(32b) Deciphering the Molecular Mechanisms of Immune Mediated Remodeling of Arterial Grafts

Objective: We developed cell-free (Acellular) Tissue Engineered Vessels (ATEVs) that were functionalized with a novel protein (H2R5) that served as vascular conduits for arterial replacement therapy. H2R5-ATEVs facilitate infiltration and capture of myeloid precursor cells under flow, which drive endothelialization and vascular wall formation, similar to native artery. However, the molecular mechanisms underlying monocyte/macrophage (MC/MΦ) driven vascular regeneration under shear stress (SS) has not been explored.

Methods: H2R5-ATEVs were implanted in the inferior abdominal aorta of CX3CR1-Confetti mice model and patency was investigated using doppler ultrasound for 4 weeks. The ATEVs explanted after 4 weeks were investigated for the presence of EC and MΦ cells by immunostaining for eNOS and CD14 respectively. To recapitulate the effect of vascular blood flow on the infiltrating MC/MΦ populating the grafts, in vitro, human THP1 cells, grown in RPMI medium containing PMA (phorbol 12-myristate 13-acetate) were subjected to fluid SS (FSS) using the IBIDI pump system at 10dyn/cm² for 24 h. The metabolic flux of the cells under SS and levels of mitochondrial oxygen consumption rate (OCR) and Extracellular Acidification Rate (ECAR) as a measure of glycolysis were determined using the Agilent Seahorse system. SS induced lipid accumulation and mitochondrial membrane potential (Ψ_m) were quantitated using BODIPY AM and TMRM staining and confocal live cell imaging. Store operated Ca²⁺ entry, and Ca²⁺ flickering, and intracellular Ca²⁺ dynamics were investigated under Yoda-1 activation of the mechanosensitive channel, Piezo1 and SS mediated activation of cell surface mechanotransducers. THP1 cells treated with Yoda1 (10μM) and those subjected to SS were stained with Fluo4-AM and the levels of cytoplasmic (c)Ca²⁺ entry and calcium oscillations and transients were determined using a fluorescence microscopy. Store operated Ca²⁺ entry (SOCE) was determined by blocking Ca²⁺ entry to ER by Thapsigargin (Tg) stimulation followed by CaCl₂ (10mM) stimulation. The stained cells were imaged using Leica Stellaris Confocal imaging and the data were analyzed using LASX software and the data were plotted using GraphPad Prism v.10.

Results and Conclusion: H2R5 ATEVs exhibited patency for 4 weeks as evidenced by complete remodeling of the neoartery populated by MC/MΦ arriving at the grafts. Endothelialization and vascular wall formation of the grafts were demonstrated by positive expression for EC markers, e.g., eNOS, and smooth muscle cell (SMC) markers (MYH11). Notably, these cells also co-expressed MC/MΦ proteins, such as CD14, in agreement with previous results from our lab. To further delineate the mechanisms behind this MC/MΦ mediated remodeling of the ATEVs, in vitro, we subjected human THP1 cells to SS to investigate MΦ polarization, and differentiation.

Effect of FSS on Mitochondrial Bioenergetics: To study the metabolic status and mitochondrial function, we subjected THP1 cells to SS (10dyn/cm² for 24h) and then quantified the levels of OCR and ECAR using the Seahorse metabolic analyzer. SS significantly reduced OCR, as evidenced by shunted basal and maximal respiration compared to M1 (LPS (100 ng/ml) + IFNγ (20 ng/ml) and M2 (IL4 (20 ng/ml) +IL13 (20 ng/ml) polarized MΦ under static conditions. The naive M0 under SS also exhibited elevated ECAR levels that were higher than M1 and M2 controls, indicating elevated glycolysis under SS. These observations suggest that MΦ under SS undergo metabolic reprogramming, tuning into M1-like polarization phenotype. We reasoned that SS mediated loss of oxidative phosphorylation and mitochondrial dysfunction could be due to impaired intracellular calcium dynamics and mitochondrial mobilization of Ca²⁺.

Simultaneous imaging of Mitochondrial Membrane potential and Lipid uptake: Confocal live cell imaging, using the membrane potential indicator, tetramethylrhodamine methyl ester (TMRM), demonstrated that THP1 cells under SS showed elevated Ψ_m, indicating a hyperpolarized state. Remarkably, THP1 cells under SS stained with the lipid marker, BODIPY AM, exhibited increased intracellular accumulation of lipid droplets. The increased lipid accumulation could be attributed to increased lipid uptake due to activation of cell surface mechanotransducers, and/or reduced clearance of intracellular lipids due to defective mitochondrial function.

Pharmacological stimulation of SS exacerbates intracellular Ca²⁺ dynamics: Activation of cell surface mechanotransducers, such as Piezo1 and through mechanical forces are established physiological cues that trigger Ca²⁺ transients in activated vascular cell types. To simulate the effect of SS on intracellular Ca²⁺ dynamics, and to activate the mechanosensitive Ca²⁺ transporter, Piezo1, THP1 cells were treated with the Yoda1 (10 uM). After 24h, the cells were stained with the fluorophore, Fluo-4 AM, that binds specifically to cytosolic Ca²⁺. Fluorescence imaging using Zeiss microscope revealed that THP1 cells treated with Yoda1 exhibited increased basal Fluo4 fluorescence, increased cytosolic Ca²⁺ oscillations and flickering. The increased Ca²⁺ oscillations could be reasoned to an intact, activated cell surface, intracellular (ER) and mitochondrial Ca²⁺ uptake component activity.

SS affects intracellular Ca²⁺ dynamics and blunts mitochondrial Ca²⁺ uptake: THP1 cells were subjected to SS and 24h later, they were stained with the fluorophore, Fluo-4 AM, that binds specifically to cytosolic Ca²⁺. Fluorescence imaging revealed that physiological activation of THP1 cells by SS resulted in elevated cytosolic Ca²⁺ upon Ca²⁺ stimulations. Strikingly, under SS, THP1 cells exhibited significantly reduced Ca²⁺ oscillations and flickering compared to static controls. We hypothesized that the elevated cytosolic Ca²⁺ under SS is due to increased cytosolic Ca²⁺ uptake, whereas the reduced Ca²⁺ oscillations could be due impaired mitochondrial uptake. To this end, we are investigating intracellular redistribution of the Ca²⁺ influx into the cells using genetic calcium sensors (GCaMP). In addition, we found that SS drives MΦ to a pro-inflammatory state as evidenced by expression of M1 genes and pro-inflammatory cytokines. Taken together, these observations, illustrate that MΦ under SS undergo metabolic reprogramming, driven by aberrant intracellular (i)Ca²⁺ dynamics. In turn this leads to secretion of cytokines, e.g., MMP1 that may serve to recruit more MC/MΦ to the luminal surface of the grafts and eventually vascular cells, leading to vascular regeneration. Our study thus delineates the role of SS and mitochondrial metabolism in MΦ-driven regeneration of ATEVs and may have significant implications in vascular graft design and applications in patients with comorbidities.