Aortic valve eln expression peaks in early postnatal stagesTo assess the timeline of Eln expression, murine WT AoVs were isolated from various embryonic stages beginning from E13.5 to postnatal stages up to adulthood (12–13 weeks old). Eln expression remains low during early embryogenesis (E13.5 to E16.5) and increases 5-fold (p < 0.0001) during late embryogenesis (E17.5 and E18). Through early postnatal stages (P0-P5), Eln expression continues to increase 12–28 fold before returning to low levels in adulthood (Fig. 1). These results indicate that Eln gene expression is limited to a short time immediately before and after birth.Fig. 1ElastinmRNA levels in developing murine aortic valve. (A) Timeline of Eln expression in AoV from early developmental stages to adulthood. qPCR fold change normalized to endogenous Gapdh gene. Mean ± SEM shown; N = 5–9 biological replicate per group; p-value determined by One-way ANOVA followed by Tukey’s multiple comparison test. In situ hybridization RNAscope with Eln probe (red) and nuclei counterstaining for DAPI (blue) of (B) P0 (C) P5 (D) P30. Dotted white line outlines AoV leaflet. Scale bar = 10 μm.Spatial transcriptomics reveals correlation of elastin with known elastogenesis genesAt P3, using spatial transcriptomic analysis we found that valve tissue could be separated from other surrounding cardiac tissue based on global gene expression patterns (Fig. 2A and B). A list of all statistically significant up-regulated differentially expressed genes in the AoV leaflet tissues compared to the surrounding tissues can be seen in Supplemental Table S2 sorted by ascending p-value.Unbiased analysis of Eln correlation to other genes resulted in 369 statistically significant (P < 0.05) genes positively correlated with Eln expression. Of these genes, 3 had a “very strong” correlation (0.8 < R < 1), 36 had a “moderate” correlation (0.6 < R < 0.8), 119 had a “fair” correlation (0.3 < R < 0.6), and 26 had a “poor” correlation (0 < R < 0.3) using Akoglu (2018) User’s guide to correlation coefficients24 and further adapted25. The list of genes, their correlation coefficient to Eln expression, and their corresponding p-value are in Supplemental Table S3 sorted by ascending p-value. The top ten correlated genes, (Fig. 2C), includes Fbln5, Lox, and Fn1, which are known key regulators of elastic fiber formation in the aorta26,27,28. The most correlated gene was Acta2, a common marker of smooth muscle cells and myofibroblasts, cell types previously reported to mediate elastogenesis.Fig. 2Aortic valve cells clusters separately from other cardiac cells by spatial transcriptomic analysis at P3. (A) Spatial transcriptomics of P3 aortic valve sections. P3 aortic valve H&E image representation with surrounding tissue spots (blue) and aortic valve leaflet spots (red). Scale bar = 1 mm. (B) 2-D t-SNE cluster plot showing aortic valve leaflet tissue cluster (red) and surrounding tissue (blue). (C) List of top ten correlated genes with Eln from spatial transcriptomics data. For each spot (131 total), gene read count values were normalized to the total number of reads for a given spot. (D) Eln expressing cells are positive for alpha smooth muscle actin (αSMA). P0 aortic valve sections assessed by in situ hybridization RNAscope for Eln (red), immunofluorescence for αSMA antibody (green) and nuclei counterstaining for DAPI (blue). Dotted white line outlines aortic valve leaflet. Scale bar = 10 μm. Yellow highlighted box corresponds to the zoom image showing high magnification of αSMA and Eln positive cells in the aortic valve (left). Scalebar = 5 μm. N = 6.
Eln is expressed in alpha-smooth muscle actin positive cellsTo further test the relationship between Eln production and αSMA positive cells in the WT AoV, in situ hybridization using RNAscope combined with immunofluorescence were performed. P0 AoVs were assessed due to the noted increase in Eln in early postnatal stages (Fig. 1A). In tissues other than heart valves, fibroblasts and smooth muscle cells are known to be responsible for elastogenesis29,30. We found that cells expressing αSMA expressed Eln in the AoV at P0 stage (Fig. 2D).Murine hypopigmentation associates with less fibers in early postnatal developmentWe previously showed that AoV elastic fiber patterning is altered in adult mice with different pigmentation phenotypes, where the AoVs of hyperpigmented (K5-Edn3) mice have disrupted elastic fibers and AoVs from hypopigmented (KitWv and Albino) mice are largely devoid of elastic fibers17,18. The K5-Edn3 transgenic mice express the cytokine endothelin 3 (End3) under the control of keratin promoter leading to excess pigmentation in cutaneous and non-cutaneous sites19. The Albino mice contain melanocytes but melanogenesis is blocked by a mutation in Tyr, which is a pigment rate limiting enzyme. Here, we investigated early postnatal stages to assess the timing of changes in the extracellular matrix fiber patterning that occurs with the variation of pigment. To assess the structural patterning of Eln and collagen, AF633 and CNA35-488, respectively, were used in wholemount AoVs at P0 and P7 stages (Supplemental Fig. 2). In WT P0, qualitative observations indicated that elastic fibers were circumferentially aligned with collagen fibers, whereas at P7 elastic fibers became more radially aligned (Fig. 3A). This orthogonal alignment was also previously observed in the three cusps of the adult murine AoV17,18.At P0 there was no significant difference in the mean fluorescence intensity of AF633 in the leaflets of K5-Edn3 (p = 0.7046 N = 3, n = 5) and Albino mice (p = 