Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension
- Internal Medicine Research Unit, Pfizer Inc, United States
- Comparative Medicine, Pfizer Inc, United States
- Drug Safety Research & Development, Pfizer Inc, United States
- Early Clinical Development, Pfizer Inc, United States
- Acceleron Pharma, United States
- Eisai Inc, United States
- Inflammation and Immunology Research Unit, Pfizer Inc, United States
- Target Sciences, Emerging Science and Innovation, Pfizer Inc, United States
- Department of Pharmacology, Vascular Biology and Therapeutics Program, Yale University School of Medicine, United States
Figures
Figure 1
Lymphatic endothelial markers are reduced in human failing heart.
(A–D) Expression of lymphatic endothelial marker genes VEGFC (A), LYVE1 (B), PDPN (C), FLT4 (D) in hearts from healthy human donors (HH) and hearts from patients with chronic heart failure (CHF). …
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Figure 1—source data 1
Clinical information.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig1-data1-v2.xlsx
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Figure 1—source data 2
Raw data supporting Figure 1.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig1-data2-v2.xlsx
Figure 2 with 1 supplement
VEGFCC156S treatment prevented angiotensin II-induced cardiac dysfunction.
(A) Experimental design for animal studies. Angiotensin II (angII) was infused to induce cardiac dysfunction, and VEGFCc156s (VEGFC) was infused as a lymphangiogenic therapy via the same …
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Figure 2—source data 1
Raw data supporting Figure 2.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Effect of VEGFCC156S on angiotensin II-induced cardiac dysfunction, cardiac lymphatics, and skin lymphatics.
(A) Pharmacokinetic analysis of VEGFCC156S in mice (n = 2 per dose group). (B) Plasma VEGFC concentration measured by ELISA at euthanasia (n = 10–17/group). (C–S) Mice were infused with saline + …
Figure 3 with 1 supplement
VEGFCC156S treatment improved cardiac lymphatic vascular function.
(A) Representative lymphangiography images of adult mouse hearts. FITC-dextran (green) indicates blood vessels, cadaverine (orange) indicates vascular leak and qdot (red) indicates injection site …
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Figure 3—source data 1
Raw data supporting Figure 3.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Single-channel lymphangiography images.
FITC-dextran (green) indicates blood vessels, cadaverine (orange, with white arrowheads) indicates vascular leak and qdot (red) indicates injection site (with a red arrowhead) and lymphatic vessels …
Figure 4 with 1 supplement
VEGFCC156S treatment ameliorated angiotensin II-induced inflammatory responses.
(A–B) Heat map visualization of all FDR-significant differential expression changes in genes comparing angII and ctrl groups (angII vs ctrl), angII+VEGFC and angII groups (angII+VEGFC vs angII), and …
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Figure 4—source data 1
Raw data supporting Figure 4.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Isolation of cardiomyocytes, lymphatic endothelial cells and blood endothelial cells using Prox1-eGFP mice for RNA-seq and additional data analysis.
(A) Design of Prox1-eGFP KI mouse model. The model was generated using CRISPR/Cas9 Technology, and an IRES-eGFP cassette was inserted into the 3’UTR of the endogenous Prox1 locus. (B) Representative …
Figure 5 with 1 supplement
VEGFCC156S treatment ameliorated angiotensin II-induced hypertension.
(A) Experimental design for telemetry study. (B) Mean blood pressure of control, angII and angII+VEGFCc156s mice after minipump implantation (a linear mixed model was used for statistics). (C–E) …
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Figure 5—source data 1
Raw data supporting Figure 5.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
VEGFCC156S treatment ameliorated angiotensin II-mediated blood pressure increase.
(A–I) Telemeters were implanted into mice followed by minipump implantation as described in Figure 5A. Systolic blood pressure at baseline (A), week 1 (B) and week 4 (C), diastolic blood pressure at …
Figure 6 with 1 supplement
VEGFCC156S treatment improved angiotensin II-induced kidney dysfunction.
