch by serum stimulation. (A) Example of lymphatic/blood mosaic vessel characterized by discontinuous LYVE–1 labeling (arrowheads) along PECAM-positive vessel segments. (B) The effect of serum treatment for 120 hours on endothelial cell identity was quantified based on the percentage of lymphatic vessels fully covered with LYVE–1 marker. (C) Example of lymphatic/blood mosaic vessel characterized by discontinuous podoplanin labeling (arrowheads) along PECAM-positive vessel segments. (D) The effect of serum treatment for 120 hours on endothelial cell identity was quantified based on the percentage of lymphatic vessels fully covered with podoplanin marker. Scale bars = 50 μm. *** represents a significant difference between Day 0 (t = 0 hr) and Day 5 (t = 120 hr) groups (p < 0.0005).