The lymphatic vasculature is involved in the pathogenesis of acute cardiac injuries, but little is known about its role in chronic cardiac dysfunction. Here, we demonstrate that angiotensin II infusion induced cardiac inflammation and fibrosis at 1 week and caused cardiac dysfunction and impaired lymphatic transport at 6 weeks in mice, while co-administration of VEGFCc156s improved these parameters. To identify novel mechanisms underlying this protection, RNA sequencing analysis in distinct cell populations revealed that VEGFCc156s specifically modulated angiotensin II-induced inflammatory responses in cardiac and peripheral lymphatic endothelial cells. Furthermore, telemetry studies showed that while angiotensin II increased blood pressure acutely in all animals, VEGFCc156s-treated animals displayed a delayed systemic reduction in blood pressure independent of alterations in angiotensin II-mediated aortic stiffness. Overall, these results demonstrate that VEGFCc156s had a multifaceted therapeutic effect to prevent angiotensin II-induced cardiac dysfunction by improving cardiac lymphatic function, alleviating fibrosis and inflammation, and ameliorating hypertension. The lymphatic vasculature plays an important role in mediating fluid balance and local inflammation in many tissues and organs throughout the body . The heaVEGFC, LYVE1, FLT4 loci are associated with cardiovascular and metabolic phenotypes including cardiac hypertrophy in humans  reduction was observed for lymphatic markers VEGFC, LYVE1, and FLT4, and a 31.6% reduction was observed in PDPN (p=0.07) in hearts from chronic heart failure patients compared with hearts from healthy donors  for single-nucleotide polymorphisms (SNPs) that were near lymphatic marker gene loci and were associated with human cardiovascular or metabolic comorbidities. Interestingly, several SNPs near lymphatic endothelial markers n humans ; Table 1y donors , suggestTo examine the role of the lymphatic vasculature in cardiac dysfunction further, we used a model of angiotensin II infusion, which promotes cardiac dysfunction directly via its effects on cardiomyocytes  and indiNppb, encoding heart failure marker B-type natriuretic peptide (BNP), in angiotensin II-treated mouse hearts compared with control hearts, while VEGFCc156s significantly decreased Nppb expression in angiotensin II-infused hearts , which stains the cell membrane. Whereas angiotensin II infusion significantly increased cardiomyocyte size and heterogeneity, VEGFCc156s treatment prevented the change in cell size heterogeneity in angiotensin II-infused mouse hearts , confirmTo understand how VEGFCc156s ameliorated cardiac dysfunction, the effect of VEGFCc156s on cardiac lymphatic vascular marker expression and VEGFR3-mediated signaling cascades was examined. Similar to what was observed in human hearts , a signiTo investigate whether the changes in podoplanin-positive lymphatic vessel density and lumen area in heart were translated into functional changes, we conducted lymphangiography to directly evaluate cardiac lymphatic transport function. FITC-dextran and cadaverine were injected intravenously to label the cardiac blood vasculature and reveal any vascular leak, respectively. Next, fluorescent qdots were injected into the apex of the heart. Qdots are macromolecules that diffuse into interstitial fluid and may be cleared by the lymphatic vasculature and transported back to the blood circulation . AngioteProx1 gene using CRISPR/Cas9 technology and confirmed the eGFP signals from lymphatic vessels in the heart and the diaphragm  analysis to profile the transcriptional changes in different cell populations from the heart. Transcriptional changes in diaphragm lymphatic endothelial cells (LECs) were also profiled to evaluate the effect of VEGFCc156s on peripheral lymphatics, as the treatment is systemic. To efficiently distinguish LECs from blood endothelial cells (BECs), we generated a Prox1-eGFP knock-in mouse model in which eGFP expression is driven by the endogenous promoter of the lymphatic endothelial marker iaphragm . The Proiaphragm  and usediaphragm . Differeiaphragm . A causaiaphragm . One comiaphragm . Notablyiaphragm . Causal iaphragm .To validate the findings from the RNA-seq analysis, we examined circulating chemokine levels as well as chemokine levels in skin, which is highly vascularized with lymphatic capillaries. Consistent with the RNA-seq analysis, a significant 43% increase in plasma KC/GRO levels was noted in angiotensin II-infused mice compared with control mice, and VEGFCc156s treatment trended to reduce plasma KC/GRO levels in angiotensin II-infused mice . FurtherAce2 mRNA, which encodes the degradation enzyme for angiotensin II, was induced in both angiotensin II-infused mice and VEGFCc156s-treated, angiotensin II-infused mice to a similar extent compared with the kidneys from control mice, indicating a similar compensatory response in kidney to cope with continuous angiotensin II infusion  stained sections by a veterinary pathologist, angiotensin II infusion induced vascular hypertrophy in the kidney of both angiotensin II-infused and VEGFCc156s-treated angiotensin II-infused animals, which is consistent with the putative action of angiotensin II as a vasoactive molecule . HoweverLyve1, Pdpn, Vegfc, and Flt4 in kidney, which is consistent with previous reports  was observed in angiotensin II-infused hearts compared with control hearts, while animals that had also been treated with VEGFCc156s demonstrated a significant reduction in the expression of these markers  protein  were used as treatments. The sample size was predicted prior to study start with a power analysis. All animal experimental procedures were carried out in accordance with regulations and established guidelines and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee (AUP # KSQ-2013–00895).10 to14 weeks old C57/BL6N (#027 purchased from Charles River laboratories) male mice were used for most of the studies, and Prox1-eGFP knock-in male mice were generated using the CRISPR/Cas9 technology and used for in vivo studies at 9–15 weeks old. Bovine serum albumin , human angiotensin II  and 20) (or Meloxicam SR) and Buprenorphine were used as pain medications for pre-op and post-op care.Animals were randomized based on body weight and implanted with a minipump (Alzet 1002 or Alzet 2006 pump) subcutaneously for continuous infusion for 7 days or 42 days. Isoflurane was used as anesthetic in the minipump implantation surgery, and Carprofen in the form of MediGel CPF (Clear HProx1-eGFP knock-in model was generated by inserting an IRES-eGFP cassette into the 3′UTR of the endogenous Prox1 locus (at 470 bp downstream of the stop codon). For gene editing in one-cell embryos collected from super-ovulated C57BL/6J donors, Cas9 (Streptococcus pyogenes) protein (IDT) or mRNA (TriLink), a pair of single guide RNAs (sgRNA) and a donor plasmid were delivered via pronuclear injection. Seed sequences for the sgRNAs were 5′tacccagctgccgagggcat, 5′gtgagctacccagctgccga, and 5′aaccaatgccctcggcagct. The seed sequences were carefully chosen to minimize potential risk of off-target effect. Specifically, these guides lack any potential off-target with off-target score over 1.5 (http://www.benchling.com) across the whole mouse genome. The donor plasmid consists of IRES-eGFP flanked by 1.0 kb and 0.8 kb homology arms on the 5′ and 3′ sides, respectively. Founders were identified by genomic PCR with the forward and reverse primers, 5TCCAGGCAACA′GTTCTACAG and 5′TGCACATCAGATTGTCTAAGG, both external to the homology arms. Germline transmission of the targeted allele was achieved by crossing the founders with wild-type C57BL/6J mice.The Animals were bled via tail snip immediately prior to subcutaneous injection of 0.1 μg/g or 0.3 μg/g human VEGF-C (Cys156Ser) protein dissolved in phosphate buffered saline containing 0.1% BSA. Blood was sampled in EDTA from the animals at 1, 2, 4, 6, and 24 hr after injection via tail snip bleeding. Plasma VEGFC concentration was measured using the human VEGFC Quantikine ELISA kit  or V-PLEX Human VEGFC kit .20) was provided to the animals 2 days prior to surgery and 2 days following surgery, and Buprenorphine was administered post-operatively, and then as needed. The animals recovered for 2 weeks following surgery and telemetry signals were evaluated for acceptable signals. Animals with functional telemeters and stable telemetry signals were used for the following minipump implantation and continuous telemetry recordings. Mean blood pressure, systolic blood pressure, diastolic blood pressure, and heart rate were recorded from the animals at 1 min intervals for 5 weeks. The data for individual day reflect the averaged value over a 24-hr reading period and the weekly data reflect the averaged values over a 3-day reading period. Except necessary post-op care and husbandry needs, any other potential disturbances were minimized. Time-course telemetry recordings were analyzed using a linear mixed model, in which animals are treated as random effects, while time point (i.e. week) and group are treated as fixed effects. Tukey’s multiple comparison adjustment was performed for pairwise comparisons in the linear mixed model when the group had a statistically significant impact on the endpoint.Telemeters  were implanted in the animals by cannulating the carotid artery. A small skin incision was made midline over the ventral surface of the neck. The carotid artery was exposed and isolated using blunt dissection. Three lengths of non-absorbable suture were passed underneath the isolated vessel section . Tension was applied to the proximal and distal sutures to occlude blood flow and elevate the vessel. A small incision was made in the vessel wall with a 25-gauge needle bevel; the catheter was inserted, and tension was released on the proximal occlusion suture. The catheter was advanced beyond the middle and proximal suture approximately 8.0 mm. The middle and proximal sutures were tied around the vessel and catheter. The skin was closed in an interrupted pattern using 6–0 absorbable monofilament suture. Carprofen in the form of MediGel CPF . B-mode images from long axis and M-mode images from short axis were obtained by one trained individual (T.S. or X.C.), and data were analyzed blindly by another trained individual (L.S.) using Vevo Lab. Pulse wave velocity doppler was conducted using a doppler flow velocity system (Indus Instruments). Simultaneous doppler velocity spectrograms were obtained from the aortic arch and the abdominal aorta using two probes, and the recordings were analyzed blindly by L.S. following manufacturer’s technical notes. Pulse wave velocity was calculated by dividing the distance between the probes over the time measurement.Animals were subjected to isoflurane anesthesia, and tissues  and blood were collected from the animals. Hearts and lungs were weighed, and hearts, chest skins, and kidneys were snap-frozen in liquid nitrogen and stored in −80°C. The tibia was isolated from the animals, and its length was measured with a caliper. Hemoglobin level and hematocrit percentage were measured using an AimStrip Hb Hemoglobin test system (Germaine Laboratories) using whole blood in EDTA. The blood was next centrifuged, and plasma was isolated and stored at −80°C until being used for plasma VEGFC concentration measurements. For histology, animals were perfused transcardially with PBS containing heparin (20 U/ml), vasodilators (adenosine 1 g/L and papaverine 4 mg/L) and 25 mM potassium chloride (to arrest the heart in diastole) followed by 10% neutral-buffered formalin perfusion. The heart, ear, diaphragm, and kidney were next collected and immersed in 10% neutral-buffered formalin at 4°C for 24–48 hr.Wet heart weights were measured immediately after euthanasia, and hearts were dried in a 37°C incubator for 5 days. Dry weights were measured after desiccation. The wet-to-dry weight ratio was calculated by dividing the wet weight over the dry weight for each animal.