Knauer, L. A. | bioRxiv Science

bioRxiv2025

Lymphatic Malformations with Activating KRAS Mutations Impair Lymphatic Valve Development Through Matrix Metalloproteinases

BACKGROUNDLymphatic malformations (LMs) are lesions due to inherited or somatic mutations that lead to a defective lymphatic vasculature. Activating KRAS mutations have been identified recently in LM patients with lymphedema, chylous ascites, or life-threatening chylothorax. In a LM mouse model, KRAS mutations are associated with a loss of lymphatic valves, which has been proposed to cause chylothorax via retrograde lymph flow into the pleural space. However, the mechanisms underlying the loss of lymphatic valves are unknown. METHODSTo investigate the mechanisms leading to valve loss, we combined the lymphatic-specific and tamoxifen-inducible Flt4CreERT2 with Kras-loxP-stop-loxP-G12D (Kras+/G12D) mice and Prox1GFP reporter mice to induce the restricted expression of KRAS-G12D and enable valve quantification in postnatal pups. Human dermal lymphatic endothelial cells (hdLECs) expressing KRAS-G12D were probed for changes in mRNA and protein expression with qRT-PCR, western blot, and gel zymography, and mechanistic studies were performed using 3D cell culture in collagen matrices. RESULTSOur data showed that lymphatic-specific expression of KRAS-G12D significantly attenuated valve development in the mesentery, diaphragm, and ear skin. qRT-PCR, western blot, and gel zymography using hdLECs expressing KRAS-G12D revealed the upregulation of the plasminogen activator (PA) pathway and matrix metalloproteinases (MMPs). The MMPs were sufficiently activated by plasmin, the product of the PA pathway, in hdLECs grown in a 3D collagen matrix, indicating a role for MMPs in the degradation of valve ECM core. Furthermore, a broad-spectrum MMP inhibitor given to Flt4CreERT2;Kras+/G12D mice rescued lymphatic valve development. CONCLUSIONSWe conclude that hyperactive KRAS signaling upregulates MMPs that become excessively activated by the upregulation of the PA pathway. MMPs then degrade the lymphatic valve ECM core preventing valve formation.

pathology↗

bioRxiv2023

Rictor induces AKT signaling to regulate lymphatic valve formation

Lymphatic valves are specialized structures of the collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress (OSS) from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves throughout life. Conventionally, in other tissue types, AKT activation requires dual kinase activity and the mammalian target of rapamycin complex 2 (mTORC2) commands this process by phosphorylating AKT at Ser473. Here we showed that embryonic and postnatal lymphatic deletion of Rictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human lymphatic endothelial cells (hdLECs) not only significantly reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions, but also abolished the upregulation of AKT activity and valve-forming genes in response to flow. We further showed that the AKT target, FOXO1, a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric LECs, in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in both mesenteric and ear lymphatics. Our work revealed a novel role of RICTOR signaling in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately allows the formation and maintenance of a normal lymphatic valve.

developmental biology↗