Figures and data
Methylene blue staining of isolated mouse IALVs
(A) Representative image of an isolated and cleaned IALV after methylene blue staining which revealed cells of various morphology. (B) is the zoomed in image of the yellow dotted box in A which contained large ovoid cells with granular staining (B, yellow asterisks). Fine cellular extensions (red asterisks) stained by methylene blue in some cells were visualized with color channel separation and division (C). (D, E) Similar as B and C, but in a separate vessel which stained with a higher density of methylene blue stained cells some of which had limited cellular processes. F) Focal reconstruction from imaging a methylene blue stained vessel using an upright microscope and immersion objective.
Staining Murine IALVs for ICLC Markers
Representative immunofluorescent max projections of half vessel confocal image stacks imaged from mouse IALVs stained for ICLC markers. DAPI (A), cKit (B), and CD34 (C) and their merged image (D). Representative max projections of the intermediate filament vimentin (E), the intermediate filament desmin (F), CD34 (G) and their merged image (H). Representative max projection of vimentin (I), cKit (J), CD34 (K) and their merged image (L). Scale bar = 100 µm for all images.
Immunofluorescent Labeling of Murine IALVs With Markers for ICLC, LMC, LEC, and Immune Cell Populations
We stained isolated mouse IALVs with cellular markers used to differentiate various cell types observed in cLVs. Half vessel image stacks were taken with confocal microscopy and the resulting representative max projections are shown. (A) CD34 stained cells and LMC staining with SMA (B) and calponin (C) and the corresponding merged (D) image. Significant overlap in (E) CD34 staining along with the fibroblast marker PDGFRα compared to LMC staining with SMA (G) and the merged (H) image. The endothelial marker CD31 (I) to delineate LECs with PDGFRα staining (J), and the LMC marker calponin (K) with the merged image (L). Mononuclear phagocyte cells were identified by anti-GFP (M) in IALVs from MacGreen mice and counter stained with PDGFRα (N) and the hematopoietic marker CD45 (O) with (P) the merged image. PDGFRβ (S) stained many cells that were CD34 (Q) and PDGFRα (R) positive in addition to signal detected in the LMC layer (U). Max projections of only the luminal frames of a z-stack at lymphatic valve locations revealed PDGFRβ, CD34, and PDGFRα labeling in bipolar shaped cells with long extensions that traveled throughout the valve leaflets (V, W). Secondary only stained control IALV (Y). Scale bar = 100 µm for all images.
PDGFRα+ Cells Reside Primarily in the Murine Lymphatic Collecting Vessel Adventitia and Some in the Subendothelial Space
Max projection of confocal imaging of an IALV stained for LECs with CD31 (A), LMCs with MYH11(B), and for PDGFRα (C) with the corresponding merge file (D). Orthogonal views of the z-stack with (E) showing a single slice in the z stack and E’ and E” the orthogonal views. White dotted boxes outline locations where PDGFRα signal is observed between LMC and LEC layers. Scale bar is 100 µm in (D) and 50 µm in (E).
Colocalization of CD34 and PDGFRα
Representative max projections and their corresponding threshold adjusted images for colocalization analysis for PDGFRα (A), CD34 (B), and their colocalized signal (C). Pearson’s coefficient and Mander’s coefficients were calculated from 3 separate stained IALVS, each from a separate mouse.
iCre-ROSA26mTmG Labelling and Fidelity to Target Putative Pacemaker Cell Populations
Stitched montages of serial max projections of GFP and tdTomato signal from live IALVs isolated from PdgfrαCre-ROSA26mTmG (A), Ng2Cre-ROSA26mTmG (B), PdgfrαCreERTM-ROSA26mTmG (C), PdgfrβCreERT2-ROSA26mTmG (D), cKitCreERT2-ROSA26mTmG (E), and Myh11CreERT2-ROSA26mTmG (F). IALVs were digested into single cells and GFP+ cells were purified via FACS from Prox1-eGFP (G), Myh11CreERT2-ROSA26mTmG (H), PdgfrαCreERTM-ROSA26mTmG (I), and PdgfrβCreERT2-ROSA26mTmG (J) mice. Representative gels demonstrating RT-PCR products corresponding to the respective gene used in the promoter of the transgenes to drive GFP signal or Cre mediated recombination of ROSA26mTmG from each GFP+ sorted population (K) to assess fidelity. Images are representative of IALVs from at least 3 separate mice. FACs and RT-PCR was repeated at least 3 times for each mouse.