(A–D) Urine was collected 4.5 weeks after minipump implantation. Urine volume (A), Urine non-esterified fatty acid (NEFA) levels (B), total urinary protein (C) and albumin to creatinine ratio (ACR) …
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Figure 6—source data 1
Raw data supporting Figure 6.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Analysis of pathological features, gene expression, and lymphatic vessel density in mouse kidneys.
(A) Kidneys from the mice described in Figure 2A were fixed, sectioned, and stained. Representative H and E stains and Picrosirius red (PSR) stains of mouse kidney sections are shown (n = 8–10/group)…
Figure 7 with 2 supplements
VEGFCC156S treatment reduced the gene expression of cardiac dysfunction, fibrosis and inflammatory markers, and alleviated cardiac fibrosis and inflammation at 1 week after treatment.
(A) Experimental design for the animal study. Angiotensin II (angII) was infused to induce cardiac dysfunction, and VEGFCc156s (VEGFC) was infused as a lymphangiogenic therapy via a …
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Figure 7—source data 1
Raw data supporting Figure 7.
- https://cdn.elifesciences.org/articles/58376/elife-58376-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Effect of VEGFCC156S on the lymphatic vessels in heart, kidney and ear skin at 1 week after treatment.
Mice were infused with saline + BSA, saline+ VEGFCC156S, angiotensin II + BSA, or angiotensin II + VEGFCC156S as described in Figure 7A. (A) Plasma VEGFC concentration measured by ELISA at …
Figure 7—figure supplement 2
VEGFCC156S treatment reduced the gene expression of inflammatory markers in heart and alleviated cardiac inflammation at 1 week after treatment.
(A–I) RNA was isolated from the mouse hearts, and quantitative RT-PCR was performed for Ptprc(Cd45) (A), Il1b (B), Il6 (C), Itgam (D), Itgax (E), Ccl3 (F), Ccl4 (G), Ccl6 (H), Cxcl10(IP-10) (I), and …
Author response image 1
Analysis of cell-specific marker expression in isolated cardiomyocytes (CMs), cardiac lymphatic endothelial cells (ECs), cardiac blood ECs and diaphragm lymphatic ECs.
The cells were isolated from the heart and diaphragm of a Prox1-eGFP mouse following the cell isolation procedure shown in Figure 4—figure supplement 1D in main manuscript. RNA was isolated from the …
Tables
Table 1
SNPs mapping to lymphatic endothelial markers associate with cardiac comorbidities.
SNPs in human genetic loci, where lymphatic endothelial marker genes VEGFC, LYVE1, FLT4 are located, are associated with cardiovascular and metabolic phenotypes. MRVI1, murine retrovirus integration …
| SNP | Nearest gene(s) | Associated phenotype | p Value | Reference |
|---|---|---|---|---|
| rs2333496 | VEGFC | Waist-to-hip ratio (WHR) adj BMI | 8.00E-11 | Lotta, et al. |
| rs7660760 | VEGFC | Left ventricle wall thickness | 4.00E-07 | Wild, et al. |
| rs309795 | VEGFC | 2 hr Glucose Adj. for BMI | 2.22E-06 | Saxena, et al. |
| rs114108584 | VEGFC | Idiopathic dilated cardiomyopathy | 9.00E-06 | Xu, et al. |
| rs11042906 | LYVE1 and MRVI1 | Systolic BP | 2.00E-14 | Kichaev, et al. |
