Unknown,Transcriptomics,Genomics,Proteomics

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Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema

ABSTRACT: Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in lymphedematous arm compared to normal arm in patients. The role of APLN in LD was confirmed in APLN-knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of non-integrative RNA-delivery LentiFlash® vector that will be evaluated for phase I/IIa clinical trial.

INSTRUMENT(S): Illumina HiSeq X

ORGANISM(S): Homo sapiens

SUBMITTER: Draia-Nicolau Ondina

PROVIDER: E-MTAB-13381 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lymphedema (LD) is characterized by the accumulation of protein-rich interstitial fluid, lipids and a significant inflammatory cell infiltrate in the limb. It causes a significant morbidity and is a common disabling disease affecting more than 150 million people worldwide, however there is no yet curative treatment. In this study, we found that LD tissues from patients exhibit inflamed gene expression profile compared to their normal arm. This was next confirmed by a lipidomic analysis that revealed severe decrease in arachidonic acid-derived lipid mediators generated by the 15-lipoxygenase (15-LO) in lymphedematous arms. Using a mouse model of lymphedema, we reproduced the etiology of the human pathology including the loss of specialized pro-resolving lipid mediators that play essential role in resolution of inflammation. This was associated with a lack of nonlymphoid PPAR-positive regulatory T cells (Treg) recruitment in the injured limb adipose tissue. Importantly, we identified the lymphatic endothelial 15-LO as responsible for the chemoattraction and survival of this Treg subpopulation. These results were confirmed by an aggravation of LD and degradation of the lymphatic network in an original transgenic mouse model in which ALOX15 gene has been selectively deleted in the lymphatic system (ALOX15lecKO). Importantly, this phenotype was rescued by the injection of ALOX15-expressing lentivectors. These results provide evidence that lymphatic 15-LO may represent a novel therapeutic target for LD by serving as a mediator of nonlymphoid Treg cell population invasion into lymphedematous adipose tissue to resolve inflammation. Experimental workflow: To broadly identify gene expression signatures associated to secondary LD, we performed bulk-RNA sequencing on dermolipectomy tissue samples from women who developed LD after breast cancer. Four patient biopsies (normal arm and LD arm in each patient) were studied and the differential expression analysis (DEseq) followed by a protein-coding RNA profiling.

Project description:To examine the role of platelets in mouse monocyte function, we administered anti-GPIba antibody to deplete platelets or IgG as a control to 48 mice. After 12 hours, mice were either exposed to LPS or PBS (as a sham control) for 3 hours. Peripheral blood mononuclear cells (PBMCs) were collected, and monocytes were sorted into QIAzol lysis buffer. The experimental setup included 4 conditions, with each condition repeated 3 times. In each experiment, the blood from 4 mice was pooled to generate biological replicates of 12 mice, distributed into 4 groups: Sham mice (IgG) treated with PBS (n = 3) Sham mice (IgG) treated with LPS (n = 3) Platelet-depleted mice (anti-GPIba) treated with PBS (n = 3) Platelet-depleted mice (anti-GPIba) treated with LPS (n = 3) After these treatments, mouse Ly6G− IA/IE− CD45+ CD11b+ CD115+ monocytes were FACS-sorted directly into Quiazol for RNA extraction. The lysed samples were sent to GENEWIZ for RNA isolation and bulk RNA-seq analysis. RNA extraction and library preparation were conducted according to the GENEWIZ (Azenta Life Sciences) pipeline. Ultra-low input RNA-Seq was performed on Illumina HiSeq PE 2x150 bp with approximately 350M reads. Subsequently, reads were mapped to the Mus musculus GRCm38 reference genome (ENSEMBL) using the STAR aligner v.2.5.2b, and unique gene hit counts were generated with featureCounts from the Subread package v.1.5.2. Standard analysis was performed by GENEWIZ, including differential gene expression analysis using DESeq2. p-values and log2 fold changes were calculated using the Wald test, with genes meeting criteria of adjusted p-value < 0.05 and absolute fold change > 2 considered significantly differentially expressed genes (DEGs). Gene ontology (GO) analysis of significant DEGs was conducted by GENEWIZ (Azenta Life Sciences) using the Fisher exact test.\\" *Our goal is to assess the alternations on transcription levels (mRNA-Seq) between the different groups. Twelve samples contain 500 - 10000 mouse monocytes that were directly sorted into 1 ml QIAzol. **Experimental setting: samples were generated from three independent experiments (biological replicates N1, N2 and N3), each on a different day. Each experiment consisted of 4 different groups: Group 1: treated with IgG + PBS Group 2: treated with IgG + LPS challenge Group 3: treated with AntiGPIb + PBS Group 4: treated with AntiGPIb + LPS challenge First run with biological replicates Nr. 1 (indicated as \\"N1\\") contained samples 1 - 4. Second run with biological replicates Nr. 2 (indicated as \\"N2\\") contained samples 5 - 8. Third run with biological replicates Nr. 3 (indicated as \\"N3\\") contained samples 9 - 12. Experimental questions/analysis: 1) what is the effect of AntiGPIb treatment w.o LPS challenge? i.e. \\"Group 3 (Treatment) vs. Group 1(Ctrl)\\" 2) what is the effect of LPS in \\"Group 2 (Treatment) vs. Group 1 (Ctrl)\\" as well as \\"Group 4 (Treatment) vs. Group 3 (Ctrl)\".

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Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema

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