Angiogenic Potential of Endothelial Cells in Response to the Stiffness and Anisotropy of Right Ventricle Mimetic Scaffolds

Introduction: Right ventricular (RV) failure is the cause of mortality in patients of pulmonary hypertension (PH) and left heart failure with preserved ejection and fraction. In the diseased RV, the tissue becomes highly stiff and anisotropic, and the remodeling leads to fibrosis and capillary rarefaction, which worsens the nutrition and blood supply to the dysfunctional organ. A significant therapeutic strategy for patients with RV failure is the promotion of angiogenesis in the myocardium to reverse the malfunction. However, the biomechanics mechanism of this process is unclear. Specifically, the effect of altered tissue mechanical properties on the angiogenic potential of endothelial cells (EC) is unknown. Here we aim to investigate the impact of RV-like substrate stiffness and anisotropy on the angiogenic potential of ECs. We hypothesize that increased stiffness and anisotropy confer opposite and divergent effects on the angiogenic potential of ECs.

Previous work has shown that ECs from distinct origins respond differently to mechanical signals, thus ensuing variations in cell behavior. Human umbilical cord endothelial cells (HUVECs) and human coronary arterial endothelial cells (HCMECs) are two cell lines originating from different anatomical locations of human body. HUVECs have been extensively studied for their cellular functions and angiogenic capabilities, but they do not reside in cardiac tissues. In contrast, HCMECs are more relevant to cardiac angiogenesis but have not been well investigated. It is unclear whether the mechanical response of these two cell lines ECs is identical. Thus, we also investigated the responses of various cell lines to RV-like substrate mechanical properties.

Materials and

Methods: We have previously developed a tissue-engineered platform to fabricate four different electrospun scaffolds with varying stiffness, representing normal (soft) and failing (stiff) RVs, and varying degree of anisotropy, representing isotropic and anisotropic substrates. The soft and anisotropic group resembles healthy RVs while the stiff and anisotropic group resembles failing RVs. The stiff and isotropic group resembles ischemic RVs composed of acellular scar tissues, and the soft and isotropic group serves as controls. In this study, 5 x 10^4 of HUVEC and HCMEC were cultured on each scaffold group (~1cm x 1cm) for 72 hr. Cell viability and adhesion were measured by Alama blue assay. Next, a tubulogenesis assay was performed. Approximately 3 x 10^4 of HUVEC cells were plated on the Geltrex and incubated with the condition medium collected from the cells in various scaffold groups. Neovessel formation was measured with bright field microscopy. Student t test was used to compare the data from two cell lines within each scaffold group. For each cell line, a one-way ANOVA with Tuckey's post hoc test was performed to compare the impact of matrix mechanics on the ECs responses.

Results, Conclusions, and Discussions: HCMECs exhibit superior growth and metabolic activity compared to the HUVEC cells from the same scaffold group (p < 0.05). This indicates that the cardiac originated ECs grow better than venous ECs in the RV-like mechanical conditions.
Next, we used cell viability assay to measure the proliferation of ECs (Fig.1). We found that in the HUVEC cells, under the same type of anisotropy, the stiff scaffold resulted in less ECs than the soft scaffold, indicating a suppressive effect of stiffness on EC growth. In the stiff scaffolds, the anisotropic group had enhanced EC proliferation than the isotropic group, indicating a beneficial effect of anisotropy on EC growth. But this trend was absent in the soft scaffolds. In the HCMEC cells, we observed similar behavior as HUVEC cells, but the effects were more significant in this cell line (with smaller p values).
Then, we examined how cell adhesion was affected by substrate mechanics (Fig.2). In the HUVEC cells, we did not observe an impact of stiffness in either isotropic or anisotropic groups. In the stiff scaffolds, we saw enhanced cell adhesion in the anisotropic group. In the HCMEC cells, in isotropic scaffolds, the stiff group had reduced cell adhesion than soft group, indicating a suppressive effect of stiffness. Moreover, in either soft or stiff scaffolds, the presence of anisotropy enhanced the cell adhesion, indicating the beneficial effect of anisotropy on EC function.
Finally, we examined the mechanical control of angiogenic potential by tubulogenesis assay (Fig.3). Overall, in soft or stiff scaffolds, the anisotropic group showed enhanced tube formation than the isotropic group, indicating a beneficial effect of anisotropy on EC angiogenic potential. Moreover, in isotropic or anisotropic scaffolds, the soft group resulted in more neovessel formation than the stiff group. This is consistent with observations of suppressive effect of stiffness in cell growth and adhesion.
This study presents original findings that larger stiffness reduced EC growth and angiogenic potential and the substrate anisotropy elicited the opposite (beneficial) effect. Our findings strongly suggest that the myocardium mechanical properties should be taken into consideration in the design of regenerative therapies to improve microvascular circulation.

Acknowledgements (Optional): We thank Sam Adhikari for assisting to quantify the cells from microscopic images.