| rs2218793 | LYVE1 and ADM | High density lipoprotein cholesterol (HDL-C) | 1.60E-11 | Sinnott-Armstrong, et al. |
| rs11042937 | LYVE1 and CTR9 | Coronary artery disease | 3.00E-10 | Van der harst, et al. |
| rs7940646 | LYVE1 and MRVI1 | Triglycerides 2017 Mostly European and East Asian | 2.93E-08 | Lu, et al. |
| rs10840457 | LYVE1 and MRVI1 | Arterial stiffness index | 3.00E-08 | Fung, et al. |
| rs11603178 | LYVE1 and MRVI1 | Diastolic BP | 9.85E-08 | Nealelab-uk-biobank |
| rs12807023 | LYVE1 and ADM | Plasma + serum IDL-TAG levels | 8.21E-07 | Kettunen, et al. |
| rs12807023 | LYVE1 and ADM | Plasma + serum XS-VLDL values (concentration, TAG) | 5.41E-06 | Kettunen, et al. |
| rs634501 | FLT4 and MGAT1 | High density lipoprotein cholesterol (HDL-C) | 9.35E-08 | Lu, et al. |
| rs142342609 | FLT4 and SCGB3A1 | WHR adjusted BMI | 7.00E-07 | Justice, et al. |
| rs12517906 | FLT4 and OR2Y1 | Body mass index (females) | 6.00E-06 | Johansson, et al. |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Mice, male | C67/BL6N | Charles River laboratories | Catalog: #027 | |
| Mice, male | Prox1-eGFP KI | This paper | eGFP gene was inserted at the end of endogenous Prox1 gene to generate this indicator line. The sperms of the animals are cryopreserved in Charles River Laboratories. | |
| Peptide, recombinant protein | Bovine serum albumin | Sigma-Aldrich | A7030 | |
| Peptide, recombinant protein | human angiotensin II | Sigma-Aldrich | A9525 | |
| Peptide, recombinant protein | recombinant human VEGF-C (Cys156Ser) protein VEGFC | R and D systems | 752-VC | |
| Other | minipump | Alzet | Alzet 1002 or Alzet 2006 | |
| Sequence-based reagent | Prox1-eGFP genotyping forward primer | This paper | PCR primer | The primer sequence is 5’TCCAGGCAACAGTTCTACAG. It can be ordered from Integrated DNA Technologies using the sequence. |
| Sequenced-based reagent | Prox1-eGFP genotyping reverse primer | This paper | PCR primer | The primer sequence is 5’TGCACATCAGATTGTCTAAGG. It can be ordered from Integrated DNA Technologies using the sequence. |
| Commercial assay or kit | human VEGFC Quantikine ELISA kit | R and D systems | DVEC00 | |
| Commercial assay or kit | V-PLEX Human VEGFC kit | MSD | K151LTD-1 | |
| Other | Telemeters | Data Sciences International | PA-C10 or HD-X10 | |
| Other | Vevo2100 or Vevo3100 | VisualSonics Inc | ||
| Other | doppler flow velocity system | Indus Instruments | ||
| Other | AimStrip Hb Hemoglobin test system | Germaine Laboratories | ||
| Other | Trizol reagent | ThermoFisher | 15596026 | |
| Commercial assay or kit | RNeasy mini kits | Qiagen | 74104 | |
| Commercial assay or kit | TaqMan RNA to Ct 1-step kit | Thermofisher | 4392938 | |
| Commercial assay or kit | high capacity cDNA reverse transcription kit | Thermofisher | 4368814 | |
| Other | TaqMan gene expression master mix | Thermofisher | 4369016 | |
| Other | Tapman probes for mouse | Thermofisher | Nppb, Mm01255770_g1; Pdpn, Mm01348912_g1; Lyve1, Mm00475056_m1; Flt4, Mm01292604_m1; Vegfc, Mm00437310_m1; Col1a1, Mm00801666; Col1a2, Mm00483888; Col3a1, Mm00802300_m1; Fn1, Mm01256744; Cd68, Mm03047343_m1; Cxcl10, Mm00445235_m1; Ptprc, Mm01293577_m1; Itgax, Mm00498701_m1; Itgam, Mm00434455_m1; Il1b, Mm00434228_m1; Il6, Mm00446190_m1; Ccl6, Mm01302419_m1; Ccl4, Mm00443111_m1; Ccl3, Mm00441259_g1; Ace2, Mm01159006_m1; Hprt, Mm03024075_m1 | |
