FAK- and TGF-β1-dependent biomechanical alterations in microvascular endothelial cells exposed to spatial fluid shear stress gradients

Technical Summary: FAK- and TGF-β1-dependent biomechanical alterations in microvascular endothelial cells exposed to spatial fluid shear stress gradients

Problem Statement
Endothelial cells (ECs) form a selective barrier regulating vascular permeability and are highly sensitive to mechanical cues, particularly shear stress (SS) from blood flow. While macrovascular ECs experience relatively uniform SS, microvascular endothelial cells (μECs) in capillary networks are exposed to highly heterogeneous flow environments characterized by steep spatial shear stress gradients (SSG). These gradients arise naturally from vessel bifurcations or curvature and are exacerbated in pathologies like atherosclerosis. Despite their physiological relevance, the direct effects of SSG on μEC kinematics, mechanics, and signaling remain poorly understood compared to studies on uniform SS. Furthermore, it is unclear whether specific mechanotransduction pathways govern the migration direction (rheotaxis) and force generation of μECs in response to these gradients.

Methodology
The study utilized a custom-built impinging flow device compatible with long-term live-cell videomicroscopy to expose human dermal (HMEC-1) and human cerebral (hCMEC/D3) μECs to controlled SSG. The device generated an axisymmetric flow pattern with defined SS and SSG profiles, mimicking (patho)physiological levels (3 mL/min flow rate).

Key experimental approaches included:

• Kinematic Analysis: Long-term time-lapse imaging (up to 35 hours) to track cell migration speed, direction, and collective motion coordination (correlation length) using particle image velocimetry (PIV).
• Mechanical Measurements: Traction Force Microscopy (TFM) on soft (3 kPa) hydrogels to quantify cellular traction stresses and strain energy. Monolayer Stress Microscopy (MSM) was employed to calculate inter- and intra-cellular monolayer stresses.
• Pharmacological Perturbation: Cells were treated with specific inhibitors to dissect signaling pathways: PF-573228 (FAK inhibitor), Y-27632 (ROCK inhibitor), and SB-525334 (TGF-β type I receptor inhibitor). Recombinant TGF-β1 was also applied to assess its sufficiency.
• Transcriptomics: RNA sequencing (RNA-seq) was performed on HMEC-1 and hCMEC/D3 monolayers after 20 hours of flow exposure to identify differentially expressed genes (DEGs) and enriched pathways.
• Immunofluorescence: Staining for cytoskeletal components (F-actin, pMLC, vinculin) and proliferation markers (Ki-67) to validate mechanical and transcriptional findings.

Key Results

1. Migration Kinematics and Rheotaxis: Exposure to SSG caused an immediate and sustained two-fold reduction in μEC migration speed. Concurrently, cells exhibited enhanced coordination of collective motion. Crucially, both μEC types demonstrated "reverse rheotaxis," migrating collectively against the flow direction toward the jet center (regions of high SSG). This behavior was specific to SSG; constant uniform SS did not induce reverse rheotaxis.
2. Mechanical Memory: Upon flow cessation, migration speed partially recovered, but the cells retained a persistent directional bias (reverse rheotaxis) and elevated coordination for at least 10 hours. Similarly, traction stresses and monolayer stresses remained elevated post-flow, suggesting a form of mechanical memory or ECM remodeling.
3. Force Generation: Flow exposure triggered an immediate ~20% increase in traction stresses and strain energy, followed by a linear, time-dependent increase over 20 hours. Monolayer tensile stresses also increased significantly.
4. Transcriptional Reprogramming: RNA-seq revealed substantial transcriptional remodeling, with hCMEC/D3 showing greater sensitivity than HMEC-1. Key upregulated pathways included focal adhesion signaling, cytoskeletal regulation (PI3K-AKT, Hippo), and TGF-β1 signaling. Genes associated with ECM remodeling and TGF-β1 activation (e.g., THBS1, JUNB, PDGFB) were upregulated, while inflammatory pathways (e.g., NF-κB) were suppressed.
5. Mechanistic Dissection:
   • FAK: Inhibition of FAK abolished reverse rheotaxis, causing cells to migrate with the flow (forward rheotaxis), despite retaining strong traction forces. FAK was required for dynamic traction adhesion turnover and directional migration but not for the magnitude of traction forces.
   • ROCK: Inhibition of ROCK reduced traction force magnitude by ~10-fold and slowed migration speed but did not prevent reverse rheotaxis. Cells still migrated against the flow, albeit slower.
   • TGF-β1: Inhibition of TGF-β1 signaling abolished the time-dependent linear increase in traction stresses observed during flow but did not affect the acute initial increase or the direction of migration. Conversely, exogenous TGF-β1 application under static conditions was sufficient to drive progressive traction strengthening.

Significance and Claims
The authors claim that spatial shear stress gradients (SSG) are critical regulators of μEC biomechanics, distinct from uniform shear stress. The study establishes that:

• SSG induces a specific "reverse rheotactic" phenotype in μECs, characterized by migration against the flow and enhanced collective coordination.
• The directionality of this migration is strictly dependent on FAK-mediated focal adhesion dynamics, independent of the absolute magnitude of traction forces.
• The progressive strengthening of traction forces over time is driven by TGF-β1 signaling and actomyosin contractility but is mechanistically decoupled from the establishment of migration direction.
• μECs retain a "mechanical memory" of flow exposure, maintaining altered mechanics and migration patterns even after the stimulus is removed.

These findings provide a mechanistic framework for understanding how microvascular endothelium adapts to complex hemodynamic environments, with potential implications for understanding vascular homeostasis and disease progression in regions prone to SSG, such as vessel bifurcations. The study suggests that targeting FAK or TGF-β1 pathways could differentially modulate endothelial migration patterns versus mechanical strengthening.