Periodic Reporting for period 1 - ECSiTe (Endothelial Cell Signature in the Total Environment)
Reporting period: 2021-07-01 to 2023-06-30
Endothelial cells (ECs) line the inner part of blood vessels and are constantly exposed to mechanical forces. Flow shear stress levels change across the vasculature ranging from 5 to 20-70 dynes/cm2 in veins and aortic valves, respectively. The sensation of shear stress by ECs depends on the tissue stiffnesses, being 0.5-4 kPa in brain, or more than 100 kPa in the aortic valves. Though ECs sense both mechanical cues simultaneously, they have been classically studied separately. Their combined effects, however, could be behind the EC heterogeneity, crucial for the proper function of some organs, but probably also responsible for the low efficiency of vascular-related treatments. Understanding how shear stress and stiffness modulate the ECs behavior would provide new tools towards more specialized tissue engineering and vascular repair approaches. The ECSiTe project addresses this by studying the interplay of fluid shear stress and tissue stiffness in the modulation of the EC gene expression (aim1, working package 1, WP1). Then, the effect of organ-specific basement membrane proteins in organ-specific ECs is studied (aim2, WP2). Finally, the acquired knowledge is integrated to differentiate the contribution of the microenvironment to the ECs heterogeneity (aim3, WP3). Across 14 combinations of shear stress and stiffness, genes being modulated by shear stress or stiffness alone were identified. Importantly, interaction effects between both mechanical cues were identified in 603 genes, related to important cardiovascular processes, such as the migration of ECs. These results correlated with morphological changes in the ECs, with implications in the transcriptional activity of YAP1, an important mechanotransducer. Now, the contribution of organ-specific basement membrane proteins on the modulation of the EC heterogeneity is being addressed. This data is the first to investigate in a large-scale and non-biased approach the role of the interaction of shear stress and tissue stiffness in the vasculature.
For the first part of the project (WP1), a flow chamber was developed to study 14 combinations of shear stress and stiffness applied on human umbilical vein ECs (HUVECs). Three levels of stiffness (1, 10, 100 kPa) were achieved using different compositions of polyacrylamide hydrogels. These where functionalized with collagen I, and HUVECs were seeded on top forming monolayers. Placed in the flow chamber, cells were exposed to five levels of shear stress (0, 5, 15, 25, 40 dynes/cm2) using peristaltic pumps with circulating media for 24h (Figure 1a). The RNA of the cells was sequenced and analyzed across conditions. Most of the variation in the data was explained by shear stress (Figure 1b). The factorial design permitted distinguishing between genes modulated by shear stress or by stiffness alone. Importantly, a list of genes modulated by the interaction of both mechanical cues was also obtained (Figure 1c), identified to be relevant in cardiovascular processes, such as angiogenesis or EC migration (Figure 1d). Interestingly, shifts in the activation of such processes across conditions suggested that ECs on stiffer substrates were more sensitive to changes in shear stress, confirming an interaction between both cues (milestone M1). The RNAseq results for migration were further correlated with in vitro data, showing that the migration of ECs at a low shear stress (1-5 dynes/cm2) is modulated by tissue stiffness, with a peak at 10 kPa. One of the genes supporting these results was YAP1, which is an important mechanotransducer known to translocate in the nuclei when cells flatten due to the higher traction forces from stiffer substrates [1]. Once in the nuclei, Yap1 promotes the transcription of numerous genes. Importantly, several of Yap1-promoted genes were identified in the list of interaction genes. Hence, the location of Yap1 and changes in cell shapes were studied across the 14 conditions. Consistent with the literature, on stiffer substrates cells flattened and Yap1 was found in the cell nuclei (Figure 1e). However, under shear stress, rounder cells flattened and Yap1 started translocating to the nuclei. Moreover, on stiffer substrates, the lowest shear stress (5 dynes/cm2) made flat cells become rounder and Yap1 was less present in the nuclei. This proves that specific levels of shear stress modify the stiffness-specific EC behavior.
The second part of the project, which constitutes WP2 and WP3, is still ongoing. However, human brain and aortic valve ECs were obtained and expanded, and the protocol for the deposition and decellularization of their basement membranes is ready to go. These constitute milestone M2 and the key elements to perform the experiments (M3 and M4).