0.2175 N = 5, n = 13) when compared to WT mice leaflets (Fig. 3B, left). At P7, leaflets of WT (p = 0.0005 N = 3, n = 6) and K5-Edn3 (p < 0.0001 N = 3, n = 5) mice had significantly increased mean fluorescence intensity of AF633 in comparison to that of Albino leaflets (N = 5, n = 13) (Fig. 3B, left). No significant differences were observed between the three groups for CNA35-488 mean fluorescent intensity at P0 and P7 stages (Fig. 3B, right). These results indicate that lack of leaflet pigmentation following peak postnatal Eln expression correlates with reduced formation of elastic fibers in AoV leaflets.Fig. 3Elastic fiber patterning in developing murine aortic valve. (A) Wholemount elastic (red) and collagen (cyan) fiber staining in WT (C57BL/6J mice), hyperpigmented (K5-Edn3) and hypopigmented (Albino) AoV leaflets at P0 and P7 stages. Leaflets in the light microscope images (inserts) correspond to those in the images of the stained leaflets. P0 images, 60X magnification and P7 images, 20X magnification. (B) Quantification of elastic fiber (left) and collagen fiber (right) staining at P0 (blue circle) and P7 (red circle) stages in WT, K5-Edn3 and Albino. (C) qPCR gene expression for elastin in K5-Edn3 and Albino at P0, P7 and P30 stages. P0 (blue circles), P7 (red circles) and P30 (black circles). Fold change normalized to endogenous Gapdh. Mean ± SEM shown; p-value determined by One-Way ANOVA followed by Tukey’s multiple comparison test; N = 5–9 biological replicates per group.Both high and low levels of pigmentation alter Eln expression in AoV leaflets in neonatal stagesTo determine if the differences in elastic fiber abundance in the AoV were due to changes in Eln expression, we performed RT-qPCR on AoV leaflets from the three mouse models. At P0, Eln expression was significantly ~ 12-fold higher in WT AoV when compared to K5-Edn3 (p = 0.0002) and Albino (p = 0.0013) AoVs. Interestingly, at P7 Eln expression trended higher in both K5-Edn3 (p = 0.25, ~ 7.66-fold change, N = 6) and Albino (p = 0.90, ~ 2.2-fold change, N = 4) compared WT leaflets (Fig. 3C), but the observed differences did not reach the established cutoff for statistical significance. Lastly, Eln expression was low at P30 in all three mouse models. These results suggest that both elevated and diminished levels of pigmentation in the leaflet affect Eln expression in early neonatal stages.Alpha-smooth muscle actin and melanocytic marker positive cells express Eln during AoV developmentPrevious analyses could not identify a single population of Eln producing cells in the AoV; however, it was hypothesized that the lack of an observable population may have resulted from Eln production prior to the P7 timepoint considered13. Given our data that Eln expression peaks between P0-P5 (Fig. 1A), we analyzed the relationship between phenotypic markers and Eln at P0. Following the observed correlation between elastic fiber patterning and pigmentation, we first assessed the relationship between Eln, αSMA, and melanocytic markers. Using Tyr and Eln RNAscope probes combined with immunolabeling for αSMA, we found a population of Eln+ and αSMA+ cells that express Tyr (Fig. 4A and B, Supplemental Fig. 3). Using immunolabeling, we next assessed other melanocytic markers, Trp1 and Mitf, (Fig. 4C and D) and found a population of Trp1+ and Mitf+ cells that are also Eln+, substantiating our finding that AoV melanocytes can produce Eln.Next, we assessed whether endothelial cells, which line the AoV surface, may contribute to elastogenesis. Using immunolabeling for VE-Cad, we observed a subset of VE-Cad positive cells to be Eln+ (Fig. 5A). Next, we used Vimentin, which labels the majority of resident VICs and was used as a gene marker along with Col1a1, Col3a1 and Versican to cluster VICs in the previously mentioned single cell RNA Sequencing study13. We also observed a population of Vimentin+ cells to be Eln+ (Fig. 5B). Finally, using CD45 antibody to label local immune cells, we found these cells to be distinct from Eln expressing cells (Fig. 5C). These findings suggest that several different cell phenotypes within the AoV can produce Eln.Fig. 4Alpha smooth muscle actin expressing cells that are positive for melanocyte specific markers express elastin in aortic valve leaflets. (A) Eln expressing cells are positive for smooth muscle actin and melanocyte markers. 3-D reconstruction of P0 aortic valve wholemount leaflet labeled with elastin (Eln, gray) and Tyrosinase (Tyr, magenta) RNAscope probes, and alpha-smooth muscle actin (αSMA, green) immunolabeling. XZ and YZ orthogonal views for merged and separate channels are shown in the bottom and side panels, respectively. N = 6. (B) In situ hybridization with Eln and Tyr probe and immunofluorescence with αSMA immunolabeling of P0 aortic valve section. Dotted white line outlines aortic valve leaflet. Scale bar = 10 μm. N = 4. In situ hybridization of wholemount P3 WT aortic valve labeled for Eln (gray) and antibody staining (magenta) for (C) Tyrosine related protein 1 (Trp1), (D) Microphthalmia-associated transcription factor (Mitf) and nuclei counterstaining for DAPI (blue). Scale bar = 50 μm. Yellow arrow heads point to markers co-localization. The yellow line shows the cross section corresponding to the orthogonal views. The XZ and YZ orthogonal views are shown in the bottom and side panels with only magenta and gray channel, respectively. N = 6. Wholemount single channels images are shown for each of the corresponding marker.
Multiple cell types including melanocytes contribute to elastogenesis in the developing murine aortic valve
