Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation

Abstract

Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA -driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA -driven VMs.

PI3K-C2β limits mTORC1 signaling and angiogenic growth

“…Ultra-rare variants in PIK3C2B cause focal epilepsy in humans ( 43 ), and on the basis of our data, it is tempting to speculate that these patients also have a predisposition to develop vascular anomalies, perhaps in an epistatic context. This may be relevant to some patients with vascular malformations associated with high PI3K/mTORC1 signaling for which the genetic cause is unknown ( 4 , 44 , 45 ).…”

Engineering Organ-on-a-Chip Systems for Vascular Diseases

“…Membrane-separated channels, 29 crossconnected parallel channels 23,100 PDMS/glass, 23,100 PDMS/PET membrane 29 PAH-ECs, PAH-SMCs, PAH-ADCs, collagen, Matrigel, fibronectin, poly D-lysine 5 parallel channels separated via micropillars were utilized to create perivascular, adventitial, medial, intimal, and luminal layers of a pulmonary artery. 23,100 Vascular dysfunctions, inflammation, and malformation Branching channels, 35 cross-connected parallel channels, 38,40,101 microwell plate format, 62,102 straight channel 39 Agarose/gelatin, 35 collagen, [38][39][40] POMaC, 62 PDMS/ glass 101 HUVECs, HDMVECs, HLMVECs, iECs, iSMCs, HBVPCs, HUASMCs, HPMECs, pericytes, fibroblasts, AECs, fibrin, collagen IPN hydrogel channels mimicked vascular stiffness, permeability, and self-healing of the endothelial barrier. 35 Laser ablation created an array of capillary-sized channels within a collagen matrix.…”

“…39 Aligned patterning may have therapeutic benefit in preventing atherosclerotic lesion formation. 39 Furthermore, other vascular conditions such as ischemia-reperfusion, 31,32,76,78,[97][98][99] Hutchinson-Gilford progeria syndrome, 117 vascular malformation, 101 and pulmonary arterial hypertension injury 23,29,100 have been modeled on a chip. For example, in a microfluidic design, microgrooves connecting parallel channels enabled neuronal segregation and axonal growth for modeling central nervous system injury-ischemia.…”

“…32 The elevated shear stress and disturbed blood flow, occurring predominantly at arterial bifurcations and branching points, were stimulated by adjusting the geometric complexity and hierarchical microstructure of microchannels, applying additional cyclic tensile and compressive forces, as well as precise control over the flow of liquids within the chips. 76,101,118,119 The benefits of adjustable parameters within these devices enable the simulation of blood-related disorders, such as vascular malformation and pulmonary arterial hypertension. 120 For instance, to study pulmonary arterial hypertension, parallel channels separated via micropillars were utilized to create perivascular, adventitial, medial, intimal, and luminal layers of a pulmonary artery.…”

“…34,36,64 Advanced approaches incorporate confocal microscopy with Z-stack imaging, delivering detailed insights into cell morphologies and surface protein distributions, particularly valuable when conducting 3D and multilayered cell culture within microfluidic setups. 36,69,101,102,104 Beyond optical microscopy, additional imaging methods like SEM and histology can be used for comprehensive analysis. 22,41,42,85,90 Furthermore, cells and media retrieved from the device enable further investigations such as RNA sequencing, secretomics, and metabolomics 16,20,78 Snapshot Imaging Snapshot imaging in OOC systems corresponds to capturing a single moment in time for in-depth analysis.…”