During the duration of the project, the results were presented in national and international conferences across Europe and the US, including the GRC on Biomechanics in Vascular Biology in South Hadley, Massachusetts. The researcher published a first author review paper on the impact of microenvironmental cues on the organ-specific heterogeneity. An article covering WP1 is in preparation and a second paper compelling WP2 and WP3 together with the cell alignment data in WP1 is expected. The RNAseq data from WP1 will be made available in a public repository after the publication of the paper. However, this is already being exploited by a collaborator from the UK working on a computational model, and by a starting project on the biofabrication of 3D vascularized structures using organ-specific spheroids.
The second part of the project, which constitutes WP2 and WP3, is still ongoing. However, human brain and aortic valve ECs were obtained and expanded, and the protocol for the deposition and decellularization of their basement membranes is ready to go. These constitute milestone M2 and the key elements to perform the experiments (M3 and M4).
During the duration of the project, the results were presented in national and international conferences across Europe and the US, including the GRC on Biomechanics in Vascular Biology in South Hadley, Massachusetts. The researcher published a first author review paper on the impact of microenvironmental cues on the organ-specific heterogeneity. An article covering WP1 is in preparation and a second paper compelling WP2 and WP3 together with the cell alignment data in WP1 is expected. The RNAseq data from WP1 will be made available in a public repository after the publication of the paper. However, this is already being exploited by a collaborator from the UK working on a computational model, and by a starting project on the biofabrication of 3D vascularized structures using organ-specific spheroids.
In the last decade, few studies have studied combined effects between shear stress and stiffness on ECs. On 2.5 kPa, for example, shear stress induced atheroprotective signals in ECs, compared to cells on stiffer substrates [2,3]. Sex-specific responses to combinations of both mechanical cues were also described, showing that male ECs were more sensitive than female ECs [4]. Here, ECSiTe unraveled, for the first time, unbiased RNAseq profiles for ECs under 14 different combinations of shear stress and stiffness. These conditions mimic healthy tissues, but also stiffened conditions due to disease, like atherosclerosis or cancer. Therefore, the ECSiTe dataset will become a reference for further investigation on the EC mechanobiology in vascular biology, oncology, and tissue engineering.
Additional results are expected on the contribution of organ-specific microenvironments in the EC behavior. Basement membranes produced by brain and aortic valve ECs will be combined with mimicking mechanical cues, and these environments will be switched from their cell type. Since aortic valve ECs align perpendicularly to flow [5], we expect that brain ECs align perpendicularly when exposed to aortic valve conditions and vice-versa. This work is ongoing and will be performed by a master’s student under the researcher’s supervision.
[1] A. Elosegui-Artola, I. Andreu, A. E. M. Beedle, D. Navajas, S. Garcia-Manyes, Cell 2017, 171, 1397.
[2] J. C. Kohn, D. W. Zhou, F. Bordeleau, A. L. Zhou, B. N. Mason, M. J. Mitchell, M. R. King, C. A. Reinhart-King, Biophys J 2015, 108, 471.
[3] P. A. Galie, A. Van Oosten, C. S. Chen, P. A. Janmey, Lab Chip 2015, 15, 1205.
[4] B. D. James, J. B. Allen, Adv Healthc Mater 2021, 10.
[5] J. T. Butcher, A. M. Penrod, A. J. García, R. M. Nerem, Arterioscler Thromb Vasc Biol 2004, 24, 1429.
Additional results are expected on the contribution of organ-specific microenvironments in the EC behavior. Basement membranes produced by brain and aortic valve ECs will be combined with mimicking mechanical cues, and these environments will be switched from their cell type. Since aortic valve ECs align perpendicularly to flow [5], we expect that brain ECs align perpendicularly when exposed to aortic valve conditions and vice-versa. This work is ongoing and will be performed by a master’s student under the researcher’s supervision.
[1] A. Elosegui-Artola, I. Andreu, A. E. M. Beedle, D. Navajas, S. Garcia-Manyes, Cell 2017, 171, 1397.
[2] J. C. Kohn, D. W. Zhou, F. Bordeleau, A. L. Zhou, B. N. Mason, M. J. Mitchell, M. R. King, C. A. Reinhart-King, Biophys J 2015, 108, 471.
[3] P. A. Galie, A. Van Oosten, C. S. Chen, P. A. Janmey, Lab Chip 2015, 15, 1205.
[4] B. D. James, J. B. Allen, Adv Healthc Mater 2021, 10.
[5] J. T. Butcher, A. M. Penrod, A. J. García, R. M. Nerem, Arterioscler Thromb Vasc Biol 2004, 24, 1429.