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), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System. The expression of every gene was normalized to the expression of housekeeping gene Hprt and then normalized to control group to be presented as fold change in the corresponding figures.Snap-frozen tissues were smashed, mixed and distributed into small portions for RNA and protein isolation. RNA was isolated from the tissues using Trizol reagent (Thermofisher) and RNeasy mini kits (Qiagen). The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes , Akt antibody , p-Akt (Ser473) antibody , p44/42 MAPK (Erk 1/2) antibody , phsopho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody , and GAPDH antibody  were used for western blots. For western blots, tissues were lysed using cell lysis buffer  containing Halt protease and phosphatase inhibitor cocktail and EDTA. Protein concentrations were measured using Pierce BCA protein assay kit and samples were denatured using NuPAGE LDS sample buffer and reducing agent at 95°C for 5 min. A total of 20–40 μg of proteins were loaded to each well of NuPAGE 4–12% Bis-Tris Midi gels and ran in MOPS buffer with Xcell4 Surelock midi gel electrophoresis system. The gels were transferred to PVDF membranes using an iblot2 gel transfer device and blocked with 5% milk  for 1 hr under room temperature followed by primary antibody incubation at 4°C overnight. The next day, membranes were washed and incubated with corresponding HRP-conjugated secondary antibody for 1 hr under room temperature. Then the membranes were washed, developed using SuperSignal west Femto maximal sensitivity substrate and imaged with Amersham Imager 600 . All reagents were from ThermoFisher unless otherwise stated. Densitometric quantification was conducted using Image J.2 was used to define the vessels and reduce the noise from podoplanin-positive cells. The count of podoplanin-positive lymphatic vessels was normalized to the total area for each section and defined as the density of podoplanin-positive lymphatic vessels. The average lumen area was calculated by dividing the lumen area of the podoplanin-positive lymphatic vessels by the density of podoplanin-positive lymphatic vessels in each section. A linear mixed model with animals treated as random effects and group and sections treated as fixed effects was used for the statistical analysis for the quantification of podoplanin-positive lymphatic vessel density and lumen area. Tukey’s multiple comparison adjustment was used for pairwise comparisons in the linear mixed model. The data for podoplanin-positive lymphatic vessel density was normalized to the average value of ctrl group and represented as fold change. The analysis for the quantification of cardiomyocyte size and variance coefficient was conducted following previously published methods and SOPs for muscle fiber size and size variability quantification  through the hilus of the kidney from each animal was stained with H and E using an automatic slide stainer (Leica) and microscopic evaluation was performed by a veterinary pathologist. Cryo-sections (10 μm thickness) through the hilus of the kidney were generated for immunohistochemistry. Cryo-section (10 μm thickness) of OCT embedded hearts at different depths spanning the whole ventricle were generated for immunohistochemistry. Cryo-section (10 μm thickness) of OCT embedded ears at different depths spanning the whole ear were generated for immunohistochemistry. Picrosirius red (PSR) stain were conducted using mouse heart or kidney frozen sections following an optimized protocol using Bouin’s solution , 1% phosphomolybdic acid , 0.1% picrosirius red solution  and 1% acetic acid . Lyve1 , podoplanin , CD68 , smooth muscle actin  and GFP  immunostaining was performed in 3% goat serum (ThermoFisher) and 2% BSA in 0.3% Triton-X (ThermoFisher) at 4°C overnight, followed by fluorescent secondary antibodies (Invitrogen). Autofluorescence were quenched using trueview autofluorescence quenching kit and sections were mounted in VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (vector laboratories). For the whole mount stain of diaphragm, the whole diaphragm of Prox1-eGFP mice was used for staining following the immunostaining protocol for cryo-sections without the autofluorescence quenching step. For the whole mount stain of ear skins, the ear skins were isolated and cleaned, and stained for Lyve1 following the immunostaining protocol for cryo-sections without the autofluorescence quenching step. The whole-mount-stained samples were imaged using a confocal microscope (ZEISS) with 20x objective. WGA stain was conducted using Alexa 594-conjugated WGA  following manufacturer’s protocol with one modification that sections were incubated with WGA overnight at 4°C instead of 10 mins under room temperature. The WGA-stained sections were washed after WGA incubation and mounted with Prolong Gold mounting medium with DAPI . Stained slides were scanned using a ZEISS Axio Scan Slide Scanner with 20x objective and whole section images were analyzed using Visiopharm and custom-designed apps. For the quantification of CD68-positive macrophages, three to six sections spanning the ventricle were used, and macrophages were defined as CD68 and DAPI double-positive cells. Macrophage density was calculated by dividing the count of macrophages to the total area for each section. The density of macrophages near arteries was defined as the total count of macrophages within 20 μm distance from any smooth muscle actin-positive artery normalized to the total area for each section. The data for macrophage density was normalized to the average value of ctrl group and represented as fold change. For the quantification of podoplanin-positive lymphatic vessel density and area, 6 to 12 sections spanning the ventricle were used. A size filter of 500 μmfication . The varLymphangiography was performed in anesthetized mice 5–6 weeks after minipump implantation following a previously reported method with modifications . The aniClostridium histolyticum , Dnase I , Hydraluronidase  and 2.5% BSA in HBSS for 45 min in 37°C waterbath with shaking. The tissue pieces were next broken up further with pipetting and the enzymes were neutralized with DMEM/F12 medium  containing 10% FBS . The solutions with digested tissues were centrifuged at 400 g for 3 min, and cardiomyocytes were pelleted from digested heart tissues. The cardiomyocytes were next washed with PBS once and lysed for RNA with RLT cell lysis buffer. The supernatant of each solution was isolated and filtered through 40-µm cell strainer. The solutions were then centrifuged at 1500 g for 5 min to pellet the cells and the cells were resuspended in red blood cell lysis buffer  to lyse red blood cells. Next, the cells were pelleted and washed with FACS buffer (1% BSA in PBS) twice and cell density was counted. 0.2 million cells were aliquoted either from wild-type animal tissues or Prox1-eGFP tissues for single stain controls. Mouse Fc block  were added to each tube to inhibit non-specific binding and the samples were stained for CD45.2 , or/and CD31  for 30 min on ice in the dark. The cells were then washed with FACS buffer twice and resuspended in sort buffer , 2 mM EDTA  and 0.5% BSA in PBS) at 20 million/ml for live cell sorting. DAPI  was added to the cells before sorting to stain the dead cells and the cells were sorted using SONY SH800 cell sorter with 100 µm chip. Blood endothelial cells were defined as DAPI negative, CD45.2 negative, CD31 positive, and eGFP negative cells and lymphatic endothelial cells were defined as DAPI negative, CD45.2 negative, CD31 positive, and eGFP positive cells. The cells were sorted into RLT lysis buffer at 4°C, and total RNA was extracted from each isolated cell population using the RNeasy MinElute cleanup kit  following manufacturer’s protocol. Extracted total RNA was stored at −80°C freezer until the library preparation step. For low-input RNA-seq, total RNA concentration for each sample was measured using high sensitivity plates on a Lunatic nanodrop instrument (Unchained Labs) before proceeding to library prep. Of total RNA, 2 ng were aliquoted from each sample from which mRNA was isolated and converted into cDNA using the Smart-Seq ultra low input RNA kit  following manufacturer’s protocol. The libraries were next prepared from converted cDNA using Nextera DNA XT Library Prep Kit  following manufacturer’s protocol. Library concentrations were quantified using Qubit 1X dsDNA HS Assay kit  and the quality of the libraries was examined using Agilent High Sensitivity D1000 Screentape System  on an Agilent 4200 Tapestation. The libraries from cardiomyocytes were pooled together and sequenced using NextSeq 500/550 High Output Kit . The libraries from endothelial cells were randomized, divided into two sets and pooled together for two rounds of sequencing using NextSeq 500/550 High Output Kits .Prox1-eGFP mice were used for cell isolation at 5–6 weeks after minipump implantation for preparing RNA-seq samples and wildtype non-carrier mice were used at similar age for preparing single stain controls. The animals were euthanized using carbon dioxide, and heart and diaphragm were isolated from the animals and transferred into a storage solution containing 2.5% BSA  in HBSS . The tissues were