RT-PCR Profiling of FACs Sorted Cells from iCre-ROSA26mTmG
Expanded RT-PCR profiling of genes to discriminate LECs, LMCs, and other cell types in our GFP+ sorted cells from Prox1-eGFP (A), Myh11CreERT2-ROSA26mTmG (B), PdgfrβCreERT2-ROSA26mTmG (C), and PdgfrαCreERTM-ROSA26mTmG (D). RT-PCR results for CD31, Cx45, Ano1, and CD45 in GFP+ cells sorted from PdgfrαCreERTM-ROSA26mTmG IALVs. RT-PCR was repeated at 2-4 times for each gene over each sorted cell population collected from different mice.
Isobaric contractile Assessment of popliteal cLV from PdgfrαCreTM-Ano1 fl/fl mice
Summary of the contractile parameters recorded from popliteal cLVs in PdgfrαCreERTM-Ano1fl/fl mice. Normalized contraction amplitude (A), ejection fraction (B), contraction frequency (C), fractional pump flow (D), end diastolic diameter (E), and vessel tone (F) were assessed. No statically significant differences observed. Mean and SEM shown, n=6 popliteal vessels from 3 mice PdgfrαCreERTM-Ano1fl/fl mice and n=10 popliteal vessels from 6 mice Ano1fl/fl mice.
Isobaric contractile Assessment of popliteal cLV from PdgfrαCreTM-Cx45 fl/fl mice
Summary of the contractile parameters recorded from popliteal cLVs in PdgfrαCreERTM-CX45fl/fl mice. Normalized contraction amplitude (A), ejection fraction (B), contraction frequency (C), fractional pump flow (D), end diastolic diameter (E), and vessel tone (F) were assessed. No statically significant differences observed. Mean and SEM shown, n=5 popliteal vessels from 3 mice PdgfrαCreERTM-CX45fl/fl mice and n=8 popliteal vessels from 11 mice CX45fl/fl mice.
Isobaric contractile Assessment of popliteal cLV from PdgfrαCreTM-Cav1.2 fl/fl mice
Summary of the contractile parameters recorded from popliteal cLVs in PdgfrαCreERTM-Cav1.2fl/fl mice. Normalized contraction amplitude (A), ejection fraction (B), contraction frequency (C), fractional pump flow (D), end diastolic diameter (E), and vessel tone (F) were assessed. Mean and SEM shown, n=6 popliteal vessels from 3 mice PdgfrαCreERTM-Cav1.2fl/fl mice and n=9 popliteal vessels from 20 mice Cav1.2fl/fl mice. The contractile data from control Cav1.2fl/fl vessels was previously published but was separated by sex (Davis et al., 2022) while they are combined here. * denotes significance at p <0.05.
PDGFRα Cells Express Multipotent Cell Makers
Representative RT-PCR results profiling purified GFP+ cells purified from IALVs isolated from PdgfrαCreERTM-ROSA26mTmG via FACS. PDGFRα cells expressed the multipotent markers Klf4, Sca1, Gli1, CD29, CD105, and CD44 (A) with total brain cDNA serving as a positive control (B). Representative RT-PCR results showing lack of expression of some of these markers in the GFP+ cells purified from Myh11CreERT2--ROSA26mTmG (C) or Prox1-eGFP mice, in contrast to the RFP+ population from Myh11CreERT2--ROSA26mTmG mice (D). RT-PCRs were repeated at least 2 times from separate purified cells populations from different mice. Representative max projections of IALVs stained for Sca1 (E), PDGFRα (E), SMA (F) or Sca1 (I), PDGFRα (K), MYH11 (J), and their corresponding merged file (H, L). Scale bar is 100 µm.
