Figure S5.
Heterogeneous LV defect in S1pr1iΔLECmice. (A) Lymphatic vessels with leaky LVs from 3-mo-old S1pr1iΔLEC (TM@P1–7) mice were fixed, and whole-mount IHC was performed for the indicated markers. Confocal imaging and 3D reconstruction were performed to identify the structural defects in the LVs. One LV appeared to have three leaflets (arrows). One or both leaflets were abnormally elongated at their insertion points in two other LVs, resulting in abnormal en face LV structure (right). (B) Back leak test revealed significant leakage in the ileal LVs of 4-mo-old S1pr1iΔLEC (TM@8w). (C) A leaky LV from a 4-mo-old S1pr1iΔLEC (TM@8w) mouse was fixed, and whole-mount IHC was performed for PROX1 (blue) and CD31 (red). Confocal imaging and 3D reconstruction revealed a single leaflet (arrow). (D) Lymphatic vessels were incubated with CellTracker Green (CMFDA) stain and imaged live under various Pins and Pouts. The confocal images were 3D reconstructed or analyzed at various planes. Control LV was open when Pin = Pout and closed when Pout > Pin. Tight overlap between leaflets can be observed in digital sections. In contrast, a leaky LV from a 4-mo-old S1pr1iΔLEC (TM@8w) mouse remained open when Pout > Pin. Digital sections revealed short leaflets that did not overlap. Statistics: (A) Live imaging followed by fixation and whole-mount IHC was performed using n = 2 LVs from 3-mo-old S1pr1f/f;Prox1-tdTomato (TM@P1–P7) and n = 3 LVs from 3-mo-old S1pr1iΔLEC;Prox1-tdTomato (TM@P1–P7) mice. Additionally, n = 3 LVs that were not imaged live from 3-mo-old S1pr1iΔLEC (TM@P1–P7) mice were directly fixed and imaged by whole-mount IHC. (B) n = 13 LVs from n = 8 4-mo-old S1pr1f/f (TM@8w) mice and n = 13 LVs from n = 5 4-mo-old S1pr1iΔLEC (TM@8w) mice were analyzed for back leak. **P < 0.01. (C and D) Following back leak test (B), n = 5 LVs from 4-mo-old S1pr1f/f (TM@8w) mice and n = 8 leaky LVs from 4-mo-old S1pr1iΔLEC (TM@8w) mice were analyzed by live imaging, followed by fixation and whole-mount IHC. In addition to those 8, the single leaflet valve from the S1pr1iΔLEC (TM@8w) mouse was assessed only by fixation and whole-mount IHC (C). (D) Under live imaging at high pressure, three control valves from 4-mo-old S1pr1f/f (TM@8w) mice were completely normal. A representative control valve is shown in D. One control valve had a small gap at the commissure at the high pressure. The remaining valve had 1 dysfunctional commissure where the annulus failed to meet and asymmetrical leaflet insertions into the sinus, which prevented it from closing in response to adverse pressure. Of the S1pr1iΔLEC (TM@8w) LVs at high pressures, four LVs failed to close while three were partly closed and one tightly closed. From the four leaky LVs that failed to close, one had short leaflets that did not reach the midpoint of the lumen (shown in D). In two of the four leaky LVs and two of the three partly leaky LVs, there was asymmetry in the leaflet’s upstream insertion site.

Heterogeneous LV defect in S1pr1 iΔLEC mice. (A) Lymphatic vessels with leaky LVs from 3-mo-old S1pr1iΔLEC (TM@P1–7) mice were fixed, and whole-mount IHC was performed for the indicated markers. Confocal imaging and 3D reconstruction were performed to identify the structural defects in the LVs. One LV appeared to have three leaflets (arrows). One or both leaflets were abnormally elongated at their insertion points in two other LVs, resulting in abnormal en face LV structure (right). (B) Back leak test revealed significant leakage in the ileal LVs of 4-mo-old S1pr1iΔLEC (TM@8w). (C) A leaky LV from a 4-mo-old S1pr1iΔLEC (TM@8w) mouse was fixed, and whole-mount IHC was performed for PROX1 (blue) and CD31 (red). Confocal imaging and 3D reconstruction revealed a single leaflet (arrow). (D) Lymphatic vessels were incubated with CellTracker Green (CMFDA) stain and imaged live under various Pins and Pouts. The confocal images were 3D reconstructed or analyzed at various planes. Control LV was open when Pin = Pout and closed when Pout > Pin. Tight overlap between leaflets can be observed in digital sections. In contrast, a leaky LV from a 4-mo-old S1pr1iΔLEC (TM@8w) mouse remained open when Pout > Pin. Digital sections revealed short leaflets that did not overlap. Statistics: (A) Live imaging followed by fixation and whole-mount IHC was performed using n = 2 LVs from 3-mo-old S1pr1f/f;Prox1-tdTomato (TM@P1–P7) and n = 3 LVs from 3-mo-old S1pr1iΔLEC;Prox1-tdTomato (TM@P1–P7) mice. Additionally, n = 3 LVs that were not imaged live from 3-mo-old S1pr1iΔLEC (TM@P1–P7) mice were directly fixed and imaged by whole-mount IHC. (B) n = 13 LVs from n = 8 4-mo-old S1pr1f/f (TM@8w) mice and n = 13 LVs from n = 5 4-mo-old S1pr1iΔLEC (TM@8w) mice were analyzed for back leak. **P < 0.01. (C and D) Following back leak test (B), n = 5 LVs from 4-mo-old S1pr1f/f (TM@8w) mice and n = 8 leaky LVs from 4-mo-old S1pr1iΔLEC (TM@8w) mice were analyzed by live imaging, followed by fixation and whole-mount IHC. In addition to those 8, the single leaflet valve from the S1pr1iΔLEC (TM@8w) mouse was assessed only by fixation and whole-mount IHC (C). (D) Under live imaging at high pressure, three control valves from 4-mo-old S1pr1f/f (TM@8w) mice were completely normal. A representative control valve is shown in D. One control valve had a small gap at the commissure at the high pressure. The remaining valve had 1 dysfunctional commissure where the annulus failed to meet and asymmetrical leaflet insertions into the sinus, which prevented it from closing in response to adverse pressure. Of the S1pr1iΔLEC (TM@8w) LVs at high pressures, four LVs failed to close while three were partly closed and one tightly closed. From the four leaky LVs that failed to close, one had short leaflets that did not reach the midpoint of the lumen (shown in D). In two of the four leaky LVs and two of the three partly leaky LVs, there was asymmetry in the leaflet’s upstream insertion site.

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