then cut into tiny pieces and digested with a digestion enzyme mixture with Collegenase A , Collegenase from Sequencing reads were de-multiplexed using the picard tool bcl2fastq, and quality control was performed on the fastq files using FastQC. The reads were then aligned to the mouse reference genome (GRCm38) using the STAR aligner (v.2.5.3a) with quantMode specified for Transcriptome SAM, out SAM type specified as BAM, out Filter Mismatch Nover Lmax as 0.05, align SJDBoverhang Min as 1, out Filter Score Min Over Lread as 0.9, out Filter Match Nmin Over Lread as 0.9, and align Intron Max as 1000000. Gene counts for each sample were generated from the BAM alignment files using Salmon (v.0.12.0) in alignment-aware mode, based on gene/isoform features annotated by Ensembl release 95. Using the log2 normalized gene counts, we performed principal components analysis across samples within each of the four different cell types to identify outlier samples and removed samples that were greater than three standard deviations from the mean value of the first two principal components. For differential expression analysis, all downstream differential expression analysis and functional enrichment analyses were performed in R (v.3.5.3). We performed differential expression analysis within each of the four different cell types among treatment groups using DESeq2, adjusting for age of the animals, and sequencing run batch for the endothelial cell experiments, as well as surrogate technical variables detected using sva analysis. We additionally performed differential expression analysis across all three endothelial cell groups to identify potential interactions with treatment using the limma-voom framework. To identify pathways and sets of genes enriched in our significantly differentially expressed results, we performed functional enrichment analysis using the topGO enrichment analysis with the weight01 method. Finally, in order to identify potential upstream regulators of the observed expression changes from our results, we performed causal reasoning analysis based on a previously reported approach . In ordeChemokine levels in plasma and native skin lysates were examined using MSD U-plex chemokine combo 1 (Ms) kit  following manufacturer’s protocol. 20 μg of native skin lysate was loaded to each well. The data was analyzed using MSD discovery workbench software (v4.0).Animals were transferred into metabolic cages for urine collection for 24 hr and returned to regular cages after collection. Non-esterified fatty acid and creatinine in urine were measured by Siemens clinical analyzer (ADVIA Chemistry XPT system). Albumin was measured using BCP albumin assay kit (Sigma-Aldrich). Total protein amount was measured using Pierce BCA protein assay kit (ThermoFisher).LYVE1, Hs00272659_m1; PDPN, Hs00366766_m1; FLT4, Hs01047677_m1; VEGFC, Hs01099203_m1; HPRT1, Hs02800695_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System. The expression of every gene was normalized to the expression of housekeeping gene HPRT1 and then normalized to the healthy heart group to be presented as fold change in the corresponding figures.Human heart tissues were commercially purchased and three commercially available healthy human heart RNA samples  were also purchased and used for the analysis. RNA was extracted from human tissues using Qiazol reagent and RNeasy plus universal kit (Qiagen), and cDNA was synthesized using high-capacity cDNA reverse transcription kit (ThermoFisher). qPCR was conducted using TaqMan probes (https://www.ebi.ac.uk/gwas/). Locus-wide variant lookups for associations to cardiometabolic-related traits were performed for genes involved in lymphatic-related pathways, including VEGFC, LYVE1 and FLT4. Methods for each GWAS analyses can be found at each reference.GWAS summary statistics were aggregated from publicly available resources, including the GWAS catalog . All the fitted models meet the model assumptions. All the samples used in the study were biological repeats, not technical repeats. No samples were excluded from the analysis unless it was determined to be a significant outlier by the Grubbs’ test.The statistical analysis used for every figure has been illustrated in the corresponding figure legends. Independent two-tailed Student’s  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The manuscript shows the importance of lymphatic vasculature in chronic cardiac dysfunction with mechanical insights. Moreover, the authors elegantly show that lymphangiogenic therapy using VEGFCc156s can alleviate pathological changes and ameliorate heart function by improving cardiac lymphatic function and mitigating inflammation and hypertension. Finally, this study provide a potential therapeutic avenue for chronic cardiac dysfunction induced by hypertension.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, including Gou Young Koh as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Matthias Barton as the Senior Editor.Thank you for submitting your article "Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension" for consideration by eLife.The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. In the light of the comments made by the reviewers, we decided to offer a "major revision" prior to publishing your manuscript in The comments of all two reviewers are in good agreement. While the reviewers found this work to be of importance as a translational research, they raised concerns about the lack of mechanistic insights on the preclinical VEGFC1563 therapy for chronic cardiac dysfunction. The authors are required to carefully address the comments point-by-point in a data-driven manner or with further analyses or discussions. Specifically, the authors are encouraged to pay attention to the major comment 1 of reviewer 1 and comments 1-3 of reviewer 2. If necessary, please provide the reasons for not implementing the suggested changes. We believe the authors could revise the manuscript successfully given their expertise.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Reviewer #1:eLife after addressing following points.The manuscript by Song et al., shows the importance and mechanistic insights of lymphatic vasculature in chronic cardiac dysfunction. The authors demonstrated pathological and functional association of cardiac lymphatics in an angiotensin infusion-induced cardiac dysfunction model. Moreover, they showed that lymphangiogenic therapy using VEGFCc156s can alleviate pathological changes and ameliorate heart function by improving cardiac lymphatic function and mitigating inflammation and hypertension. The intriguing findings of this study suggest a potential therapeutic strategy for chronic cardiac dysfunction induced by hypertension. Therefore, I consider this work suitable for publication in (1) VEGFC – VEGFR3 signaling pathway is the most well-known signaling axis regulating development and growth of lymphatic vasculature. The authors showed that VEGFCc156s infusion reduced the loss of cardiac lymphatic vasculature as demonstrated by increased podoplanin+ lymphatic vessel density and area in Figure 2J and Figure 2—figure supplement 1M. However, an interesting observation was made in kidney lymphatic vasculature, where the VEGFCc156s administration reduced lymphatic endothelial cell marker genes and lymphatic vessel density in Figure 6E, F and Figure 6—figure supplement 1B, C, D. What is the authors' explanation for such an opposite effect of VEGFCc156s, and what is the effect of VEGFCc156s on other organ lymphatics? For example, "skin" which is related to pathogenesis of hypertension.2) How are cardiac lymphatic valves affected in AngII and AngII+VEGFC groups?3) In Figure 2I and Figure 4G, what is the image showing? Is it ctrl, angll or angll+VEGFC group? The authors should provide a representative image for each group.4) In Figure 4—figure supplement 1B, eGFP signal shown is quite not clear. Co-staining with LEC marker genes are required.5) In Figure 2—figure supplement 1G and H – representative images for each group is required. In Figure 2—figure supplement 1L and Figure 6—figure supplement 1D, P-value for the quantification is omitted.6) Results – LYVE1 is highly expressed in a subset of macrophages. How could the authors exclude the involvement of LYVE1 sequence from LYVE1+ macrophages when analyzing the SNP of the genome from whole human heart tissue?7) Results – What is the source of VEGF-C?8) Results – PDPN expression can be upregulated in inflamed heart, but the authors stated that the PDPN expression was reduced in chronic heart failure patients. What could be the reason for this discrepancy?9) More defined analyses are required to visualize interstitial mononuclear cell infiltrate and tubular basophilia in the kidney.10) Figure 1A can be arranged by Table 1.11) Figure 2 – What is an implication of the lung weight change? Does it imply a reduction in lung edema?12) In Figure 3 – The injection site and cardiac lymphatics are unclear.13) Overall, the data presentation should be improved. It is hard to follow them.Reviewer #2:Song et al. report that continuous infusion of Vegfr-3 ligand VEGF-C156S prevents development of chronic cardiac dysfunction using angiotensin II (AngII) infusion-induced model. Continuous delivery of VEGFC156s increased heart lymphatic vascular density and lymphatic drainage, reduced AngII-induced cardiac and lung hypertrophy and improved cardiac contractibility together with a decreased Nppb expression The authors also analysed the transcriptional changes in LECs, BECs and cardiomyocytes isolated from the heart and diaphragm LECs. They report that AngII produces global changes at the transcriptomes of LECs, BECs and cardiomyocytes and many of the changes are resolved upon VEGFC156s, specifically in LECs of heart and diaphragm. AngII induced genes involved in inflammation and immune cell chemotaxis, changes that were reversed upon VEGFC156s treatment. They also suggest that TLR, Hif1a and Gli1 pathways drive LEC transcriptional changes in cardiac dysfunction, which are then ameliorated upon VEGF-C treatment. Since hypertension is one of the characteristics of chronic cardiac disease, the authors analysed the effect of VEGFC156s and demonstrate by that it reduces blood pressure. Finally, the authors report kidney dysfunction (by Urine volume and ACR ratio) recovery after VEGFC156s treatment.Finding that lymphangiogenic therapy improves resolution of myocardial edema and inflammation, and prevents cardiac dysfunction and fibrosis is not new, as reported in several previous studies . However, here the authors convincingly show that co-administration of VEGF-C improves multiple pathological parameters