Scallan, J. P. | Search bioRxiv

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↗

bioRxiv2020

Ex vivo Demonstration of Functional Deficiencies in Popliteal Lymphatic Vessels from TNF-Tg Mice with Inflammatory Arthritis

BackgroundRheumatoid arthritis (RA) is a progressive immune-mediated inflammatory disease characterized by intermittent episodes of pain and inflammation in affected joints, or flares. Recent studies demonstrated lymphangiogenesis and expansion of draining lymph nodes during chronic inflammatory arthritis, and lymphatic dysfunction associated with collapse of draining lymph nodes in RA patients and TNF-transgenic (TNF-Tg) mice experiencing arthritic flare. As the intrinsic differences between lymphatic vessels afferent to healthy, expanding, and collapsed draining lymph nodes are unknown, we characterized the ex vivo behavior of popliteal lymphatic vessels (PLVs) from WT and TNF-Tg mice. We also interrogated the mechanisms of lymphatic dysfunction through inhibition of nitric oxide synthase (NOS). MethodsPopliteal lymph nodes (PLNs) in TNF-Tg mice were phenotyped as Expanding or Collapsed by in vivo ultrasound and age-matched to WT littermate controls. The PLVs were harvested and cannulated for ex vivo functional analysis over a relatively wide range of hydrostatic pressures (0.5 to 10 cmH2O) to quantify the end diastolic diameter (EDD), tone, amplitude (AMP), ejection fraction (EF), contraction frequency (FREQ) and fractional pump flow (FPF) with or without NOS inhibitors Data was analyzed using repeated measures two-way ANOVA with Bonferronis post hoc test. ResultsReal time videos of the cannulated PLVs demonstrated the predicted phenotypes of robust versus weak contractions of the WT versus TNF-Tg PLV, respectively. Quantitative analyses confirmed that TNF-Tg PLVs had significantly decreased AMP, EF and FPF versus WT (p<0.05). EF and FPF were recovered by NOS inhibition, while the reduction in AMP was NOS independent. No differences in EDD, tone, or FREQ were observed between WT and TNF-Tg PLVs, nor between Expanding versus Collapsed PLVs. ConclusionThese findings support the concept that chronic inflammatory arthritis leads to NOS dependent and independent draining lymphatic vessel dysfunction that exacerbates disease, and may trigger arthritic flare due to decreased egress of inflammatory cells and soluble factors from affected joints.

pathology↗