Age-Related Changes in Urethral Structure and Responds to Injury: Single-Cell Atlas of a Rat Model of Vaginal Birth Injury induced Stress Urinary Incontinence - PubMed (original) (raw)
Age-Related Changes in Urethral Structure and Responds to Injury: Single-Cell Atlas of a Rat Model of Vaginal Birth Injury induced Stress Urinary Incontinence
Liangyu Zhao et al. Res Sq. 2024.
Abstract
Stress urinary incontinence (SUI) greatly affects the daily life of numerous women and is closely related to a history of vaginal delivery and aging. We used vaginal balloon dilation to simulate vaginal birth injury in young and middle-aged rats to produce a SUI animal model, and found that young rats restored urethral structure and function well, but not the middle-aged rats. To identify the characteristics of cellular and molecular changes in the urethral microenvironment during the repair process of SUI. We profiled 51,690 individual female rat urethra cells from 24 and 48 weeks old, with or without simulated vaginal birth injury. Cell interaction analysis showed that signal networks during repair process changed from resting to active, and aging altered the distribution but not the overall level of cell interaction in the repair process. Similarity analysis showed that muscle, fibroblasts, and immune cells underwent large transcriptional changes during aging and repair. In middle-aged rats, cell senescence occurs mainly in the superficial and middle urothelium due to cellular death and shedding, and the basal urothelium expressed many Senescence-Associated Secretory Phenotype (SASP) genes. In conclusion, we established the aging and vaginal balloon dilation (VBD) model of female urethral cell anatomy and the signal network landscape, which provides an insight into the normal or disordered urethra repair process and the scientific basis for developing novel SUI therapies.
Keywords: Senescence-Associated Secretory Phenotype (SASP); Stress urinary incontinence (SUI); single cells RNA sequence; urethra; vaginal balloon dilation (VBD).
Conflict of interest statement
Additional Declarations: There is NO Competing Interest. The authors declare no competing interests.
Figures
Figure 1. Global expression profiling of normal and urinary incontinence rat urethra cells by single-cell RNA-seq.
(a) Schematic illustration of the experimental workflow in this study. (b) Urodynamic functional study. Leak point pressure (LPP). (c-e) t-distributed stochastic neighbour embedding (tSNE) plots of all urethra cells from normal and urinary incontinence rat. Cells are coloured according to their (c) cell types, (d) treatment groups, or (e) age. (f) Heatmap of the top 30 DEGs in each major cluster (left panel), with the GO analysis (biological process) according to the DEGs of each major cluster (right panel). The top2 specific marker genes are labelled in parentheses. A gradient of light blue to dark red indicates low to high expression levels in the heatmap. (g) Dissimilarity of 6 major urethra cell clusters between groups are shown on bubble diagram. The gradient of bubbles sizes indicates low to high scaled Jaccard distance, and the gradient of red indicates low to high scaled Bary values. (h) Bar plot showing the cell count proportion of 8 major cell clusters in each group. VBD: vaginal balloon dilation; BAPN: b-aminopropionitrile + VBD; Epic: epithelial cell; EndoC: endothelial cells; Mus: muscle cells; Fib: fibroblast; Neu: neuroendocrine cells; Leu: leukocytes.
Figure 2. Transcriptional changes of cell microenvironment during urethral injury repair.
(a) The barplot showing the total interaction count and strength within urethral microenvironment of young normal and VBD rat. (b) Circle plots showing the up- or down-regulation of cell–cell communication network between young normal and VBD rat. The red line represents up-regulated interaction and the blue line represents down-regulated. (c-d) Violin plot showing the expression level of the genes that are associated with (c) energy metabolism and (d) cell cycle. (e) Uniform manifold approximation and projection (UMAP) dimensionality reduction plot of all rat striated muscle of urethra. Cells are coloured according to their types. (f) Bubble diagram showing the marker genes of each striated muscle cluster. The gradient of bubbles sizes indicates low to high expressed percent, and the gradient of blue to red indicates low to high average expression level. (g) Bar plot showing the cell count proportion of 3 striated muscle clusters in each group. (h) Heatmap of the DEGs in 3 striated muscle clusters. A gradient of light blue to dark red indicates low to high expression levels in the heatmap. (i) Bar plot showing the GSEA (biological process and KEGG pathways) terms according to the DEGs between sham24 and VBD24 striated muscles. Statistical analysis was based on Fisher's exact test; two-tailed; the confidence interval is 95%. A gradient of light blue to red indicates negative to positive enrichment of the term. The length of the bar indicates the P value. (j) Violin plot showing the expression level of genes belong to “muscle system process” terms among sham24, VBD24 and BAPN24 striated muscles. VBD: vaginal balloon dilation; BAPN: b-aminopropionitrile + VBD; Epic: epithelial cell; EndoC: endothelial cells; Mus: muscle cells; Fib: fibroblast; Neu: neuroendocrine cells; Leu: leukocytes.
Figure 3. Cell–cell communication network within the urethra microenvironment of young and old VBD rat.
(b) The barplot showing the total interaction count and strength within urethral microenvironment of young and old VBD rat. (c-d) Circle plots showing the total (c) interaction count and (d) strength of cell–cell communication network between young and old VBD rat. The red line represents up-regulated interaction and the blue line represents down-regulated. (e) The barplot showing the relative information flow between young and old VBD rat urethra microenvironment. Green or purple fonts represent signals with statistical differences. (f) Heatmap of the outcoming signals of each cluster between young and old VBD rat urethra microenvironment. A gradient of white to dark red indicates low to high expression weight values in the heatmap. (g) Heatmap of the outcoming signals of each cluster between young and old VBD rat urethra microenvironment. A gradient of white to dark blue indicates low to high expression weight values in the heatmap.