in chronic cardiac dysfunction model. It is therefore a clinically relevant result, especially because the authors deliver the protein systemically and show that relatively modest two-fold increase in circulating levels of VEGF-C156S produces substantial therapeutic benefits. Unfortunately, the study is rather descriptive and the authors did not pursue further molecular analysis of cardiac and diaphragm lymphatic endothelial cells to better characterize the molecular changes imposed by failing heart and treatment with VEGF-C.1) An important distinguishing feature of the current manuscript is systemic delivery of VEGFR-3 ligand, as opposed to cardiac delivery in previous publications. Therefore the authors should provide better characterization of the effects of systemic VEGF-C administration on lymphatic vasculature both in heart and other organs, such as skin. Notably, they should establish whether their infusion method leads to the expansion of initial capillary network or, perhaps enlargement of pre-collecting or collecting vessels. At least one publication demonstrated that recombinant VEGF-C156S promoted lymphatic contractility , hence improved lymphatic draining function upon VEGF-C156S administration can be potentially uncoupled from its lymphangiogenic activity. Indeed, in the lymphoangiography images of Figure 3 the angII effects on the heart and the vessel leakiness are clear, however the Qdot images do not show much expansion of lymphatic vascular network. Furthermore, increased skin lymphangiogenesis has been shown to improve outcomes in salt-induced hypertension model , therefore lymphatic vessel expansion in organs other than heart is potentially directly relevant.2) The beneficial effects of VEGFC156S infusion in AngII-infusion model are encouraging. However, AngII and VEGF-C156S were infused simultaneously. To know if lymphangiogenic treatment can be used as therapy, ideally it would be important to know whether VEGFC-156S administration helps after establishment of cardiac dysfunction. In the very least, the timeline of cardiac and other organ lymphangiogenesis should be documented in more detail.3) Do the authors check the effect of VEGFC156s alone? It would have been great added value to include it as control in all in vivo experimentation.Prox1, Flt1 and Nrp1 , Ptprc (immune cells), collagen 6 subunits (fibroblasts) and confirm that at least some of the functionally relevant transcripts, identified as differentially expressed in LECs by bioinformatics analyses, are indeed expressed in lymphatic endothelial cells by immunostaining. Similarly, the suggestions that N-Myc drives VEGF-C effects and that AngII induces and Vegf-C repress activation of TLR, Hif1A and Gli1 pathways in LECs, are intriguing, but they are not substantiated by any other means, and therefore remains speculative.4) The authors made a number of broad conclusions based on bulk RNAseq data of cardiac and diaphragm LECs. However, I am not convinced that they monitor cell-autonomous effects in their analyses. For example, several genes that they report to be modulate in LECs in their models are to the best of my knowledge, expressed in immune cells and not in LECs, such as PF4, ITGAM, CD276 etc. One potential explanation, and we observed this effect also in our models, is that bulk sorted LECs can be contaminated with small number of immune cells . Please provide evidence of the purity of isolated populations by analysing the expression of established markers, such as CD31 and CDH5 , 5) Since patients with hypertension harbour lower VEGFC serum levels, did the authors analysed what were the serum levels of endogenous VEGF-C in their hypertension model for angII and angII+ VEGFC156s conditions?6) The authors show that in concordance with reduction of hypertension, VEGFC156s improved kidney dysfunction. But what is the effect of VEGFC156s in renal lymphatics? Only expression levels of lymphatic markers and density analysis was performed. The authors confirm previous studies showing that in response to hypertension there is expansion of lymphatic vessels in kidneys and observe a decrease LEC markers expression upon VEGFC156s. This part is controversial, how do the authors explain the decrease on lymphatic markers expression upon VEGF156s? The authors suggest this is a result of the low dose used, however this dose was enough to recover lymphatic function, inflammation and hypertension and poor retention in the kidney would justify no differences, however there is a decrease of lymphatic endothelial cell markers when comparing angII and angII+ VEGFC. Indeed, in the kidney the effect of angII and angII +VEGFC156s on lymphatic markers is opposite to what is observed in the heart. How do the authors explain this point?7) AngII infusion is known to induce extensive interstitial fibrosis. What was the effect of VEGF-C156S administration on this important pathophysiological parameter? Reviewer #1:[…] (1) VEGFC – VEGFR3 signaling pathway is the most well-known signaling axis regulating development and growth of lymphatic vasculature. The authors showed that VEGFCc156s infusion reduced the loss of cardiac lymphatic vasculature as demonstrated by increased podoplanin+ lymphatic vessel density and area in Figure 2J and Figure 2—figure supplement 1M. However, an interesting observation was made in kidney lymphatic vasculature, where the VEGFCc156s administration reduced lymphatic endothelial cell marker genes and lymphatic vessel density in Figure 6E, F and Figure 6—figure supplement 1B, C, D. What is the authors' explanation for such an opposite effect of VEGFCc156s, and what is the effect of VEGFCc156s on other organ lymphatics? For example, "skin" which is related to pathogenesis of hypertension.We agree with the reviewer that systemic administration of VEGFCc156s could have a multitude of effects outside of the heart. To address this concern, we examined the effect of VEGFCc156s on ear skin lymphatic vessels 6 weeks post-angiotensin II infusion, and observed that similar to the effects of VEGFCc156s on the lymphatic vessels in heart at this timepoint, VEGFCc156s treatment significantly increased ear skin lymphatic vessel diameter and lymphatic vascular density, supporting a systemic lymphangiogenic response induced by VEGFCc156s treatment . We agree that the changes in skin lymphatics could contribute to the effect of the treatment on hypertension and we have added text to the Discussion accordingly.lyve1 staining data in the kidney at the 6-week timepoint are intriguing. As new data with this revision, we conducted in vivo studies to profile the time-course of the protective effects of VEGFCc156s on angiotensin II-induced cardiac dysfunction and characterized the changes induced by the treatment at 1 week after treatment. In this new study, VEGFCc156s treatment in healthy and angiotensin II-infused animals induced a trend towards increased lymphatic vessel density in skin and a significant increase in podoplanin-positive lymphatic vessel density in heart, indicating that VEGFCc156s can indeed induce lymphangiogenesis . However, at this time point, Lyve1 expression and Lyve1-positive lymphatic vessel density was not altered in the kidney of animals treated with VEGFCc156s alone , confirming that the VEGFCc156s treatment did not induce lymphangiogenesis in kidney at the dose we use for our studies. These new data along with the data on kidney lymphatics at 6 weeks after angiotensin II infusion  suggest that the increase in renal lyve1 gene expression and lymphatic vessel density in this model could be secondary to the hypertension status of the animals. At 1 week, the hypertension is not sufficient to drive this change, and by 6 weeks, hypertension has caused an adaptation in angiotensin II-infused animals to induce lymphangiogenesis that is ameliorated by the reduced hypertension in VEGFCc156s-infused, angiotensin II-treated animals. In addition, it is possible that the systemic infusion of VEGFCc156s did not significantly affect kidney lymphatics due to the low dose and poor local kidney retention, whereas transgenic overexpression of VEGFD specifically in kidney as in previous reports would sustain high local expression and therefore produce a greater local effect. We have discussed these hypotheses in the Discussion section.We agree with the reviewer that the lymphatic marker qPCR and 2) How are cardiac lymphatic valves affected in AngII and AngII+VEGFC groups?Unfortunately, we made cross-sections of the heart for immunofluorescence staining and we did not have additional heart tissue to perform whole mount imaging of the lymphatic valves. Therefore, we were unable to evaluate this parameter. This is discussed as a limitation in the manuscript Discussion and will be an area of interest for us to investigate in the future.3) In Figure 2I and Figure 4G, what is the image showing? Is it ctrl, angll or angll+VEGFC group? The authors should provide a representative image for each group.We have now added representative images for podoplanin and smooth muscle actin staining for each treatment group in Figure 2I as well as representative images of CD68, Smooth muscle actin and DAPI staining for each treatment group in Figure 4G.4) In Figure 4—figure supplement 1B, eGFP signal shown is quite not clear. Co-staining with LEC marker genes are required.Prox1-eGFP mice with GFP and Lyve1 co-staining . The GFP stain overlapped with the Lyve1 stain, indicating that the eGFP signal came from the lymphatic vessels in the Prox1-eGFP mice.We are pleased to provide new immunofluorescence staining data using mouse heart cross sections and whole mount staining using diaphragm tissue from 5) In Figure 2—figure supplement 1G and H – representative images for each group is required. In Figure 2—figure supplement 1L and Figure 6—figure supplement 1D, P-value for the quantification is omitted.We have added representative WGA stain images from heart cross-sections in Figure 2—figure supplement 1H. We have also added the p-value for the quantifications previously shown in Figure 2—figure supplement 1L and Figure 6—figure supplement 1D .6) Results – LYVE1 is highly expressed in a subset of macrophages. How could the authors exclude the involvement of LYVE1 sequence from LYVE1+ macrophages when analyzing the SNP of the genome from whole human heart tissue?Lyve1 and another gene are located or close to, and the results do not indicate the exact gene that mediates the effect or the exact cell type that mediates the effect. We agree with the reviewer that it is possible that several types of cells including both Lyve1+ macrophages and Lyve1+ lymphatic vessels may mediate the effect, and