Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis

Abstract

Background: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed.

Methods: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements.

Results: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls.

Conclusions: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Keywords: echocardiography; endothelial cells; heart valve diseases; inflammation; lymphangiogenesis.

Figure 1.. Identification of lymphatic vessels in inflamed mitral valves with in vivo lineage-tracing.

A. Fluorescence images of mTom and mGFP labeling in 12-week-old B.g7 and K/B.g7 lineage-traced mice. Boxes highlight identified mGFP+ vessels which are displayed at higher magnification in the right-most boxes. Scale bars are 50 μm. LA: left atrium, LV: left ventricle. B. Immunostaining of endothelial markers CD31 and ERG (first and second rows), lymphatic marker LYVE1 (middle row), hematopoietic marker CD45 (fourth row), and fibroblast marker Periostin (bottom row) in 20 week old K/B.g7 lineage-traced mitral valves. Arrows point to mGFP+ vessels. C. Quantification of mGFP+ vessel numbers in mitral valves of 4-, 8-, 12-, and 20-week old B.g7 and K/B.g7 lineage-traced mice. Each data point represents a different mouse. Statistics were calculated with Mann Whitney U tests. D. Quantification of mGFP+LYVE1+ vessel numbers in mitral valves of 4-, 8-, 12-, and 20-week old B.g7 and K/B.g7 lineage-traced mice (top graph) and proportion of mGFP+ vessels that were LYVE1+ in each genotype and age group (bottom graph). In C-D, circles indicate female mice and triangles indicate male mice. E. Paired bar graphs of the distance of mGFP+ vessels from the atrial surface and total valve thickness at the location of each vessel for 12- and 20-week old B.g7 and K/B.g7 lineage-traced mice. Each data point represents a different mouse and lines connect paired metrics per mouse. Schematic depicts how measurements in Figure 1F were taken: a line oriented perpendicularly to the valve length was drawn through each mGFP+ vessel and both the total length of the line (valve thickness) and the distance from the middle of the vessel to the atrial surface along that line (atrial distance) were quantified. F. Whole mounted image of a 17-week-old K/B.g7 lineage-traced mitral valve. Anatomical features of the valve are labeled with an area of lymphatic vessels enlarged in the righthand image. LA: left atrium, LV: left ventricle.

Figure 2.. Single-cell RNA sequencing reveals a heterogeneous cell population including Lymph-VEC subsets differentially localized in the MV.

A. UMAP plot of all VEC clusters in 3-, 8-, and 25-week old B.g7 and K/B.g7 mice (n = 2 per age and genotype, one male and one female in each experimental group) overlaid with Clusters 0-6. B. Table reporting the top 10 differentially expressed genes in each VEC cluster. C. Genes enriched in Hulin et al’s VEC, coapt-VEC, and Lymph-VEC populations overlaid on the UMAP plot. D. Prox1 and Lyve1 transcript expression levels overlaid on UMAP plots with respective immunostaining of PROX1 and LYVE1 in serial sections of a 12-week-old K/B.g7 MV immediately below. Arrowheads delineate PROX1+LYVE1^neg cells. Scale bars are 100 μm. A: atrium, V: ventricle. E. Ccl21a and Flt4 transcript expression levels overlaid on UMAP plots with respective immunostaining of CCL21 and VEGFR3 in serial sections of a 12-week-old K/B.g7 EC-lineage-traced MV immediately below. Scale bars are 50 μm.

Figure 3.. A distinct population of Lymph-VEC 2 cells adopt fibrotic characteristics during chronic inflammation.

A. Quantification of Lymph-VEC 1-3 frequencies amongst total VECs (top) and total Lymph-VECs (bottom) in 3-, 8-, and 25-week old K/B.g7 valves from the scRNA seq dataset. B. Individual pseudotime trajectories of B.g7 and K/B.g7 VECs superimposed with clusters (top) or mouse age (bottom). Trajectories were created using slingshot. “*1” indicate the location of Lymph-VEC 1 cells in each plot. Dotted circles highlight the subset of Lymph-VEC 2 cells that emerge in aged K/B.g7 valves. C. Pseudotime trajectories of Lymph-VECs from B.g7 or K/B.g7 valves superimposed with clusters (right) or mouse age (left). D. Bar plot of the negative log10-transformed p-values of the 10 most significant enriched pathways that are up-regulated (green) or down-regulated (red) in K/B.g7 Lymph-VECs found at the end of the trajectory shown in C. Tradeseq was used to identify genes that were differentially expressed at the end of the K/B.g7 Lymph-VEC trajectory compared to the start of the trajectory using a start-vs-end test. Significantly up-regulated (160) or down-regulated (158) genes were determined as those with an adjusted p-value < 0.05. Represented pathways are from Gene Ontology’s Biological Processes.

Figure 4.. Blockade of VEGFR3 signaling inhibits mitral valve lymphatic growth.

A. MAZ51 treatment scheme. B. Representative immunostaining of VEGFR3, LYVE1, and CD31 in mitral valves of MAZ51- (n = 9) or control- (n = 9) treated mice. A: atria, V: ventricle. Scale bars in left, lower magnification panels are 100 μm and in right, higher magnification panels are 25 μm. C. Bar graph of the number of VEGFR3+LYVE1+ lymphatic vessels in mitral valves of K/B.g7 mice treated with MAZ51 or vehicle control. D. Bar graph of the average mitral valve (MV) thickness of each mouse treated with control or MAZ51. E. Bar graph of the largest MV thickness measurement from each mouse treated with control or MAZ51. F. VEGF-C Trap treatment scheme. G. Representative immunostaining of VEGFR3, LYVE1, and CD31 in mitral valves of VEGF-C Trap- (n = 7) or control- (n = 8) treated mice. A: atria, V: ventricle. Scale bars in left, lower magnification panels are 100 μm and in right, higher magnification panels are 25 μm. H. Bar graph of the number of VEGFR3+LYVE1+ lymphatic vessels in mitral valves of K/B.g7 mice treated with VEGF-C Trap or control. I. Bar graph of the average mitral valve (MV) thickness of each mouse treated with control or VEGF-C Trap. J. Bar graph of the largest MV thickness measurement from each mouse treated with control or VEGF-C Trap. Mann Whitney U tests were used and circles indicate female mice and triangles indicate male mice in C-E and H-J.

Figure 5.. Valve thickness and lymphatic density correlate with worsened indices of cardiac function in aged K/B.g7 mice.

A-D. Echocardiograph was used to quantify the left ventricular ejection fraction (A), E/A ratio (B), E/e’ ratio (C), and PA/MV dimensionless index (D) in K/B.g7 and uninflamed controls aged 6-9 months. Using histological approaches, valve thickness (E) and lymphatic density (F) were quantified in each mouse. Circles indicate female mice and triangles indicate male mice. Mann Whitney U tests were used to calculate statistics.

Figure 6.. Lymphatic vessels identified more frequently in human rheumatic valve samples.

A. Images of pentachrome staining on rheumatic and non-rheumatic human valve sections. Scale bars of lower magnification images are 500 μm and higher magnification are 100 μm. B. Immunostaining of case/rheumatic and control valves for LYVE1, VEGFR3, and VWF using serial sections from the same region of each indicated valve. Rightmost images are enlarged from the area delineated with a white box in each merged image. Scale bars are 100 μm. C. Immunostained rheumatic and non-rheumatic valves were reviewed for presence of VEGFR3+ or LYVE1+ lymphatic vessels and percentage of valves with lymphatics present were calculated.