lyve 1 pe  (R&D Systems)

Journal: Nature Communications

Article Title: Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

doi: 10.1038/s41467-021-23546-6

Figure Lengend Snippet: a – b Analysis of lymphatic morphology in the ear of Prox1 GFP lymphatic reporter mice injected with 1 µg of Poly(C) or VEGFC mRNA-LNPs. Representative images 22 days ( a ) and 60 days ( b ) after the ear treatment of 15 ( a ) and 5 ( b ) mice in each group are shown by whole-mount fluorescent stereo microscopy (upper panels; bars, 1000 µm ( a ) and 500 µm ( b )) and Prox1-GFP signal and LYVE1 immunostaining of slides processed by paraffin-based histology are shown (lower panels; bars, 50 µm). Arrows indicate Prox1-GFP and LYVE1 double positive lymphatic vessels. c Assessment of the time-dependent effect of intradermal administration of 1 µg of Poly(C) or VEGFC mRNA-LNPs in the ear at days 5, 12, 17, 22, 35, and 60. Quantitative data for the length of lymphatic network, average lymphatic vessel diameter, and number of branching points per field of view are represented as mean and SEM from Poly(C) or VEGFC mRNA-LNP-injected ears of 5–15 mice in each group (two-tailed, paired T -test, for lymphatic network length P = 0.0005 after 22 days for 15 mice and P = 0.0005 after 60 days for 10 mice; for average lymphatic vessel diameter P = 0.000005 after 22 days for 15 mice and P = 0.0002 after 60 days for 10 mice; for number of branching points P = 7.48 × 10 −7 after 22 days for 15 mice and P = 0.0001 after 60 days for 10 mice). d Monitoring the dose-dependent effect of Poly(C) or VEGFC mRNA-LNPs (0.04, 0.2, and 1 µg) 20 days after intradermal treatment of the ear. Quantitative data for the length of lymphatic network, average lymphatic vessel diameter, and number of branching points per field of view are represented as mean and SEM from LNP complex-injected ear of 6–17 mice in each group. (two-tailed Wilcoxon signed-rank test for lymphatic network length P = 7.04 × 10 −5 for 17 mice, two-tailed, paired T -test for average lymphatic vessel diameter P = 4.61 × 10 −7 for 17 mice and two-tailed, paired T -test for number of branching points P = 6.84 × 10 −7 for 17 mice when injected with 1 µg of Poly(C) or VEGFC mRNA-LNP). e Monitoring the effect of intradermal injection into ears of Prox1 GFP mice with 1 µg of Poly(C) or VEGFC mRNA-LNPs shown by flow cytometry analysis. Quantitative data for GFP positive cell number are represented as mean and SEM from Poly(C) or VEGFC mRNA-LNP-injected ears of 6 mice in each group, 22 days after mRNA-LNP injection (two-tailed, paired T -test, P = 0.0010 for 6 mice). f Monitoring the effect 1 µg of locally injected Poly(C) or VEGFC mRNA-LNPs on lymphatic growth in Prox1 GFP mice in the diaphragm 22 days after intraperitoneal injection, in the lungs 22 days after intratracheal treatment, and in the musculus gastrocnemius 22 days after intramuscular injection. Quantitative data for number of the lymphatics are shown as mean and SEM of Poly(C) or VEGFC mRNA-LNP-injected organs of 3 mice in each group. Asterisks indicate P < 0.05 compared with control. (two-tailed, unpaired T -test, diaphragm: P = 0.0354 for 3 mice, lung: P = 0.0222 for 3 mice, two-tailed, paired T -test m. gastrocnemius: P = 0.0145 for 3 mice).

Article Snippet: Lymphatic endothelial cells were shown by GFP signal and immunostaining with PE anti-mouse LYVE1 (R&D Systems, FAB2125P) in a dilution of 1:200.