unfortunately the GWAS analysis will not provide us with the information concerning cell types due to the nature of the analysis. We have revised our description on the GWAS data to make it more accurate (Discussion).We appreciate this helpful comment from the reviewer. The associations we observed in GWAS analysis are the associations between a specific loci with a disease phenotype where 7) Results – What is the source of VEGF-C?The VEGFCc156s we used for our in vivo studies is the recombinant human VEGF-C (Cys156Ser) protein purchased from R&D systems. It has been used previously to induce lymphangiogenesis in vivo in mice , and the sequence of this human VEGFC short-form is identical to the mouse VEGFC short-form.8) Results – PDPN expression can be upregulated in inflamed heart, but the authors stated that the PDPN expression was reduced in chronic heart failure patients. What could be the reason for this discrepancy?We are aware of previous publications  indicating that podoplanin expression could be upregulated in inflamed hearts due to an increase in podoplanin-expressing cells that are not lymphatic endothelial cells. This upregulation would be most significant in the highly inflamed/fibrotic infarcted area due to the local accumulation of podoplanin-expressing cells that facilitate local inflammation. To that end, we used chronic heart failure human hearts and a chronic cardiac dysfunction model, which have a very different pathogenesis compared with a myocardial infarction model, which represents acute injury. In addition, compared with the previous reports that looked specifically at the infarcted/fibrotic site in heart, we sampled from the whole heart and also utilized immunohistochemistry staining for podoplanin-positive lymphatic vessels, in which we excluded podoplanin-positive cells that were not part of a vessel from the vessel quantification using a size filter in our visiopharm-based image analysis (subsection “Histology and immunohistochemistry”). Thus, we suggest that the changes in podoplanin expression we observed in our disease model largely come from the lymphatic vasculature but not podoplanin-expressing cells, and the discrepancy between our findings and previous reports is due to the differences in disease models and disease pathogenesis. We have added the references into our revised manuscript and added a Discussion point concerning this.9) More defined analyses are required to visualize interstitial mononuclear cell infiltrate and tubular basophilia in the kidney.We appreciate the comment from the reviewer. The pathologist that evaluated the H&E staining in the kidney used this nomenclature to describe the pathological findings. To improve visualization of these endpoints, we first enlarged the H&E image in Figure 6—figure supplement 1A to improve the image resolution. To further evaluate kidney pathology, we conducted a PSR stain using the same kidney sections to add an additional evaluation of kidney fibrosis. The results indicated that angiotensin II infusion induced renal fibrosis, which was significantly reduced by VEGFCc156s treatment . We feel that the new PSR staining results will strengthen the kidney datasets reported at 6-week post-angiotensin II infusion and our overall conclusions.10) Figure 1A can be arranged by Table 1.We were informed by the editor that the format of table supplements is not preferred by the journal and therefore we have rearranged Figure 1 and converted the original Figure 1A  into Figure 1—figure supplement 1.11) Figure 2 – What is an implication of the lung weight change? Does it imply a reduction in lung edema?An increase in lung weight is commonly observed in severe cardiac dysfunction or heart failure and it does reflect a change in lung edema . We regularly use both heart weight and lung weight to evaluate cardiac function of the animals at euthanasia, and it’s possible that the reduction in the lung weight of VEGFCc156s-treated, angiotensin II-infused animals compared with animals treated with angiotensin II alone implied a reduction in lung edema. However, definitive measurement of lung edema requires measurement of the wet/dry ratio using fresh lungs and unfortunately, we no longer have fresh lungs to perform this measurement. We have added text to denote this possibility to the Results section.12) In Figure 3 – The injection site and cardiac lymphatics are unclear.We have revised Figure 3—figure supplement 1 and added a red arrowhead to the injection site (apex) and have used white arrow heads to delineate the cardiac lymphatics.13) Overall, the data presentation should be improved. It is hard to follow them.We appreciate that the complex data package has made the data somewhat difficult to follow. With this revision, we have added new data and refined the data presentation. We have additionally carefully edited the manuscript for additional clarity. We feel that these revisions have improved the data flow, and we thank the reviewer for calling this to our attention.Reviewer #2:[…] Finding that lymphangiogenic therapy improves resolution of myocardial edema and inflammation, and prevents cardiac dysfunction and fibrosis is not new, as reported in several previous studies . However, here the authors convincingly show that co-administration of VEGF-C improves multiple pathological parameters in chronic cardiac dysfunction model. It is therefore a clinically relevant result, especially because the authors deliver the protein systemically and show that relatively modest two-fold increase in circulating levels of VEGF-C156S produces substantial therapeutic benefits. Unfortunately, the study is rather descriptive and the authors did not pursue further molecular analysis of cardiac and diaphragm lymphatic endothelial cells to better characterize the molecular changes imposed by failing heart and treatment with VEGF-C.1) An important distinguishing feature of the current manuscript is systemic delivery of VEGFR-3 ligand, as opposed to cardiac delivery in previous publications. Therefore the authors should provide better characterization of the effects of systemic VEGF-C administration on lymphatic vasculature both in heart and other organs, such as skin. Notably, they should establish whether their infusion method leads to the expansion of initial capillary network or, perhaps enlargement of pre-collecting or collecting vessels. At least one publication demonstrated that recombinant VEGF-C156S promoted lymphatic contractility , hence improved lymphatic draining function upon VEGF-C156S administration can be potentially uncoupled from its lymphangiogenic activity. Indeed, in the lymphoangiography images of Figure 3 the angII effects on the heart and the vessel leakiness are clear, however the Qdot images do not show much expansion of lymphatic vascular network. Furthermore, increased skin lymphangiogenesis has been shown to improve outcomes in salt-induced hypertension model , therefore lymphatic vessel expansion in organs other than heart is potentially directly relevant.We appreciate this constructive comment from the reviewer and have significant additional data to include to address this point in the manuscript revision. To address this comment, we first conducted an additional analysis on the podoplanin-positive lymphatic vessel quantification in heart to evaluate the average podoplanin-positive lymphatic vessel lumen area. Indeed, as the reviewer pointed out, there was a significant enlargement of the podoplanin-positive pre-collector vessel lumen in VEGFCc156s-treated angiotensin II-infused hearts compared with hearts treated with angiotensin II alone , which was consistent with the significant improvement we observed in cardiac lymphatic transport in VEGFCc156s-treated hearts using lymphangiography  and indicated a functional change in the collecting vessels.In addition, we examined the effect of VEGFCc156s on the lymphatic vessels in ear skin 6 weeks after treatment. Similar to the effects of VEGFCc156s on the lymphatic vessels in heart at this timepoint, VEGFCc156s treatment significantly increased lymphatic vessel diameter and lymphatic vascular density in the ear skin of angiotensin II-infused animals, supporting a systemic lymphangiogenic response induced by VEGFCc156s treatment . We agree that the changes in the lymphatics in skin induced by VEGFCc156s treatment could contribute to the effect of the treatment on hypertension, and we have added a point on this in the Discussion.2) The beneficial effects of VEGFC156S infusion in AngII-infusion model are encouraging. However, AngII and VEGF-C156S were infused simultaneously. To know if lymphangiogenic treatment can be used as therapy, ideally it would be important to know whether VEGFC-156S administration helps after establishment of cardiac dysfunction. In the very least, the timeline of cardiac and other organ lymphangiogenesis should be documented in more detail.We agree with the reviewer that a therapeutic reversal of the cardiac disease using VEGFCc156s would be a very compelling experiment. However, due to the minipump administration of angiotensin II and VEGFCc156s, this is very difficult to achieve, as our IACUC limits the number of surgical procedures on mice . Therefore unfortunately, we are unable to conduct a reversal experiment using the infusion method. We also agree with the reviewer that a more careful assessment of the lymphangiogenesis timeline should be performed. Following this suggestion, we conducted in vivo studies to characterize the timecourse of the beneficial effect of VEGFCc156s on angiotensin II-induced cardiac dysfunction. We specifically examined the effect of VEGFCc156s 1-week post-infusion, at which time, the blood pressure was equally high in both angiotensin II-infused and VEGFCc156s-treated angiotensin II-infused mice based on the telemetry study result from Figure 5. We found that at this early time point, whereas VEGFCc156s does not have an effect on blood pressure, it significantly reduced angiotensin II-induced gene expression in pathways involved in cardiac hypertrophy, fibrosis, and inflammation. Furthermore, VEGFCc156s infusion alleviated angiotensin II-induced cardiac fibrosis and inflammation as assessed by histological staining at this early 1-week timepoint. These new results are included in Figure 7 and Figure 7—figure supplement 1 and 2. We feel that these new data significantly improve the quality of the manuscript and solidify the conclusions drawn. We thank the reviewer for the constructive advice.3) Do the authors check the effect of VEGFC156s alone? It would have been great added value to include it as control in all in vivo experimentation.We agree