Figure 4. Overview of aging characteristics of urethra microenvironment.
(a) The Venn diagram shows the up-regulated or down-regulated genes with age that are common or unique to each cell cluster. (b) The table showing the up-regulated or down-regulated genes with age that are common to some or all clusters. (c-d) Bubble diagram showing the GO analysis terms according to the (c) up-regulated DEGs or (d) down-regulated DEGs with age in each cluster. Statistical analysis was based on Fisher's exact test; two-tailed; the confidence interval is 95%. The gradient of bubbles sizes indicates low to high enrichment score, and the gradient of colour ranges from light to dark indicates high to low _P_value. (e) Bubble diagram showing the SASP genes of each cluster between young and old rat. The gradient of bubbles sizes indicates low to high expressed percent, and the gradient of blue to red indicates low to high average expression level.
Figure 5. The aging characteristics of urethra muscle clusters.
(a) Bubble diagram showing the SASP genes of each muscle cluster among young and old rat with or without VBD treatment. The gradient of bubbles sizes indicates low to high expressed percent, and the gradient of blue to red indicates low to high average expression level. (b-c) The GO analysis terms according to the up-regulated DEGs or down-regulated DEGs with age in (b) smooth muscle or (c) striated muscle cell cluster. Statistical analysis was based on Fisher's exact test; two-tailed; the confidence interval is 95%. (d) The UMAP plots showing the transcription pattern of genes belong to “regulation of neuron projection development” term (green) and striated muscle subtype characteristic genes (red). (e) Immunohistochemical co-staining of α-BTX (green) and Pha (red) in normal young and old urethra tissue paraffin sections. The right panel showing the statistics of neuron muscle junction count per muscle cell. The scale bar represents 10 µm.
Figure 6.. The aging characteristics of urethra fibroblast.
(a) The GO analysis terms according to the up-regulated DEGs or down-regulated DEGs with age in urethra fibroblast. Statistical analysis was based on Fisher's exact test; two-tailed; the confidence interval is 95%. (b-e) The UMAP dimensionality reduction plot of rat urethra fibroblast cluster. Cells are coloured according to their (b) sample group, (c) pseudotime, (d) expression level of 4 models and (e) subclusters. (f) Bar plot showing the cell count proportion of 3 fibroblast clusters in each group. (g) The GO and KEGG analysis terms according to the up-regulated DEGs in each fibroblast subcluster. Statistical analysis was based on Fisher's exact test; two-tailed; the confidence interval is 95%. (h) Violin plot showing the expression level of genes belong to GO term “negative regulation of cholesterol storage” (Pparg), “wound healing” (Col1a1, Col1a2, Col3a1, Dcn, Gsn, Fgfr1) and “tight junction assembly” (Marveld2, Mpp7, Actn4) among 3 fibroblast subclusters. (i) The link plot showing the main regulators in C2 (green) and C3 (blue) subcluster. (j) Violin plot showing the expression level of main regulators among 3 fibroblast subclusters.
Figure 7. The aging state of urethra immune microenvironment.
(a-c) The UMAP dimensionality reduction plot of rat urethra fibroblast cluster. Cells are coloured according to their (a) expression level of Ptprc, (b) expression level of each immune cell markers, and (c) subclusters. (d) Bubble diagram showing the top DEGs of each immune cell cluster. The gradient of bubbles sizes indicates low to high expressed percent, and the gradient of blue to red indicates low to high average expression level. (e) Bar plot showing the cell count proportion of 9 immune subclusters in young and old rat urethra. (f) Bar plot showing the GSEA (biological process and KEGG pathways) terms according to the DEGs between sham24 and VBD24 immune cells. Statistical analysis was based on Fisher's exact test; two-tailed; the confidence interval is 95%. A gradient of light blue to red indicates negative to positive enrichment of the term. The length of the bar indicates the _P_value. (g) Violin plot showing the expression level of genes belong to GO term “leukocyte activation” (upper and middle panel) and “PPAR signaling pathway” (bottom panel) between sham24 and VBD24 immune cells.
Figure 8. The heterogeneity of urothelium aging process.
(a) Heatmap of the DEGs in 4 urothelium subclusters. A gradient of light blue to dark red indicates low to high expression levels in the heatmap. (b) The UMAP dimensionality reduction plot of rat urothelium cells. Cells are coloured according to their subcluster and plot is split by their groups. (c) Immunohistochemical staining of uroplakin (green) in normal young and old urethra tissue paraffin sections. (d) β-gal staining of normal young and old urethra tissue paraffin sections. The lower panel is the magnification of the upper field. The scale bar represents 50 µm (upper panel) and 10 µm (lower panel). (e) Violin plot showing the expression score of SASP and frailty genes between sham24 and VBD24 in different urothelium subclusters. (f-g) The Venn diagram shows the (f) up-regulated or (g) down-regulated genes with age that are common or unique to each urothelium subcluster. (h) The table showing the up-regulated or down-regulated genes with age that are common to some or urothelium subclusters. (i-j) Bubble diagram showing the GO analysis terms according to the (i) up-regulated DEGs or (j) down-regulated DEGs with age in urothelium subcluster. Statistical analysis was based on Fisher's exact test; two-tailed; the confidence interval is 95%. The gradient of bubbles sizes indicates low to high enrichment score, and the gradient of colour ranges from light to dark indicates high to low _P_value.
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