Techniques: Injection, Microscopy, Immunostaining, Two Tailed Test, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

doi: 10.1038/s41467-021-23546-6

Figure Lengend Snippet: a – b Assessment of lymphatic proliferation 5 days after intradermal injection with 1 µg of Poly(C) or VEGFC mRNA-LNPs. EdU staining and anti-LYVE1 immunostaining of slides processed by paraffin-based histology (bars, 50 µm) shown by widefield ( a ) and confocal imaging ( b ). Arrows indicate EdU and LYVE1 double positive lymphatic endothelial cell (LEC) nuclei. Representative images of 3 ears of 3 mice in each group are shown. c Number of EdU positive LECs (two-tailed, unpaired T -test, P = 0.0260 for 3 mice) and mitotic index (two-tailed, unpaired T -test, P = 0.0266 for 3 mice) are shown 5 days after intradermal injection with 1 µg of Poly(C) or VEGFC mRNA-LNPs. Quantitative data for lymphatic proliferation are represented as mean and SEM from 3 injected ears of 3 mice in each group.

Article Snippet: Lymphatic endothelial cells were shown by GFP signal and immunostaining with PE anti-mouse LYVE1 (R&D Systems, FAB2125P) in a dilution of 1:200.

Techniques: Injection, Staining, Immunostaining, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

doi: 10.1038/s41467-021-23546-6

Figure Lengend Snippet: a 1 µg of Poly(C) or VEGFC mRNA-LNPs were intradermally injected into the back skin of C57Bl/6 mice. Growth of lymphatic vessels in back skin, lung, and small intestine was monitored 22 days after the injection. Representative images 22 days after the treatment of the back skin of 4 mice per group are shown by anti-LYVE1 immunostaining of slides processed by paraffin-based histology (bars, 125 µm). Arrows indicate LYVE1 positive lymphatic vessels. b 5 µg of Poly(C) or VEGFC mRNA-LNPs were intradermally injected into the ear of C57Bl/6 mice. Growth of lymphatic vessels in the injected and non-injected ears, lung, and small intestine was assessed 22 days after the injection. Representative images after the ear treatment of 4 mice per group are shown by anti-LYVE1 and anti-Podoplanin immunostaining of slides processed by paraffin-based histology (bars 125 µm). Arrows indicate LYVE1 and Podoplanin double positive lymphatic vessels. c Monitoring the effect of intradermal injection into ears of Prox1 GFP mice with 5 µg of Poly(C) or VEGFC mRNA-LNPs shown by flow cytometry analysis. 5 µg of Poly(C) or VEGFC mRNA-LNPs were intradermally injected into the ear. Injected and non-injected ears, lungs, and small intestines were harvested and digested into single cell suspension. Quantitative data for GFP positive cell number are represented as mean and SEM from Poly(C) or VEGFC mRNA-LNP-injected ears, non-injected ears, non-injected lungs, and non-injected small intestines of 4 mice in VEGFC mRNA-LNP group and 3 mice in control group 22 days after mRNA-LNP injection (two-tailed, unpaired T -test for injected ears of Poly(C) RNA-LNP injected vs VEGFC mRNA-LNP injected mice P = 0.0402, two-tailed, unpaired T -test for contralateral non-injected ears of Poly(C) RNA-LNP injected vs. VEGFC mRNA-LNP injected mice P = 0.4006, two-tailed, unpaired T -test for lungs of Poly(C) RNA-LNP injected vs VEGFC mRNA-LNP injected mice P = 0.3168, two-tailed, unpaired T -test for small intestines of Poly(C) RNA-LNP injected vs VEGFC mRNA-LNP injected mice P = 0.1252). Asterisks indicate P < 0.05 compared with control. All cell nuclei are labeled with DAPI (blue) in paraffin-embedded tissues.

Article Snippet: Lymphatic endothelial cells were shown by GFP signal and immunostaining with PE anti-mouse LYVE1 (R&D Systems, FAB2125P) in a dilution of 1:200.