that a VEGFCc156s alone arm would have added value in our studies. Following this advice, we added a treatment arm in new data added here that assesses one week of infusion of VEGFCc156s alone. In this assessment, a lymphangiogenic response in heart was observed in VEGFCc156s-alone infused animals, which was similar to animals that were infused with VEGFCc156s and angiotensin II concurrently. These new results are included in Figure 7 and Figure 7—figure supplement 1 and 2.4) The authors made a number of broad conclusions based on bulk RNAseq data of cardiac and diaphragm LECs. However, I am not convinced that they monitor cell-autonomous effects in their analyses. For example, several genes that they report to be modulate in LECs in their models are to the best of my knowledge, expressed in immune cells and not in LECs, such as PF4, ITGAM, CD276 etc. One potential explanation, and we observed this effect also in our models, is that bulk sorted LECs can be contaminated with small number of immune cells . Please provide evidence of the purity of isolated populations by analysing the expression of established markers, such as CD31 and CDH5 , Prox1, Flt1 and Nrp1 , Ptprc (immune cells), collagen 6 subunits (fibroblasts) and confirm that at least some of the functionally relevant transcripts, identified as differentially expressed in LECs by bioinformatics analyses, are indeed expressed in lymphatic endothelial cells by immunostaining. Similarly, the suggestions that N-Myc drives VEGF-C effects and that AngII induces and Vegf-C repress activation of TLR, Hif1A and Gli1 pathways in LECs, are intriguing, but they are not substantiated by any other means, and therefore remains speculative.Ptprc) negative cells, which should ensure that we are not identifying immune cells, and we set stringent gates to ensure the sorted cell populations were expressing the correct markers . We conducted multiple tests to optimize the cell isolation workflow and performed qPCR to assess various marker genes in the sorted cell populations to assess purity. We provide a figure to the reviewer here from one of our test runs , whereas endothelial cells  did not express Ptprc (CD45) and expressed Pecam1 (CD31). In addition, lymphatic endothelial cell populations  expressed high levels of lymphatic endothelial markers   expression and detected high counts of Pecam1 (CD31)  and Cdh5  in our isolated blood and lymphatic endothelial cell populations. Moreover, the lymphatic endothelial cell populations showed high counts/expression of lyve1(TPM count 1300-4200), Prox1 (TPM count 159-254), Pdpn (TPM count 63-131) and Flt4 (TPM count 428-1073) compared with other cell populations . The cardiomyocyte population  showed a relatively high expression of collagen 6 subunits  while lymphatic endothelial cell populations showed a low expression of collagen 6 subunits . We could only isolate 800-1200 lymphatic endothelial cells/heart and 1000-2000 lymphatic endothelial cells/diaphragm with the stringent cell isolation protocol, and the cell amount was just enough for a low-input RNAseq method for the transcriptional profiling of the distinct cell populations. Therefore unfortunately, we don’t have extra cells for additional immunostaining tests to confirm the identity of each isolated cell population. However, based on our stringent isolation and gating procedures described above as well as the RNAseq TPM count data, we suggest that the cell populations we isolated for RNAseq were relatively pure. We agree with the reviewer that the RNAseq results and pathway analysis need to be validated with more experiments, and that will fall into the scope of future studies.For our RNAseq analysis, a PCA analysis indicated that each isolated cell population clustered together and showed distinct expression profiling . In addition, consistent with our pilot optimization test result, our RNAseq analysis could not detect 5) Since patients with hypertension harbour lower VEGFC serum levels, did the authors analysed what were the serum levels of endogenous VEGF-C in their hypertension model for angII and angII+ VEGFC156s conditions?VEGFC concentration was assessed in plasma of the animals from our in vivo studies at both the 1-week and 6-week timepoints, and the results are included in Figure 2—figure supplement 1B and Figure 7—figure supplement 1A. There was no significant difference in serum VEGFC levels between ctrl animals ang angiotensin II-infused animals. However, the human study we cited in the Discussion indicated that low serum VEGFC level was associated with a higher risk of hypertension, which is consistent with our finding that increasing serum VEGFC level could be beneficial for hypertension. In addition, the data from the human study we cited were from patients with refractory hypertension that doesn’t respond to regular anti-hypertensive therapy and there could be differences in pathogenesis and disease mechanisms between refractory hypertension in human and our angiotensin II model. We agree that further investigations are needed to understand the detailed mechanisms underlying these observations.6) The authors show that in concordance with reduction of hypertension, VEGFC156s improved kidney dysfunction. But what is the effect of VEGFC156s in renal lymphatics? Only expression levels of lymphatic markers and density analysis was performed. The authors confirm previous studies showing that in response to hypertension there is expansion of lymphatic vessels in kidneys and observe a decrease LEC markers expression upon VEGFC156s. This part is controversial, how do the authors explain the decrease on lymphatic markers expression upon VEGF156s? The authors suggest this is a result of the low dose used, however this dose was enough to recover lymphatic function, inflammation and hypertension and poor retention in the kidney would justify no differences, however there is a decrease of lymphatic endothelial cell markers when comparing angII and angII+ VEGFC. Indeed, in the kidney the effect of angII and angII +VEGFC156s on lymphatic markers is opposite to what is observed in the heart. How do the authors explain this point?lyve1 staining data in the kidney at the 6-week timepoint are intriguing. As new data with this revision, we conducted in vivo studies to profile the time-course of the protective effects of VEGFCc156s on angiotensin II-induced cardiac dysfunction and characterized the changes induced by the treatment at 1 week after treatment. In this new study, VEGFCc156s treatment in healthy and angiotensin II-infused animals induced a trend towards increased lymphatic vessel density in skin and a significant increase in podoplanin-positive lymphatic vessel density in heart, indicating that VEGFCc156s can indeed induce lymphangiogenesis . However, at this time point, Lyve1 expression and Lyve1-positive lymphatic vessel density was not altered in the kidney of animals treated with VEGFCc156s alone , confirming that the VEGFCc156s treatment did not induce lymphangiogenesis in kidney at the dose we use for our studies. These new data along with the data on kidney lymphatics at 6 weeks after angiotensin II infusion  suggest that the increase in renal lyve1 gene expression and lymphatic vessel density in this model could be secondary to the hypertension status of the animals and not a direct effect of VEGFCc156s on the kidney. At 1 week, the hypertension is not sufficient to drive this change, and by 6 weeks, hypertension has caused an adaptation in angiotensin II-infused animals to induce lymphangiogenesis that is ameliorated by the reduced hypertension in VEGFCc156s-infused, angiotensin II-treated animals. In addition, it is possible that the systemic infusion of VEGFCc156s did not significantly affect kidney lymphatics due to the low dose and poor local kidney retention, whereas transgenic overexpression of VEGFD specifically in kidney as in previous reports would sustain high local expression and therefore produce a greater local effect. We have discussed these hypotheses in the Discussion section.We agree with the reviewer that the lymphatic marker qPCR and 7) AngII infusion is known to induce extensive interstitial fibrosis. What was the effect of VEGF-C156S administration on this important pathophysiological parameter?We are pleased to provide significant new data to address this point. PSR staining of mouse kidney sections indicated angiotensin II infusion induced significant kidney fibrosis after 6 weeks of infusion, which was significantly reduced in VEGFCc156s-treated animals, indicating a beneficial effect of VEGFCc156s on kidney fibrosis and supporting an overall beneficial effect of VEGFCc156s on angiotensin II-induced kidney dysfunction at this timepoint. In addition, we also observed that VEGFCc156s treatment significantly reduced angiotensin II-induced pro-fibrotic gene expression and trended to reduce the level of cardiac fibrosis in the heart as early as 1-week post-treatment . These new results indicate that VEGFCc156s improved the cardiac function in angiotensin II-infused animals by alleviating cardiac fibrosis in addition to the other mechanisms proposed in this manuscript. We thank the reviewer for this suggestion.


