02685nas a2200205   4500000000100000008004100001260001500042100001600057700001700073700001700090700002700107700001800134700001800152700001500170245006400185856005300249300001600302490000800318520215300326       2026                            d  c2026-07-141 aSina Kheiri1 aJessica Shah1 aPeiyuan Chai1 aShashaank A. Venkatesh1 aRyan A. Flynn1 aRoger D. Kamm1 aRitu Raman00a4D force patterning enables spatial control of angiogenesis  uhttps://www.pnas.org/doi/10.1073/pnas.2532667123  ae25326671230 v1233 aEngineering organized microvascular networks remains a critical challenge in tissue engineering and regenerative medicine. While biochemical approaches for patterning angiogenesis via growth factor delivery have shown promise, their inability to pattern sustained growth factors with spatiotemporal control limits effectiveness. Here, we demonstrate that dynamically patterned mechanical forces enable precise spatiotemporal control over angiogenic sprouting. We developed a magnetically actuated human vessel-on-a-chip platform that integrates a perfusable endothelialized microchannel within a collagen matrix and allows noninvasive and tunable mechanical stimulation across three spatial dimensions and time (4D). Using an automated 3-axis actuator, we systematically investigated how strain magnitude, frequency, and direction modulate endothelial cell behavior and vessel morphogenesis. Dynamic mechanical stimulation at physiological strain magnitudes (5 to 15%) enhanced endothelial alignment and barrier function while promoting angiogenesis in a strain magnitude–dependent manner: lower dynamic strain (5%) maximized sprout initiation, whereas higher dynamic strain (15%) promoted elongation of sprouts. Sequential reorientation of strain direction reprogrammed sprouting trajectories along X, Y, and Z directions, generating complex sprout geometries such as L-shaped branches. RNA sequencing revealed mechanically induced transcriptional profiles distinct from unstimulated controls, characterized by upregulation of genes associated with angiogenesis, mechanotransduction, and extracellular matrix remodeling. Functional perturbation of PIEZO1 reduced strain-induced sprouting without altering barrier function, indicating that dynamic mechanical stimulation engages multiple mechanotransduction pathways to regulate angiogenesis. Collectively, these findings establish a strategy for spatiotemporally controlled angiogenesis through 4D force patterning to program vascular morphogenesis while preserving function. This approach provides a foundation for engineering hierarchically organized vascular networks for tissue regeneration.