Techniques: Injection, Immunostaining, Flow Cytometry, Suspension, Control, Two Tailed Test, Labeling

Journal: Nature Communications

Article Title: Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

doi: 10.1038/s41467-021-23546-6

Figure Lengend Snippet: a 1 µg of Poly(C) or VEGFC mRNA-LNPs was intradermally injected into the ear of C57Bl/6 mice and the growth of blood and lymphatic vessels were examined 22 days after the injection. Representative images of anti-CD31, anti-LYVE1, anti-Podoplanin, and anti-vWF stained paraffin-embedded ear samples are shown of 5 mice in each group (bars, 25 µm (anti-CD31, anti-LYVE1), 50 µm (anti-Podoplanin, anti-vWF)). Yellow arrows indicate LYVE1 positive lymphatic vessels, white arrows indicate CD31 positive and LYVE1 negative blood vessels. b The number of vWF high and LYVE1 negative blood vessels was determined 22 days after the administration of 1 µg of Poly(C) or VEGFC mRNA-LNPs. Data are represented as mean and SEM from slides of ears of 10 mice per group (two-tailed, paired T -test, P = 0.6344 for 10 mice). c Monitoring the effect on lymphatic endothelial cells and immune cells after intradermal injection of 1 µg of Poly(C) or VEGFC mRNA-LNPs into ears of Prox1 GFP mice shown by flow cytometry analysis. Quantitative data for LYVE1+ , CD45+ , Ly6G/C (GR1)+, CD206+, CD3+, B220+ and CD11b+ Ly6G/C- cell numbers are represented as mean and SEM from Poly(C) or VEGFC mRNA-LNP-injected ears of 5–7 mice in each group, 22 days after mRNA-LNP injection (two-tailed, unpaired T -test, P = 0.0450 for LYVE1+ for 5 mice injected with Poly(C) and for 5 mice injected VEGFC mRNA-LNP, P = 0.5365 for CD45+ for 5 mice injected with Poly(C) and for 6 mice injected VEGFC mRNA-LNP, P = 0.1689 for Ly6G/C+ and CD45+ for 5 mice injected with Poly(C) and for 6 mice injected VEGFC mRNA-LNP, P = 0.7211 for CD206+ for 5 mice injected with Poly(C) and for 5 mice injected VEGFC mRNA-LNP, P = 0.8947 for CD3+ for 6 mice injected with Poly(C) and for 7 mice injected VEGFC mRNA-LNP, P = 0.7748 for B220+ for 6 mice injected with Poly(C) and for 7 mice injected VEGFC mRNA-LNP and P = 0.6922 for CD11b+ Ly6G/C- for 5 mice injected with Poly(C) and for 6 mice injected VEGFC mRNA-LNP comparing cell number). d Ly6G/C (GR1) positive immune cells were visualized after intradermal administration of 1 µg of Poly(C) or VEGFC mRNA-LNPs into the ears shown by anti-GR1 and anti-LYVE1 22 days after the injection. Representative images of anti-GR1 stained paraffin-embedded ear samples are shown of 3 mice per group (bars, 50 µm). Arrows indicate LYVE1 positive lymphatic vessels. Asterisks indicate P < 0.05 compared with control. All cell nuclei are labeled with DAPI (blue) in paraffin-embedded tissues.

Article Snippet: Lymphatic endothelial cells were shown by GFP signal and immunostaining with PE anti-mouse LYVE1 (R&D Systems, FAB2125P) in a dilution of 1:200.

Techniques: Injection, Staining, Two Tailed Test, Flow Cytometry, Control, Labeling

