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Valve Interstitial Cells Act in a Pericyte Manner Promoting Angiogensis and Invasion by Valve Endothelial Cells

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Abstract

Neovascularization is an understudied aspect of calcific aortic valve disease (CAVD). Within diseased valves, cells along the neovessels’ periphery stain for pericyte markers, but it is unclear whether valvular interstitial cells (VICs) can demonstrate a pericyte-like phenotype. This investigation examined the perivascular potential of VICs to regulate valve endothelial cell (VEC) organization and explored the role of Angiopoeitin1-Tie2 signaling in this process. Porcine VECs and VICs were fluorescently tracked and co-cultured in Matrigel over 7 days. VICs regulated early VEC network organization in a ROCK-dependent manner, then guided later VEC network contraction through chemoattraction. Unlike vascular control cells, the valve cell cultures ultimately formed invasive spheroids with 3D angiogenic-like sprouts. VECs co-cultured with VICs displayed significantly more invasion than VECs alone; with VICs generally leading and wrapping around VEC invasive sprouts. Lastly, Angiopoietin1-Tie2 signaling was found to regulate valve cell organization during VEC/VIC spheroid formation and invasion. VICs demonstrated pericyte-like behaviors toward VECs throughout sustained co-culture. The change from a vasculogenic network to an invasive sprouting spheroid suggests that both cell types undergo phenotypic changes during long-term culture in the model angiogenic environment. Valve cells organizing into spheroids and undergoing 3D invasion of Matrigel demonstrated several typical angiogenic-like phenotypes dependent on basal levels of Angiopoeitin1-Tie2 signaling and ROCK activation. These results suggest that the ectopic sustained angiogenic environment during the early stages of valve disease promotes organized activity by both VECs and VICs, contributing to neovessel formation and the progression of CAVD.

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Abbreviations

AKT:

RAC-alpha serine/threonine-protein kinase

Ang1:

Angiopoetin1

CAVD:

Calcific aortic valve disease

DLL4:

Delta like ligand four

EndMT:

Endothelial to mesenchymal transformation

LaFMI:

Lagrangian corrected forward migration index

MCEC:

Mouse cardiac endothelial cells

NO:

Nitrous oxide

ROCK:

Rho-associated protein kinase

VEC:

Valve endothelial cell

VIC:

Valvular interstitial cell

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Acknowledgments

The authors thank Jennifer Connell, PhD, for editorial assistance and Cindy Farach-Carson, PhD, for use of her microscopy equipment.

Funding

Funding was provided by AHA 11IRG5550052 and an NSF Graduate Research Fellowship to C.A.A.

Conflict of interest

The authors have nothing to disclose.

Author information

Correspondence to K. Jane Grande-Allen.

Additional information

Associate Editor Aleksander S. Popel oversaw the review of this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Figure S2: Early network formation is seen in VEC (green)/VIC (red) co-culture in the first 4 hours after seeding on Matrigel. Images were taken from 1 to 5.5 hours after seeding. Scale bar represents 100 µm. (WMV 5968 kb)

Figure S3: VEC (green)/VIC (red) are seen during network contraction into spheroids during the time period 14-19 hours after seeding onto Matrigel. During this time period, a subpopulation of VICs can be appreciated for influencing VECs organization during network collapse. Scale bar represents 100 µm. (WMV 4042 kb)

Figure S4: VEC (green)/VIC (red) collapse into spheroids by 24 hours after seeding onto Matrigel. Images were taken from 24 to 28 hours post seeding. Scale bar represents 100 µm. (WMV 3785 kb)

Figure S7: 140 hours after seeding, VECs(green) and VICs organize into large, complex, highly branched, interconnected, and invasive sprouts from the core of the spheroid. It is noteworthy the dynamic movement of the VECs along the sprouts and inside the spheroid. Scale bar represents 100 µm. (MP4 3290 kb)

Figure S8: Higher magnification of the tip of the sprouts from supplemental Figure 7. Further appreciation of the dynamic movement of the VECs (red) and VICs along the sprout is possible in this video as different cells take turns taking the role of tip or stalk cell as is seen in vascular angiogenesis. Scale bar represents 100 µm. (MP4 6400 kb)

Figure S9: An example of a VIC (red) demonstrating a LaFMI > 0.17, a chemoattractive effect on local VECs (green), during early network reorganization. Each cell's tracking tracing demonstrates the variability in the directionality of each local VEC that were or were not affected by the VIC in focus. Images were taken from 14 to 19 hours post seeding. Scale bar represents 25 µm. (WMV 942 kb)

Figure S11: 3D panoramic view along the y-axis of the z-stack reconstructed image of aSMA staining from Figure 5B. Most cells in VEVISs displayed low levels of unpolarized aSMA as opposed to the polarized aSMA found in VICs but not in CD31+ VECs during the network formation at earlier time points (Figure 1C). aSMA (red). F-actin (green). DAPI (blue). (WMV 7942 kb)

Figure S12: 3D panoramic view along the y-axis of the z-stack reconstructed image of DLL4 staining from Figure 5E. VEVIS sprouts displayed DLL4+ polarization from tip to stalk similar to what is found during vascular angiogenesis. DLL4 (green). F-actin (red). DAPI (blue). (WMV 3895 kb)

