Vasculogenic “Code Switching”: Exploring the Varying Potential of Vascular Smooth Muscle Cells (SMCs) during Vasculogenesis

Introduction: Vascular smooth muscle cells (SMCs) are essential for vascular homeostasis, maintaining vasomotor tone, and vessel structural integrity. They interact with endothelial cells (ECs) to regulate vasculogenesis and initiate vascular repair through remodeling and angiogenesis. SMCs exhibit significant plasticity, changing phenotypes in response to environmental cues during pre-vasculogenesis, vasculogenesis, and vessel maturation. Initially, SMCs are highly migrative, proliferative, and synthetic, producing ECM components like collagen and elastin. Post-vasculogenesis, they become less migratory and synthetic, forming gap junctions to support vessel function. In mature vessels, SMCs express contractile proteins and have minimal proliferation and ECM synthesis. Upon vessel injury, SMCs can revert to a phenotype that supports vasculogenesis.

Co-culturing SMCs with human umbilical vein endothelial cells (HUVECs) generally results in lumenized endothelial vasculature, though variability exists in SMC support of vascularization, potentially linked to donor status and tissue source. We hypothesize that SMC monocultures exhibit semi-contractile phenotypes, and after co-culture with HUVECs, they switch to synthetic and migrative/proliferative phenotypes to support vasculogenesis. This phenotypic modulation is crucial for vascular repair and diseases like atherosclerosis. Our study investigates the differential gene expression in SMC cultures that support or do not support vasculogenesis, exploring potential drivers of diminished SMC viability and functionality by examining modulators of signaling pathways related to SMC plasticity. This research aims to uncover mechanisms behind SMC variability and improve understanding of their role in vascular health and disease.

Materials and

Methods: To investigate the mechanisms by which SMCs contribute to vessel formation, we performed bulk RNA sequencing on SMCs that either support or do not support vessel formation when cocultured with HUVECs. After performing gene differential analysis and filtering genes based on fold-change and p-value, we selected the top 100 genes of supporting and non-supporting SMCs and ran gene ontology enrichment analysis to identify genes that have essential roles in vasculogenesis. All SMC groups were cultured similarly in T25 flasks, with regular feeding using SMGM media every two days, over a span of 7-15 days to achieve sufficient confluency. Once confluency reaches 85-100%, a portion of the SMCs is co-cultured with HUVECs, while another portion is lysed and collected in Eppendorf tubes at a density ranging from 1-2.5x10^6 cells/ml (250 ul of cell suspension) for both RNA-seq and qPCR.

Results, Conclusions, and Discussions: o Results
The results of co-cultures of different SMCs with HUVECs are shown in Figs.1A. Comparing the morphology of SMCs shows that the supportive SMCs are normally stretched and have spindle shapes. From the results of the RNA-seq analysis, the PCA analysis shows clear separation of the groups (Fig. 1B). In Fig. 1C, the Volcano plot depicting gene expression in supportive SMCs compared to non-supportive SMCs reveals that in the pre-vasculogenesis phenotype, supportive SMCs exhibit upregulation of crucial genes like ADM, ENPP2, and IGFBP6, which play significant roles in vasculogenesis and angiogenesis (as shown in the network and dot plots in Figs. 1D and E). Conversely, there is downregulation of genes such as SOX4 and MANF. The decreased expression of ADM, ENPP2, and IGFBP6 in non-supportive SMCs, points towards reduced proliferation and functionality of SMCs during the pre-vasculogenesis stage. The results of vasculogenesis by co-culturing stable supportive SMCs and HUVECs in a 96-well plate, after 4 days of co-culture are shown in Fig. 1F.
ratio of genes in biological processes related to angiogenesis and vasculogenesis. F) vasculogenesis results of supportive SMCs, vessels, and collagen expression after 4 days. The scale bar is equal to 500 um.
o Conclusions
• Analyzing gene expression of SMC to verify phenotypic state before co-culture with HUVECs can improve vasculogenesis and enhance vessel stability.
• Key SMC genes that regulate vasculogenesis are ENPP2, IGBP6, and ADM.
• SOX4 and MANF levels are higher in non-supportive SMCs.
• In mono-culture conditions, SMCs with genes related to synthetic phenotypes and very low expression of contractile genes are able to form vessel beds.
o Discussion
The efficiency and stability of vasculogenesis has been probed by focusing on SMC gene expression and function. SMCs, being highly adaptable, transition between synthetic, migrative/proliferative, and contractile phenotypes during vasculogenesis. Understanding the drivers of SMC plasticity is essential for improving experimental reproducibility. This knowledge will facilitate the development of reliable strategies for engineering vascularized tissues. Our results suggest that supportive SMCs are more synthetic in mono-culture (pre-vasculogenesis), synthetic and migratory/proliferative during vasculogenesis, and contractile post-vasculogenesis.

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