Journal: Nature Communications

Article Title: Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

doi: 10.1038/s41467-021-23546-6

Figure Lengend Snippet: a Monitoring active lymphatic function in the ears of Prox1 GFP mice after intradermal injection of 1 µg of Poly(C) or VEGFC mRNA-LNPs. Twenty-two days after treatment, 70 kDa Rhodamine dextran (Rh-D) was injected into the ear and the transport of the molecule was monitored by fluorescent microscopy 60 min post Rh-D administration. Representative images are shown of 10 injected mouse ears per group (bars, 1000 µm). Arrows indicate Prox1-GFP positive and Rh-D positive lymphatic vessels. b Monitoring active lymphatic function in the paws of mice after intradermal injection of 1 µg of Poly(C) or VEGFC mRNA-LNPs. Seventy-five days after treatment, 70 kDa Rh-D was injected into the paws and the transport of the molecule was monitored by fluorescent microscopy 90 min post Rh-D administration. Representative images are shown of 2 mouse hind limbs in each group (bars, 1000 µm). Green rectangles show the magnified area which represent the area of popliteal lymph nodes. c Analysis of lymphatic morphology in the ear of mice injected with 1 µg of Poly(C) or VEGFC mRNA-LNPs. Representative images 22 days after the treatment of 3 mouse ears per group are shown by anti-LYVE1 and anti-α-SMA immunostaining of slides processed by paraffin-based histology (bars, 50 µm). Arrows indicate LYVE1 low lymphatics surrounded by α-SMA positive cells. d – e Assessment of lymphatic proliferation 5 days after intradermal injection with 1 µg of Poly(C) or VEGFC mRNA-LNPs. Representative images of 3 mouse ears are shown in each group with EdU staining and anti-α-SMA immunostaining of slides processed by paraffin-based histology (bars, 50 µm) and shown by widefield ( d ) and confocal imaging ( e ). Arrows indicate EdU positive cells surrounded by α-SMA positive cells. All cell nuclei are labeled with DAPI (blue) in paraffin-embedded tissues.

Article Snippet: Lymphatic endothelial cells were shown by GFP signal and immunostaining with PE anti-mouse LYVE1 (R&D Systems, FAB2125P) in a dilution of 1:200.

Techniques: Injection, Microscopy, Immunostaining, Staining, Imaging, Labeling

Journal: Nature Communications

Article Title: Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

doi: 10.1038/s41467-021-23546-6

Figure Lengend Snippet: a Monitoring paw thickness and paw clinical score in tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice after treatment with PBS or Diphtheria toxin. Quantitative data are represented as mean and SEM for paw thickness and median and IQR for clinical score from 4–18 mouse paws in each group (two-tailed, paired T -test for paw thickness P = 4.21 × 10 −12 on day 8 for 16 mice, P = 5.05 × 10 −6 on day 16 for 14 mice, P = 1.62 × 10 −5 on day 30 for 18 mice, P = 0.0008 on day 60 for 13 mice, and P = 0.0112 on day 75 for 4 mice. Two-tailed Wilcoxon signed-rank test for clinical score P = 3.05 × 10 −5 on day 8 for 16 mice, P = 0.0002 on day 16 for 14 mice, P = 6.10 × 10 −5 on day 30 for 18 mice, P = 0.0020 on day 60 for 13 mice, and P > 0.9999 on day 75 for 4 mice). b Haematoxylin and Eosin staining of the paws of Flt4-CreER T2 ; iDTR fl/fl tamoxifen-treated mice 30 and 75 days after treatment with PBS or Diphtheria toxin. Representative images are shown of 5 mouse paws per group (bars, 200 µm). c Paw fibroadipose area of tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice 30 and 75 days after intradermal paw injection with PBS or Diphtheria toxin. Quantitative data are represented as mean and SEM from Haematoxylin and Eosin stained slides of 3–4 mouse paws in each group (two-tailed, paired T -test, P = 0.0469 on day 30 for 4 mice and P = 0.0172 on day 75 for 3 mice). d Representative images of anti-LYVE1 and anti-Podoplanin immunostaining of the paws of Flt4-CreER T2 ; iDTR fl/fl tamoxifen-treated mice 30 and 75 days after treatment with PBS or Diphtheria toxin. Representative images are shown of 4–5 mouse paws in each group (bars, 50 µm). Arrows indicate LYVE1 and Podoplanin double positive lymphatic vessels. e Number of lymphatic vessels of tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice 30 and 75 days after intradermal paw injection with PBS or Diphtheria toxin. Quantitative data are represented as mean and SEM from 4–5 mouse paws in each group (two-tailed, paired T -test, P = 0.0319 after 30 days for 4 mice and P = 0.0434 after 75 days for 5 mice). f Representative images of anti-Collagen I immunostaining of the paws of Flt4-CreER T2 ; iDTR fl/fl tamoxifen-treated mice 30 and 75 days after treatment with PBS or Diphtheria toxin. Representative images are shown of 5 mouse paws in each group (bars, 50 µm). Asterisks indicate P < 0.05 compared with control. All cell nuclei are labeled with DAPI (blue) in paraffin-embedded tissues.

