(H-320) Holotomographic microscopy - a platform to investigate the role of the adhesome in endothelial cell metabolism

Endothelial cell (EC) metabolism and mechanotransduction, including their ability to sense shear stress, are crucial factors determining their ability to endothelialize biomaterials for synthetic vascular grafts. EC alignment and phenotype are strongly influenced by the type of luminal fluid flow and therefore shear stress that they sense. Unidirectional laminar flow with shear stresses between approximately 0.5 dyne/cm2 and 20 dyne/cm2 promote favorable EC alignment, homeostasis, and reduce the risk of vascular pathologies such as neointimal hyperplasia1. Such alignment favors the activation of β1 integrins at focal adhesion, cellular junctions and luminal surface, indicating good cell attachment and flow.

Integrins, acting as basal EC mechanosensors, converts mechanical forces into biochemical signals regulating cell responses and are involved in metabolic processes, including glycolysis, in a shear-dependent maner2,3. Holotomographic imaging is a new imaging technique that allows us to visualize the refractive index of each voxel in a sample of live ECs. This powerful tool enables us to study intracellular components of the ECs such as mitochondria and lipids, as well as integrins involved in endothelial cell mechanotransduction under luid shear stresses.

During my undergraduate research, I established a platform to verify the presence of β1 integrin in endothelial cells at specific sites, confirming EC attachment. Building upon this platform, I aim to further explore the effects of shear stress on endothelial cell metabolism and the adhesome. By leveraging these new tools, we hope to gain a deeper understanding of the interplay between shear stress, metabolism, and integrin-related processes in endothelial cells.

Human Umbilical Veins Endothelial Cells (HUVECs) were thawed, plated, and passaged using DMEM Media, PBS, TrypLe and 5% FBS in PBS. HUVECs were cultured and passaged three times to P5 before imaging. 

Fluorescence of β1 integrin was achieved by fixing the cells with 4% Formaldehyde solution in PBS and rinsed with Wash Solution containing Triton X100. Blocking buffer composed of 5% BSA in PBS and 0.05% Tween 20 was applied. β1 integrin antibody was diluted in a buffer for 10e7 cells / 100μL. For labeling, CD29 + FITC (Integrin beta 1) Monoclonal Antibody (TS2/16), PE, eBioscience™ was used. Antibody dilution buffer was made with 30μL Triton X100, 10mL PBS, and 0.1g BSA. Cells were washed and passaged with PBS containing Ca and Mg, and treated with formaldehyde for 30 minutes at room temperature. HUVECs underwent then three rounds of PBS washes for 5min. For permeabilization, the cells were washed with washing buffer for 20 minutes. Another 3 - 5-minute PBS washes was performed before blocking them for 60 minutes with blocking buffer. Lastly, cells were rinsed with PBS for 5min, 20μL of CD29 Antibody was added, and cells sit overnight at 4°C.

Imaging was performed using two different microscopes, the Andor Dragonfly spinning disk confocal microscope and the Holographic Microscope CX-F from Nanolive. Imaging using the confocal microscope was performed by setting and staining HUVEC cells on a glass cover slide. Holotomographic images were taken label-free using live cells cultured on a 30 mm Ibidi dish with a glass bottom.

HUVECs images using: (A) Holotomographic Microscopy or (B) Confocal Microscopy. Important organelles and subcellular components are also labeled: (i) Mitochondria (ii) Lipid vesicles (iii) Lamellipodia (iv) β1 integrin fluorescently stained in red, nuclei in blue and F-actin in green.

Figure 1A is an image obtained through holotomographic microscopy showing distinct organelle structures like mitochondria (i) and subcellular components of ECs like lipids (ii) and lamellipodia (iii). Figure 1B shows HUVECs labeled with fluorescent markers and imaged using confocal microscopy, with the red fluorescence representing β1 integrin staining. By employing holotomographic and spinning disk confocal microscopies, these images provide valuable insights into the ECs’ structure.

The confocal microscope images of HUVEC cells and their components confirmed the success of our immunofluorescence protocol. We targeted β1 integrins using a monoclonal antibody labeled with a FITC fluorophore, and validated their presence and their role in sensing the surroundings and HUVECs movements. Figure (A) represents images of live HUVECs cells and their subcellular components which are difficult to image using standard fluorescence and brightfield microscopy. These label-free images obtained with the CXF Nanolive Holotomographic microscope demonstrated our ability to capture the mitochondrial network and lipids in real-time and will enable us to further investigate EC’s mechanotransduction through integrins. We will use the holotomographic microscope's powerful imaging and fluorescence capabilities to colocalize β1 integrins with subcellular components of the ECs’ adhesome under flow conditions. Doing so, we hope to gain a deeper understanding of how endothelial cell mechanotransduction through integrins, combined with shear stress in flow, influences their metabolism during endothelialization. 

The initial results provided insights into integrins role in ECs and demonstrated that the CX-F Nanolive Holotomographic microscope is a valuable and unique tool for studying the relationship between integrins and EC’s adhesome. Moving forward, we will focus on imaging β1 integrins under unidirectional flow and laminar shear stress conditions to validate their mechanotransduction function and their role in ECs' adhesome and metabolism. This comprehensive approach will advance our knowledge of the complex interactions in endothelialization process and potentially lead to significant advancements in the development of improved graft materials for Coronary Artery Disease.

1. 
   

   Brian G. Coon. et al A mitochondrial contribution to anti-inflammatory shear stress signaling in vascular endothelial cells. J Cell Biol (2022)

   
   

2. 
   

   Peng, H. et al. Metabolic Reprogramming of Vascular Endothelial Cells: Basic Research and Clinical Applications. Frontiers in Cell and Developmental Biology 9, (2021).

   
   

3. 
   

   Wang, L. et al. Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of unidirectional shear flow. Nature 540, 579–582 (2016).