ChR2-Mediated Depolarization in LMCs Only Triggers Contraction
Representative max projections of tdTomato-ChR2 signal in popliteal cLVs isolated from cKitCreERT2-ChR2-tdTomato (A), PdgfrαCreERTM-ChR2-tdTomato (C), and Myh11CreERT2-ChR2-tdTomato (E) with their corresponding brightfield image (B, D, F) respectively. Time-lapse brightfield images every 0.5 s starting at stimulation t=0 for cKitCreERT2-ChR2-tdTomato (G-J), PdgfrαCreERTM-ChR2-tdTomato (K-N), and Myh11CreERT2-ChR2-tdTomato (O-R). The I bar denotes the inner diameter at t=0 over time and white asterisks denote the contraction. Representative diameter trace for the popliteal cLV demonstrate spontaneous contractions with the dotted boxes indicating the optical stimulation event in the respective brightfield images of the time lapse images. Isolated cLVs from cKitCreERT2-ChR2-tdTomato (S), PdgfrαCreERTM-ChR2-tdTomato (T), and Myh11CreERT2-ChR2-tdTomato (U) were stimulated with light pulses (red dashed lines) and the summation of contraction triggering for each genotype (V). Mean and SEM are shown, **** denotes p<0.0001. Contraction recorded from at least 6 popliteal cLVs from 3 mice per genotype.
cKitCreERT2 Drives GCaMP6f Expression in Primarily Mast Cells in Murine IALVs Representative max projection of GCaMP6f signal over time in an IALV isolated from a cKitCreERT2-GCaMP6f mouse with ROI indicated around individual cells, primarily large ovoid cells, but also including a circumferential LMC (Cell10) and a horizontal LEC (Cell 11). Of cells 1-9, only cell 7 had any Ca2+ activity (red arrows) during the recording time as indicated by the STMs from each ROI (B) and their normalized F/F0 plots in (C). In contrast, the LMC in ROI 10 had both rhythmic global Ca2+ events (D) that spanned the cell axis (vertical axis) in the STM (E) in addition to localized Ca2+ events intervening the time between global events (green arrows). Representative max projection of GCaMP6f signal over time after stimulation with C48-80 (F) with many large ovoid cells displaying long lasting global Ca2+ events (G,H) while not immediately affecting the LMC Ca2+ dynamics (I,J).
Lack of coordinated Ca2+ Activity Across Contraction Cycle in PDGFRα Cells
Representative max projections of GCaMP6f signal over time in an IALVs isolated from PdgfrαCreERTM-GCaMP6f mice (A, D). ROIs were made around cells and GCaMP6f recorded over time to generate the corresponding STMs (B, E) for each cell and plots (C, F) respectively. Once again, incidental recombination occurred in a LMC which displayed rhythmic Ca2+ flashes (C) while the slight undulation in the other cells is due to movement artifact (B). Red arrows indicate the limited local Ca2+ activity observed in two cells from a PdgfrαCreERTM-GCaMP6f IALV.
Heterogeneous Diastolic Ca2+ Transient Activity in LMCs
Representative max projections of GCaMP6f signal over time in an IALVs isolated from Myh11CreERT2-GCaMP6f mice (A). LMCs were outlined with ROIs to assess GCaMp6F signal over time. Rhythmic global flashes (B) were entrained across all the LMCs in the FOV (C) with many cells exhibiting diastolic Ca2+ release events. Cells exhibiting at least one diastolic Ca2+ event, within the context of our focal plane constraints, over the recorded time were denoted by the red asterisks. The plot in (D) magnifies the first diastolic period, seconds 1-3, of C to assist in visualizing the lack of coordination of the diastolic events. (E) Max projection of the pseudo-linescan analysis across the axis of the vessel to highlight diastolic Ca2+ transients in all cells in the field of view and their lack of coordination across the cells (x-axis). The white dotted box shows the first diastolic period plotted in (D).
Pressure Dependency of Murine LMC Diastolic Ca2+ Transients
Representative max projection of GCaMP6f signal over 20 s in an IALVs isolated from Myh11CreERT2-GCaMP6f mice in the presence of the L-type blocker nifedipine (1μM) (A) pressurized to 0.5 cmH2O, 2 cmH2O, 5 cmH2O. The local diastolic Ca2+ transients persist in the presence of nifedipine and increase with increasing pressure as demonstrated in the whole vessel STMs (B). Particle occurrence maps highlight the Ca2+ activity in each LMC as pressure is raised (C). Representative particle analysis plots for particle area (D) and particle counts/frame at each pressure (E). Summary files for particle area (F) and count /frame (G0. * denotes p<0.05, Mean and SEM shown with n=12 separate IALVs from 8 MYH11-CreERT2-GCaMP6f mice.