Article Snippet: Lymphatic endothelial cells were shown by GFP signal and immunostaining with PE anti-mouse LYVE1 (R&D Systems, FAB2125P) in a dilution of 1:200.

Techniques: Two Tailed Test, Staining, Injection, Immunostaining, Control, Labeling

Journal: Nature Communications

Article Title: Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

doi: 10.1038/s41467-021-23546-6

Figure Lengend Snippet: a Monitoring paw thickness and clinical score in Diphtheria toxin and tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice intradermally injected with 1 µg of Poly(C) or VEGFC mRNA-LNPs. Quantitative data are represented as mean and SEM for paw thickness and box shows median and 25 th to 75 th percentiles and whiskers show 10 th –90 th percentiles for clinical score in 15–31 mouse paws in each group (two-tailed, paired T -test, for paw thickness P = 0.0090 on days 30 for 31 mice, P = 0.0053 on day 60 for 25 mice and P = 0.0082 on day 75 for 15 mice. Two-tailed Wilcoxon signed-rank test for clinical score P = 0.0050 on day 30 for 31 mice, P = 0.0278 on day 60 for 25 mice, and P = 0.0469 on day 75 for 15 mice). b Haematoxylin and Eosin histology of the paws of tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice 30 and 75 days after treatment with Diphtheria toxin and 1 µg of Poly(C) or Diphtheria toxin and 1 µg of VEGFC mRNA-LNPs intradermally. Poly(C) or VEGFC mRNA-LNPs were injected 8 days after Diphtheria toxin treatment. Representative images are shown of 5 mouse paws in each group (bars, 200 µm). c Paw fibroadipose area of tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice 30 and 75 days after treatment with Diphtheria toxin and 1 µg of Poly(C) or Diphtheria toxin and 1 µg of VEGFC mRNA-LNPs. Poly(C) or VEGFC mRNA-LNPs were injected 8 days after Diphtheria toxin treatment. Quantitative data are represented as mean and SEM in 8–9 mouse paws in each group stained with Haematoxylin and Eosin (two-tailed, paired T -test, P = 0.0001 on day 30 for 9 mice and P = 0.0047 on day 75 for 8 mice). d Anti-LYVE1, anti-Podoplanin immunofluorescent histology of the paws of tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice 30 and 75 days after treatment with Diphtheria toxin and 1 µg of Poly(C) or Diphtheria toxin and 1 µg of VEGFC mRNA-LNPs. Poly(C) or VEGFC mRNA-LNPs were injected 8 days after Diphtheria toxin treatment. Representative images are shown of 5 mouse paws in each group (bars, 50 µm). Arrows indicate LYVE1 and Podoplanin double positive lymphatic vessels. e Number of lymphatic vessels of tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice 30 and 75 days after the paw treatment with Diphtheria toxin and 1 µg of Poly(C) or Diphtheria toxin and 1 µg of VEGFC mRNA-LNPs. Poly(C) or VEGFC mRNA-LNPs were injected 8 days after Diphtheria toxin treatment. Quantitative data are represented as mean and SEM in 7–11 mouse paws in each group (two-tailed, paired T -test, P = 0.0024 after 30 days for 11 mice and P = 0.0008 after 75 days for 7 mice). f Anti-Collagen I immunofluorescent histology of the paws of tamoxifen-treated Flt4-CreER T2 ; iDTR fl/fl mice 30 and 75 days after treatment with Diphtheria toxin and 1 µg of Poly(C) or Diphtheria toxin and 1 µg of VEGFC mRNA-LNPs. Poly(C) or VEGFC mRNA-LNPs were injected 8 days after Diphtheria toxin treatment. Representative images are shown of 5 mouse paws in each group (bars, 50 µm). Asterisks indicate P < 0.05 compared with control. All cell nuclei are labeled with DAPI (blue) in paraffin-embedded tissues.

Article Snippet: Lymphatic endothelial cells were shown by GFP signal and immunostaining with PE anti-mouse LYVE1 (R&D Systems, FAB2125P) in a dilution of 1:200.

Techniques: Injection, Two Tailed Test, Staining, Control, Labeling