We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3–7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation  but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring. Wound healing interruption at any stage or ablation of its critical participants may result in non-healing wounds, excessive scarring or subsequent neoplasms [Healing of damaged tissue is a dynamic process regulated by cytokines and growth factors, proteolytic enzymes and extracellular matrix (ECM). They serve as regulatory cues for cells and tissue structures, including blood vessels, nerves etc. Different stages wound healing involve blood cells (platelets and immune cells), stromal cells , parenchymal and stem/progenitor cells . After peoplasms .In cutaneous wounds of different natures , acceleration of closure is an important point as far as it prevents infection of the wound bed and its potential life-threatening complications. Another crucial endpoint is the limitation of scarring—a naturally developed response to deep or vast injury of skin . After sThus, in cutaneous injury, a therapeutic approach that can stimulate rapid defect closure and reduce fibrosis and potentially provides ground for more effective regeneration of skin and its appendages. In recent decades, promising methods using multipotent mesenchymal stromal cells (MSC) from different sources acquired significant attention and addressed skin-healing issues . FeasiblFinally, the ability of MSC to form tissue-like constructs composing of viable cells and their native ECM resulted in the development of cell sheets (CS)—minimal tissue-engineered constructs that found application for many conditions. Our previous experience with MSC-based CS showed their efficacy for stimulation of angiogenesis, nerve repair and cardiac regeneration and proved their superiority over routinely used local injection ,12,13,14Pressure-induced lesions are a widely spread condition that remains a clinical problem with few cell-based products for its treatment. Due to the specific nature of damage that induces pressure, published studies on ulceration using surgical wound models can hardly be considered relevant evidence, and filling this gap became a practical rationale for the present study.We were also intrigued by changes in secretome and transcriptome of MSC that may occur within CS, which is believed to present a more “tissue-like” environment for cells ,16. HoweOur work was intended to provide data expanding our view on CS as a tissue-engineered construct for acceleration of cutaneous defect healing and addresses the pressure ulcer issue. Going beyond applied task of MSC-based therapies we obtained data on transcriptome and secretome changes to illustrate the shift in MSC status induced by formation of CS.2 size and was significantly lower (p = 0.063). However, only in the “Cell Sheet” group did we find animals (3/4 mice followed up by Day 21) with hair growth indicating partial recovery of skin appendages  of defect surface area normalized to initial defect in photos taken at Days 3, 7, 14 and 21 of the experiment. No difference between groups was found at days 3 and 7; however, starting from Day 14, acceleration of ulcer closure was observed in CS application group (“Cell Sheet”) A. By Daypendages A. This opendages  and detapendages  that disp = 0.0082) and Day 21 . MSC secretome injections yielded results comparable with untreated control throughout the experiment, not reaching statistical significance at endpoint , which suggested early rejection of CS along with scab by natural detachment of its mass or during removal of wound dressing. Assessment of secretome retention by this method was not possible, but published studies describe the half-life of delivered active fraction ranging from several hours to 1–2 days [Signal from labeled CS was detected only in the first evaluation point—Day 3. In certain images from the “Cell Sheet” group, PKH26 was not co-localized with DAPI-positive nuclei, which may hint at a residual nature of PKH26 signal due to its leakage during early apoptosis of MSC. We failed to detect PKH26-positive CS in any specimen taken at Day 7  was used to minimize donor-dependent variability. Assembly of CS was carried out in AdvanceStem medium as previously described  and densp-value are presented in RNA-sequencing of MSC monolayer and CS provided libraries that were normalized within each sample as reads per kilobase million (RPKM), and obtained data were used to calculate fold change for each transcript. Using Gene Ontology (GO), we mapped all transcripts with ≥2-fold increment or decrement in CS. The top 10 annotated biological process (BP) and molecular function (MF) GOs with statistically significant Additional analysis of trancriptional factor (TF) activity signature was performed using HOCOMOCO database to predict potential transcriptional regulators that may increase their activity due to microenvironment changes during assembly of CS. This provided an array of putative TFs showing increased activity that correlates with their target genes upregulation obtained from our RNA-sequencing data .Animal test demonstrated the efficiency of MSC-based sheets to accelerate pressure ulcer closure. The “Cell Sheets” group was the only one to reach statistical significance vs. untreated control at Day 21 and was clearly superior to suspended MSC  despite The most striking finding was that CS delivery induced profound recovery of dermis structure with its appendages, resulting in obvious hair growth at the site of pressure ulcer and histology, confirming the presence of glands within the dermis . This waThis finding is yet to be clarified using a more accurate method to establish whether hair has re-grown, indicating full-scale regeneration via activation of progenitors or reprogramming. Another option is that dermal layers were drawn towards the center of the defect which has not been occupied by scar mass due to specific CS influence on remodeling of GT. Thus, at the moment, we may claim successful healing induced by CS application, yet the degree of involvement of antifibrotic and regenerative mechanisms, cell proliferation or reprogramming is subject to further investigation. Injection of MSC suspension failed to stimulate ulcer closure compared to untreated control group . InitialAnalysis of Masson stain supported trigger effect of CS application. We found that CS application may accelerate active growth of GT at initial phases of healing , and by Thus, absence of CS  startingTaking into account the data from later time points (Days 14 and 21) where residual GT was marginally absent in CS-treated animals , we suggObserved changes in GT growth and remodeling depend on its vascularization. Indeed, until GT matures into a scar, it has abundant numbers of blood vessels to facilitate immune cell migration, nutrition and clearance of debris . Thus, wThe pattern of GT vascularization in the CS group had an incline towards formation of large vessels with a lumen (up to 50 µm in diameter) and a layer of endothelial cells surrounded by αSMA-positive mural smooth muscle cells B. These Explanation of how maturation of blood vessels may contribute to less scarring mainly relates to mural cells (pericytes and smooth muscle cells) localization and functional status. When they are attracted to endothelial cells basal membrane formation begins by active production of laminins and collagen IV by both cell types . After dThis explanation was concordant with the analysis of angiogenesis-related factors produced in vitro by MSC and MSC-based CS . AngiogeOther important participants are angiopoietins that activate, maintain and harness angiogenic response when blood vessel is formed. We found angiopoietin-2 (Ang-2) strongly increased in CS secretome (5.1-fold vs. monolayer), and typically Ang-2 is presented as a sprouting factor. Ang-2 acts via Tie-2 receptor to destabilize pericyte–endothelium interaction, increasing the endothelial cells’ sensitivity to inflammatory and angiogenic factors. However, the peculiar ability of Ang-2 to block sprouting and induce apoptosis of endothelial cells is described under the low production of VEGF165 ,38. BasiOverall, this profile of angiogenesis regulators produced by CS suggested a shift of its biological potency towards “stabilizing” rather than “stimulating” action on endothelial cells. Supporting our hypothesis, we also found HGF and G-CSF significantly upregulated in CS secretome (approx. 2.6-fold for both factors). These factors contribute to blood vessel maturation and leakage stop  and may p-value that contain proteins important for cell–ECM interactions (GO:000518 “Collagen binding” and GO:0098634 “Protein binding involved in cell-to-matrix adhesion”). A high number of secreted proteolytic enzymes and their inhibitors were annotated to BP clusters related to ECM maturation, turnover and change of its composition in normal during healing after injury (GO:0030198 “ECM organization” and GO:0022617 “ECM disassembly”). Secreted proteases  and upregulated CXCL-motif activators of cell migration (GO:0030334 “Reg of cell migration”) may potentially contribute to observed rapid thickening and remodeling of GT in early stages after CS transplantation. Some of GO:0030198 “ECM organization” have also been downregulated with a high combined score for this cluster. This is a typical consequence of GO annotation redundancy when the same GO may be significantly up- and downregulated during analysis with different transcripts falling into one of these categories due to high fold increment or drop. However, the majority of downregulated transcripts were related to MSC integrins and ECM proteins  that comprise CS structure rather than those that can be secreted and influence underlying GT during healing.Our conclusion regarding the shift in MSC biological potency induced by CS formation was supported by analysis of RNA-sequencing data . ResultsAmong upregulated transcripts, peculiar clusters were annotated as GO:0045540 “Reg of cholesterol biosynthesis process” and GO:0090181 “Reg of cholesterol metabolic process” rich with enzymes controlling cholesterol biosynthesis and alcohol biogenesis. More detailed analysis showed the majority of them were responsible for fatty acid metabolism known as negative regulators of angiogenic response in normal tissue and tumors including suppression of VEGF-induced capillary sprouting.Analysis of downregulated genes mapped multiple proteins known to drive angiogenesis and active endothelial proliferation. Among all downregulated BPs, GO:2001046 “Reg of EC chemotaxis” containing NOTCH1 and FGFs 1 and 2 showed the highest combined score . AnotherHOCOMOCO dataset  showed sA potential limitation of our in vitro data obtained in human MSC is that its extrapolation to animal model might be challenging, and validation of proposed mechanisms would require a more detailed study based on presented screening of changes induced by CS assembly. However, our study intended to lay ground for pre-clinical development of a cell-based product for pressure ulcer patients, so we performed tests in human MSC from adipose tissue, which is the most feasible source of allogeneic MSC. This shift creates a certain limitation, yet we believe that we also provide a physiological rationale for a novel product that can be applied in cell therapy.In vitro tests also should be evaluated keeping in mind that alterations in the repertoire of MSC secretome and transcriptome that may occur after transplantation to damaged tissue are hard to study and reproduce ex vivio. Nevetheless, this point must be taken into account while developing further hypotheses on mechanisms of impact rendered by MSC delivered via injection or CS application. g; 10 min). After supernatant was aspirated, the pellet was resuspended in complete growth medium at 5 × 103 cells/cm2 and seeded in Petri dishes. Cells were cultured at 37 °C and 5% CO2. Upon reaching 80–90% of monolayer density, cells were re-seeded at a 1:4 ratio using 0.05% Trypsin-EDTA . Mouse MSC were used for in vivo experiment using a syngeneic model to avoid immune rejection while