209 - Effect of metformin on single cell transcriptomics in hyperoxia exposed mouse lung endothelial cells

Publication Number: 209.4767

Sunil K. Sati, Medical College of Wisconsin, Brookfield, WI, United States; Girija G. Konduri, Medical College of Wisconsin, Milwaukee, WI, United States; Chien-Wei Lin, Medical College of Wisconsin, Brookfield, WI, United States; Ujala Rana, Medical College of Wisconsin, Wauwatosa, WI, United States

Background: Hyperoxia (HOX) exposure during early postnatal days 1-10 (P1-10) induces bronchopulmonary dysplasia (BPD) phenotype in mouse pups. Our recent studies demonstrated that metformin, an AMP Kinase agonist, protects angiogenesis and lung growth when given during HOX.

Objective: This study aimed to describe the effect of metformin on single-cell transcriptomics in lung endothelial cells (LEC) in HOX and normoxia (NOX) exposed mouse pups.

Design/Methods: We exposed mice to either NOX or 75% O2 (HOX) from P1-P10. We gave either saline (S) or metformin (Met) intraperitoneally to pups in both groups from P1-P10. All pups recovered in NOX from P10-P21. Lungs from 6 pups in each condition were harvested at P21. CD31+ EC were isolated and single-cell RNA (Sc-RNA) sequencing was done using the 10x Chromium Next GEM Single Cell 3’ Reagent Kits to prepare single-cell RNA libraries followed by sequencing. Data were filtered, and EC was selected using the expression of the pan EC marker, Cdh5. Uniform Manifold Approximation and Projection (UMAP) was used for dimension reduction. Heat maps were created for differentially expressed genes.

Results: A total of 18 Cdh5 positive EC clusters were identified (Fig 1a). We identified EC expressing Gpihbp1, Plvap, Edn1, and Aplnr as Cap1 capillary EC, and EC expressing Apln, Ednrb, Car4, Emp2, and Kitl as Cap2 EC (Fig 1b). We also found a cluster of EC-expressing markers for both Cap1 and Cap2 EC and labeled them Cap1+2 cells; this cluster appeared between the Cap1 and Cap2 EC clusters on UMAP (Fig 1c). As previously reported at P7-14, we found an increase in the proportion of Cap2 EC and Cap1+2 EC at P21 after hyperoxia exposure (HOX+saline vs NOX+saline) (Fig 1d). HOX+met had a similar effect on the EC but was more pronounced with almost doubling of Cap1+2 EC compared to the HOX+saline (Fig 1d). Analysis of the transcriptomes at P21 with Sc-RNA seq showed several genes (Gzmb, Sparc, Cxcl12, Sema3c) related to inflammation and fibrosis upregulated in Cap1 EC during recovery in HOX+saline (Fig 2a). A few lung injury genes (Cd74, S100a9) were downregulated in Cap2 EC in HOX+saline at P21 suggesting recovery (Fig 2b). Importantly, we found several genes related to EC proliferation, angiogenesis, and cell survival upregulated in Cap1 (Zfp36I1, Rhoj, Klf6, Jun), Cap2 (Rspo3, Cd24a) and Cap1+2 (Tmem158) EC with HOX+Met (Fig 2d-f).

Conclusion(s): Our study revealed novel transcriptome changes in LEC at P21 after HOX exposure in mouse pups. Metformin promotes proliferation and survival while decreasing inflammation in LEC during recovery from HOX.

Figure 1
Figure 1.pdfFig 1. 1-a shows the CDH5+ EC clusters on Uniform Manifold Approximation and Projection (UMAP). 1-b is a dot plot showing EC cell markers expression on X-axis and EC type on Y-axis. Average expression of the markers is shown with the color and percent of EC expressing the marker is shown with the size of the dot as shown in the figure. 1-c shows 3 cap EC clusters on UMAP. 1-d shows changes in the proportions of Cap EC according to the condition. NxS: Normoxia+saline, NxM: normoxia+metformin, HxS: hyperoxia+saline, HxM: hyperoxia+metformin.


Figure 2
Figure 2.pdfFigure 2 a-e shows the differentially expressed genes in the three Cap EC types according to the condition. A-c shows a comparison of HxS with NxS in cap1, cap2, and cap1+2 EC respectively. Fig d-f shows a comparison of HxM with HxS in cap1, cap2, and cap1+2 EC respectively. NxS: Normoxia+saline, NxM: normoxia+metformin, HxS: hyperoxia+saline, HxM: hyperoxia+metformin.