Figure S1: In order to compare the longer term co-culture of VICs and VECs to a known endothelial-pericyte co-culture, MCECs and 10T1/2 cells (to serve as pericytes) were co-cultured on Matrigel. A) Similar to previous reports of vascular endothelial/pericyte co-cultures26, the MCEC (green) and 10T1/2 (red) cells formed interwoven vasculogenic networks. Scale bar represents 100 µm. B) Quantification of MCEC/10T1/2 network size over time. MCEC-only cultures formed nascent networks at 7 hours, with a structure similar to VECs alone23. By 24 hours, the MCEC networks had consolidated with smooth node-to-tubule transitions. By 48 hours, the MCEC-only network structure had regressed substantially. Co-culturing with the 10T1/2 cells maintained network stability over this time period as demonstrated by the slowed network size decrease over time. * represents p-value <0.05. (PDF 4131 kb)

Figure S2: Early network formation is seen in VEC (green)/VIC (red) co-culture in the first 4 hours after seeding on Matrigel. Images were taken from 1 to 5.5 hours after seeding. Scale bar represents 100 µm. (WMV 5968 kb)

Figure S3: VEC (green)/VIC (red) are seen during network contraction into spheroids during the time period 14-19 hours after seeding onto Matrigel. During this time period, a subpopulation of VICs can be appreciated for influencing VECs organization during network collapse. Scale bar represents 100 µm. (WMV 4042 kb)

Figure S4: VEC (green)/VIC (red) collapse into spheroids by 24 hours after seeding onto Matrigel. Images were taken from 24 to 28 hours post seeding. Scale bar represents 100 µm. (WMV 3785 kb)

Figure S5: Initiation of angiogenic-like sprouts from VEVIS core 30 to 36 hours post seeding. As shown in the movie, VICs (red) are the first cell type to change into an invasive phenotype and begin sprouting into the Matrigel. VECs (green) can be appreciated to begin following behind the leading VICs. Scale bar represents 25 µm. (MP4 742 kb)

Figure S6: Dynamic sprouts are seen emerging from a VEVIS from 32 to 38 hours after seeding. During this time period, invading VECs (green) following VICs (red) begins to become appreciated. Scale bar represents 100 µm. (MP4 1289 kb)

Figure S7: 140 hours after seeding, VECs(green) and VICs organize into large, complex, highly branched, interconnected, and invasive sprouts from the core of the spheroid. It is noteworthy the dynamic movement of the VECs along the sprouts and inside the spheroid. Scale bar represents 100 µm. (MP4 3290 kb)

Figure S8: Higher magnification of the tip of the sprouts from supplemental Figure 7. Further appreciation of the dynamic movement of the VECs (red) and VICs along the sprout is possible in this video as different cells take turns taking the role of tip or stalk cell as is seen in vascular angiogenesis. Scale bar represents 100 µm. (MP4 6400 kb)

Figure S9: An example of a VIC (red) demonstrating a LaFMI > 0.17, a chemoattractive effect on local VECs (green), during early network reorganization. Each cell's tracking tracing demonstrates the variability in the directionality of each local VEC that were or were not affected by the VIC in focus. Images were taken from 14 to 19 hours post seeding. Scale bar represents 25 µm. (WMV 942 kb)

Figure S10: After 140 hours in culture, VEVIS sprouts formed complex geometries spreading in 3D. The continued dynamic plasticity of the sprout edge after 7 days of co-culture is demonstrated. Note how VICs (red) can be appreciated wrapping around and sustaining the VEC sprout while other VICs can be found on the leading edge of sprouts. VECs can also be found on the leading edge of sprouts at this time period. Scale bar represent 25 µm. (MP4 2067 kb)

Figure S11: 3D panoramic view along the y-axis of the z-stack reconstructed image of aSMA staining from Figure 5B. Most cells in VEVISs displayed low levels of unpolarized aSMA as opposed to the polarized aSMA found in VICs but not in CD31+ VECs during the network formation at earlier time points (Figure 1C). aSMA (red). F-actin (green). DAPI (blue). (WMV 7942 kb)

Figure S12: 3D panoramic view along the y-axis of the z-stack reconstructed image of DLL4 staining from Figure 5E. VEVIS sprouts displayed DLL4+ polarization from tip to stalk similar to what is found during vascular angiogenesis. DLL4 (green). F-actin (red). DAPI (blue). (WMV 3895 kb)

Figure S13: Representative images of VEC-only spheroid formation over 7 days in culture. Compared to the VEVIS co-cultures, the VEC networks slowly receded over 48 hours in culture. By 7 days in culture, VEC-only spheroids sparsely displayed limited sprouting. (PDF 2540 kb)

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Arevalos, C.A., Berg, J.M., Nguyen, J.M.V. et al. Valve Interstitial Cells Act in a Pericyte Manner Promoting Angiogensis and Invasion by Valve Endothelial Cells. Ann Biomed Eng 44, 2707–2723 (2016). https://doi.org/10.1007/s10439-016-1567-9

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Keywords

  • Aortic valve
  • Angiogenesis
  • CAVD
  • Angiopoietin
  • Valve endothelial cell
  • Valve
  • Interstitial cell