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Lymphatic Malformations With Activating KRAS Mutations Impair Lymphatic Valve Development Through Matrix

Diandra M Mastrogiacomo¹, Abbigail Price¹, Yuting Fu¹

¹Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa (D.M.M., A.P., Y.F., R.B., L.A.K., K.L., Y.Y., G.E.D., J.P.S.).

Arteriosclerosis, Thrombosis, and Vascular Biology

|March 26, 2026

Abstract

Background:

Lymphatic malformations are lesions that can be due to inherited or somatic mutations, and they lead to a defective lymphatic vasculature. Activating KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations have been identified recently in patients with lymphatic malformation with lymphedema, chylous ascites, or life-threatening chylothorax. In a lymphatic malformation 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.

Methods:

To investigate the mechanisms leading to valve loss, we combined the lymphatic-specific and tamoxifen-inducible Flt4CreER^T2 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 expressing KRAS-G12D were probed for changes in mRNA and protein expression with quantitative real-time polymerase chain reaction, Western blot, and gel zymography, and mechanistic studies were performed using 3-dimensional cell culture in collagen matrices.

Results:

Our data showed that lymphatic-specific expression of KRAS-G12D significantly attenuated valve development in the mesentery, diaphragm, and ear skin. Quantitative real-time polymerase chain reaction, Western blot, and gel zymography using human dermal lymphatic endothelial cells expressing KRAS-G12D revealed the upregulation of the PA (plasminogen activator) pathway and MMPs (matrix metalloproteinases). The MMPs were sufficiently activated by plasmin, the product of the PA pathway, in human dermal lymphatic endothelial cells grown in a 3-dimensional collagen matrix, indicating a role for MMPs in the degradation of valve ECM (extracellular matrix) core. Furthermore, a broad-spectrum MMP inhibitor given to Flt4CreER^T2;Kras^+/G12D mice rescued lymphatic valve development.

Conclusions:

We 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.

Insights

Activating KRAS mutations cause lymphatic valve loss by upregulating matrix metalloproteinases (MMPs) that degrade the valve's extracellular matrix. Inhibiting MMPs rescues lymphatic valve development, revealing a key mechanism in lymphatic malformations.

Area of Science:

Vascular biology
Molecular mechanisms of disease
Lymphatic system development

Background:

Lymphatic malformations result from genetic mutations affecting lymphatic vasculature.
Activating KRAS mutations are linked to lymphatic malformations and associated conditions like lymphedema and chylothorax.
The precise mechanisms causing lymphatic valve loss in these conditions remain unclear.

Purpose of the Study:

To elucidate the molecular mechanisms underlying lymphatic valve loss in the context of KRAS mutations.
To investigate the role of specific signaling pathways and enzymes in lymphatic valve development and degradation.

Main Methods:

Utilized a mouse model with lymphatic-specific, inducible KRAS-G12D expression and a Prox1GFP reporter.
Analyzed human dermal lymphatic endothelial cells (HDLECs) with KRAS-G12D expression using qPCR, Western blot, and gel zymography.
Performed mechanistic studies in 3D collagen matrices and tested MMP inhibitors in vivo.

Main Results:

Lymphatic-specific KRAS-G12D expression attenuated lymphatic valve development in mice.
KRAS-G12D induced upregulation of the plasminogen activator (PA) pathway and matrix metalloproteinases (MMPs) in HDLECs.
MMPs, activated by plasmin, degraded the extracellular matrix (ECM) core of lymphatic valves, and MMP inhibition rescued valve formation.

Conclusions:

Hyperactive KRAS signaling promotes lymphatic valve loss through MMP-mediated ECM degradation.
The PA pathway plays a crucial role in activating MMPs involved in valve development.
Targeting MMPs represents a potential therapeutic strategy for lymphatic malformations.

Read full analysis

Keywords:

endothelial cells lymphatic vessels mitral valve prevalence subclavian vein

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