| Other | QuantStudio 7 Flex Real-Time PCR System | ThermoFisher | ||
| Antibody | Anti-mouse Podoplanin antibody (Goat polyclonal) | R and D systems | AF3244 | 1:500 dilution for Western blot |
| Antibody | anti-Akt antibody (Rabbit polyclonal) | Cell signaling | #9272 | 1:1000 dilution for Western blot |
| Antibody | anti- p-Akt (Ser473) antibody (Rabbit polyclonal) | Cell signaling | #9271 | 1:1000 dilution for Western blot |
| Antibody | p44/42 MAPK (Erk 1/2) antibody (Rabbit polyclonal) | Cell signaling | #9102 | 1:2000 dilution for Western blot |
| Antibody | phsopho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Rabbit monoclonal) | Cell signaling | #4370 | 1:2000 dilution for Western blot |
| Antibody | GAPDH antibody (Rabbit monoclonal) | Cell signaling | #5174 | 1:5000 dilution for Western blot |
| Antibody | Lyve1 antibody (Rabbit polyclonal) | Abcam | ab14917 | 1:500 dilution for IF |
| Antibody | podoplanin antibody (Hamster monoclonal) | Abcam | ab11936 | 1:250 dilution for IF |
| Antibody | CD68 antibody (Rat monoclonal) | ThermoFisher | MA5-16674 | 1:300 dilution for IF |
| Antibody | smooth muscle actin antibody (Mouse monoclonal) | Abcam | ab7817 | 1:100 dilution for IF |
| Antibody | GFP antibody (Chicken polyclonal) | Abcam | ab13970 | 1:100 dilution for IF |
| Commercial assay or kit | trueview autofluorescence quenching kit | Vector laboratories | SP-8400–15 | |
| Other | Alexa 594-conjugated WGA | ThermoFisher | W11262 | 1:200 dilution for IF |
| Software, algorithm | Pathology image analysis software Visiopharm | Visiopharm | ||
| Other | FITC-Dextran | Sigma-Aldrich | FD500S | |
| Other | alexa Fluor 555 Cadaverine | ThermoFisher | A30677 | |
| Other | Qdot 655 | ThermoFisher | Q21021MP | |
| Other | Hamilton neuros micro-injection syringe | Hamilton | 6546006 | |
| Antibody | PE-Cy7 Mouse Anti-Mouse CD45.2 antibody (Mouse monoclonal) | BD Pharmingen | 560696 | 1:100 dilution for FACS |
| Antibody | APC Rat Anti-Mouse CD31 Clone MEC 13.3 (RUO) antibody (Rat monoclonal) | BD Pharmingen | 561814 | 1:100 dilution for FACS |
| Other | DAPI | Thermo Fisher | D1306 | 1:5000 dilution for FACS |
| Commercial assay or kit | RNeasy MinElute cleanup kit | Qiagen | 74204 | |
| Commercial assay or kit | Smart-Seq ultra low input RNA kit | Takara | 634890 | |
| Commercial assay or kit | Nextera DNA XT Library Prep Kit | Illumina | FC-131–1096 | |
| Commercial assay or kit | NextSeq 500/550 High Output Kit | Illumina | 20024907 | |
| Software, algorithm | R | R-project | v.3.5.3 and v.3.6.1 | |
| Commercial assay or kit | U-plex chemokine combo 1 (Ms) kit | MSD | K15321K-1 | |
| Other | Siemens clinical analyzer | ADVIA Chemistry XPT system | ||
| Commercial assay or kit | BCP albumin assay kit | Sigma-Aldrich | MAK125-1KT | |
| Other | Taqman probes for human | Thermo Fisher | LYVE1, Hs00272659_m1; PDPN, Hs00366766_m1; FLT4, Hs01047677_m1; VEGFC, Hs01099203_m1; HPRT1, Hs02800695_m1 | |
| Software, algorithm | Graphpad Prism | Graphpad |