human MSC were used for in vitro studies  as described below.Human adipose-derived MSC immortalized cell line (ASC52telo) was purchased from ATCC  and cultured using AdvanceStem culture medium . Mouse adipose-derived MSC were obtained from subcutaneous adipose tissue of male C57Bl/6 mice as previously described . Briefly2 incubator as described in g for 10 min to remove cell debris and used for BioPlex assay or animal tests without freezing.To obtain secretome samples, human MSC were cultured in a 100 mm dish in complete growth medium to 80% monolayer. Upon reaching the designated density, cells were washed by Hanks’ solution , and 10 mL of serum-free DMEM was added. Cells were cultured for 7 days in a CO4 cells/cm2 and cultured for 7 days in complete growth medium supplemented by ascorbic acid (50 μg/mL) to promote ECM deposition and assembly. Before the detachment growth medium was removed and CS was washed with warm PBS. After that, CS edges were carefully detached from the plastic using a micropipette tip used as a scrapper, and then whole-mount CS was moved for further transplantation to mice. Preparations for BioPlex assay and RNA-sequencing of human MSC cultures are described in corresponding sections below.Mouse primary adipose-derived MSC isolated as described above were used at passage 2–3 for animal test in pressure ulcer model. Animal manipulations and euthanasia procedures were performed in compliance with National and European Union regulations and were approved by the Institutional  Animal Care and Use Committee . For RNA-sequencing and growth factor BioPlex assay, human immortalized line ASC52telo  was used at passage 4–5. For both purposes, MSC were seeded in a 12-well uncoated culture plate at 8 × 10® 200 system . Assay was performed following the manufacturer’s instructions. Briefly, 9 distinct sets of fluorescent-dye-conjugated beads with capture monoclonal antibodies specific for each factor assayed were used. Samples or standards were incubated with pre-mixed bead sets in pre-wet 96-well microplate. After incubation and washing, fluorescent detection antibody mixture was added, and after incubation, the samples were washed and resuspended in assay buffer. All values were normalized with respect to DNA concentration and measured with Quant-iT PicoGreen dsDNA Assay Kit  according to manufacturer’s manual. DNA concentration was a directly proportional function of the number of cells within study limits .MSC-derived secretome samples were collected from cultures at the state of confluent monolayer or CS and were used for multiplex ELISA. For normalization to DNA concentration, cells that remained in the plate after medium collection were lysed by TRIzol reagent . Concentrations of Angiopoietin-2, G-CSF, HGF, PDGF-BB and VEGF were simultaneously evaluated with a multiplex bead-based sandwich immunoassay kit  using Bio-Plex® mRNA Purification Kit , and Illumina library was made from polyA RNA with NEBNext® Ultra™ II RNALibrary Prep  according to manufacturer’s protocol. Concentrations of nucleic acids in obtained libraries were analyzed by Qubit dsDNA HS Assay Kit  using Qbit 2.0 equipment. RNA-sequencing was performed on HiSeq1500 at 50 b.p. read length according to manufacturer’s protocols.Immortalized human MSC (ASC52telo) were cultured in AdvanceStem medium to obtain monolayer see  or CS d. After chttps://amp.pharm.mssm.edu/Enrichr/ accessed on 1 July 2020).Quality control was carried out using the FastQC program, and while analyzing, the source data of the adapters were not found; therefore, programs for their removal were not used. Mapping was performed using bowtie2–2.4.0; to assess changes in the level of gene expression samples were normalized as reads per kilobase million (RPKM). GO gene clustering and annotation of up- and downregulated transcripts was performed using the David 6.8 resource and Enrichr web-interface (https://hocomoco11.autosome.ru accessed on 3 July 2020). To establish the potential for interaction of mapped transcription factors with the putative genes, bash scripts were used to obtain a unique list.For mapping of putative transcription factors that overlap with transcripts obtained after RNA-seq, we used resources of online database HOCOMOCO  Animal Care and Use Committee .For in vivo studies, we used male C57Bl/6 mice 12–14 weeks of age. The pressure ulcer defect was formed according to the protocol described by Stadler et al.  with modn = 12–16 animals per group):(1)“Suspension”—injection suspended MSC to the edges and bottom of skin defect.(2)“Secretome”—injection of MSC secretome into the edges and bottom of skin defect.(3)“Cell Sheet”—application of CS to ulcer surface.(4)“Untreated”—animals without therapy.At the end of 3 cycles of ischemia/reperfusion, test material was transplanted to animals (day 0). The animals were divided into the following groups (6 cells in 150 μL of PBS per defect). Secretome injections were performed in a similar volume . CS was transplanted to the defect in a drop of PBS, straightened with tweezers and allowed to adhere to lesion surface.The MSC suspension was administered in the amount of 4–5 injections into the edges and bottom of the wound  × 10To preserve the cellular material (primarily the CS on the defect surface), transplantation sites in all groups were covered for 3 days with Tegaderm film -breathable, sterile, transparent and waterproof stickers for closing wounds. All animals were housed in individual cages for 21 days until the end of experimentSkin defect area was measured at days 3, 7, 14 and 21 in photographs of wound surface at each time-point. Area was calculated using ImageJ software .After animal euthanasia, whole-mount specimen of defect  were isolated and bisected for histological assessment as follows; one half was fixed in formalin, paraffin-embedded, sectioned at 5 µm and stained with hematoxylin/eosin. The other half was placed in Tissue-Tek O.C.T. Compound and frozen in liquid nitrogen and stored at −70 °C, and frozen sections at 6–7 µm were obtained for immunofluorescent staining.Sections stained by hematoxylin-eosin/Masson’s Trichrome were used for morphometry to assess thickness of granulation tissue on days 3 and 7 . Since the composition of the defect was qualitatively different in the groups on Days 14 and 21, collagen deposition was evaluated by calculating its percentage of dyed collagen fibers (blue) to the total defect area using an ImageJ-associated color deconvolution plugin . AdditioFrozen tissue sections (at days 14 and 21) were fixed in 4% formaldehyde, blocked by 10% normal goat serum, washed and incubated with rat-anti-mouse CD31  and rabbit-anti-αSMA  antibodies. Then, slides were washed in PBS and incubated in a mixture of goat anti-mouse AlexaFluor 594 and goat anti-rabbit AlexaFluor 488 conjugated antibodies. At the end of incubation, slides were washed, counterstained with DAPI and mounted.Microphotographs of sections were taken under ×100/×200 magnification in 3 random fields of view (FOV) per section . Vessel counts included CD31-positive structures per FOV; αSMA staining was used to detect large vessels and myofibroblasts in granulation tissue. Structure counts were obtained using ImageJ software . Classification of blood vessels from Müller et al. with adaptations was used for analysis of blood vessel type [Cells were labeled by a commercial PKH26 dye , according to manufacturer’s protocol. The cell suspension was labeled immediately before the transplantation; for the CS group, cells were labeled before assembly of CS. To detect a fluorescent signal, tissue samples were frozen in Tissue-Tek O.C.T. Compound medium and sectioned. Immediately after microscopy, slides were dried at RT, and we visualized PKH26-positive cells using a fluorescence microscope . For a more detailed study of cells’ localization, sections were fixed and were immunolabeled to detect blood vessels and myofibroblasts followed by nuclear stain by DAPI.p < 0.05. Analysis was performed using Prism 7.03 (GraphPad Software).Data are expressed as mean ± standard deviation. To compare >2 groups, we used analysis of variance (ANOVA) and Tukey criterion for multiple comparisons, or Kruskal–Wallis criterion supplemented by Dunn criterion (as a nonparametric ANOVA). Differences were considered significant at Pressure ulcer healing is effectively induced by MSC secretome or CS application, while suspended cells performed poorly despite its long-lasting retention in the tissue. The most peculiar finding is that CS was the only group that showed minimal scarring with growth of hair and dermal glands in histological sections of pressure ulcer site at Day 21. The nature of this phenomenon should be established including the mechanism of defect healing via activation of regenerative capacity or healthy skin elements being drawn to the site of resorbed ECM.We propose that CS acts via a trigger mechanism mediated by its paracrine factors released at early stages of healing to induce recovery of skin structure after pressure ulcer. Effects of CS involve stabilization of vasculature and rapid GT growth followed by a specific pattern of remodeling that results in ECM resorption, but not scarring. This anti-fibrotic influence facilitates full closure of defect and observed healing of dermis.The spectrum of growth factors and cytokines produced by MSC after CS assembly can be described as a pattern for activation of blood vessel stabilization  rather that sprouting (VEGF165), which was concordant with vascular patterns in corresponding study groups.The proposed mechanism relies on changes of secretome, yet transcriptomic profile and TFs activity hint at future evaluation of potential MSC reprogramming within CS. RNA-sequencing profiles also show that transcriptome of MSC in CS is enriched for biological processes and molecular function that support the expression of factors stabilizing blood vessels and limiting endothelial sprouting. Future studies will address our findings regarding the potential role of ECM remodeling induced by delivered CS that may explain changes of GT maturation and its quick resorption resulting in the recovery of skin structure with its appendages (hair and glands).A conceptual study limitation is a lack of understanding of immune cells interaction with CS and how it may modulate the response of immunocompetent cells as far as they present important generators of angiogenic stimuli in damaged tissue ,48. SuccThe findings are potentially of importance for both fundamental understanding of MSC participation in regeneration as well as for development in tissue engineering methods addressing an unmet medical need—pressure ulcer.