Complex lymphatic anomalies (CLAs) are rare diseases characterized by the abnormal development of lymphatic vessels. CLAs can be caused by somatic activating variants in KRAS (e.g. KRASG12D), which stimulate MAPK and PI3K signaling. Although KRAS-MAPK signaling is known to play a critical role in CLA pathogenesis, the contribution of KRAS-PI3Kα signaling remains unclear. To investigate the role of RAS activation of PI3Kα in CLAs, we analyzed mice carrying two missense mutations in the RAS-binding domain of p110α (encoded by Pik3ca), the catalytic subunit of PI3Kα. These two mutations block the interaction between p110α and RAS but do not affect its kinase activity. Disruption of RAS-mediated PI3Kα activation in lymphatic endothelial cells reduced lymphatic vessel branching but did not affect lymphatic valve formation. In KrasG12D-mutant mice, blocking RAS activation of p110α reduced the pathological enlargement of lymphatic vessels, but failed to prevent KrasG12D-induced lymphatic valve loss. Similar results were observed following p110α deletion in KrasG12D-mutant mice. Together, these findings demonstrate that KrasG12D drives distinct disease phenotypes through separate downstream pathways. KRAS-PI3Kα signaling promotes pathological lymphatic vessel enlargement, whereas KRAS-MAPK signaling disrupts lymphatic valve formation.

A recent study reported the existence of lymphatic vessels in normal bone and suggested their involvement in bone regeneration after injury. However, this conclusion was based on approaches that do not allow unequivocal identification of the spatial localization of lymphatic endothelial cells (LECs). Here, we employed a Prox1-based genetic tool and a dual-recombinase-mediated LEC-specific labeling system to trace lymphatic vessels with high specificity. We found that LECs are present in the connective tissues, including the periosteum surrounding the bone. However, they do not reside within the bone itself, nor do they penetrate the periosteum to facilitate bone regeneration after injury. By contrast, hyperplastic LECs on the bone surface breach the periosteum and invade bone tissue in mouse models of generalized lymphatic anomaly and Gorham-Stout disease. These data demonstrate that lymphatic vessels are absent from bone during homeostasis and regeneration after injury but invade bone during disease. This Matters Arising paper is in response to Biswas et al. (2023), published in Cell. See also the response by Yang et al. (2026), published in this issue.

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 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 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 Flt4CreERT2;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.

Although mammals generally demonstrate limited regenerative capacity compared with amphibians, the digit tip retains remarkable regenerative potential, providing a useful model to study successful mammalian regeneration. This process involves coordinated immune cell activity, vascular remodeling, and tissue reconstruction, yet the molecular checkpoints controlling regenerative versus fibrotic outcomes remain poorly understood. In mammals, regeneration of the digit tip (P3) proceeds through myeloid cell migration, early osteoclast-mediated osteolysis of the distal bone, and subsequent blastema-mediated regeneration. Here we test the hypothesis that lymphatic vessels regulate regenerative capacity by modulating local immune cell dynamics and osteoclast function. Using a lymphatic system-specific reporter line, we discovered that lymphatic vessels grow toward the nail region from the ventral side of the digit during quiescence and after amputation. These lymphatics closely surround, but do not invade, the native or regenerated bone. Unexpectedly, genetic, pharmacological, and surgical inhibition of lymphangiogenesis accelerated early osteolysis through enhanced transition of myeloid cells to osteoclasts, resulting in faster and more robust regeneration. These findings reveal a mechanism linking lymphatic vessel, immune regulation, and bone remodeling, suggesting that targeted manipulation of lymphatics dynamics may enhance regenerative outcomes after musculoskeletal injury.

Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific processes that drive KRAS-mediated CLAs have yet to be fully elucidated. Here, we used single-cell RNA sequencing to construct an atlas of normal and KrasG12D-malformed lymphatic vessels. We identified 6 subtypes of lymphatic endothelial cells (LECs) in the lungs of adult wild-type mice (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes were specified during development, we integrated our data with data from 4 stages of development. We found that proliferating and Ptx3 LECs were prevalent during early lymphatic development and that collecting and valve LECs emerged later in development. Additionally, we discovered that the proportion of Ptx3 LECs decreased as the lymphatic network matured but remained high in KrasG12D mice. We also observed that the proportion of collecting and valve LECs was lower in KrasG12D mice than in wild-type mice. Last, we found that immature lymphatic vessels in young mice were more sensitive to the pathologic effects of KrasG12D than mature lymphatic vessels in older mice. Together, our results expand the current model for the development of the lymphatic system and suggest that KRAS mutations impair the maturation of lymphatic vessels.

While local nanoparticle delivery to lymph nodes is well studied, there are few design criteria for intravenous delivery to the entire lymph node repertoire. In this study, we investigated the effect of NP pH transition on lymph node targeting by employing a series of ultra-pH-sensitive (UPS) polymeric micelles. The UPS library responds to pH thresholds (pKa 6.9, 6.2, and 5.3) over a range of physiological pH. We observed a dependence of intravenous lymph node targeting on micelle pH transition. UPS6.9 (subscript indicates pKa) shows poor lymph node delivery, while UPS5.3 delivers efficiently to lymph node sets. We investigated targeting mechanisms of UPS5.3, observing an accumulation among lymph node lymphatics and a dependence on lymph node-resident macrophages. To overcome the pH-threshold barrier, which limits UPS6.9, we rationally designed a nanoparticle coassembly of UPS6.9 with UPS5.3, called HyUPS. The HyUPS micelle retains the constitutive pH transitions of each polymer, showing stepwise responses to discrete pH thresholds. We demonstrate that HyUPS improves UPS6.9 delivery to lymph nodes, extending this platform for disease detection of lymph node metastasis.

Objectives: Complex lymphatic anomalies (CLAs) are rare diseases with variable clinical manifestations caused by the abnormal development of lymphatic vessels. The Lymphatic Malformation Institute (LMI), Lymphangiomatosis & Gorham’s Disease Alliance (LGDA), and LGD Alliance Europe sponsored an international conference on CLAs so leaders in the field could discuss recent advances in research on CLAs. Methods: The conference occurred in Dallas, Texas, between September 29th and September 30th, 2023. The event had 22 distinguished speakers and 38 attendees. Additional researchers, clinicians, and patients attended the conference virtually. Results: In this article, we summarize the key takeaways from the meeting. The conference emphasized the crucial role of the patient voice in guiding research, and attendees heard stories from patients and parents of children with CLAs. The conference covered topics such as the genetic causes of CLAs, preclinical models, clinical advances, new technologies for CLA research, lymphatic imaging, and patient-centered research. Videos of all the presentations can be found on the LGDA website. Conclusions: The meeting revealed that while progress has been made, many challenges still exist surrounding the diagnosis and treatment of CLAs. Sustained research efforts are necessary to fill the gaps in knowledge and improve the care and quality of life of CLA patients.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

Supplementary Figure 3 PDF file - 76K, This Figure shows the knockdown efficiency of shRNAs against KRAS and that MEK1/2 inhibition suppresses RHOA

Supplementary Figure 1. (a) Representative images of metastasis to the liver of control mice in Colo357 model was shown by H&E staining. Total magnifications are 100X and 200X. Scale bar, 50μm. (b) Colo357 human pancreatic cancer cells (1 × 106) were injected orthotopically into the pancreas of SCID mice. Treatment began when the established tumor was visible by ultrasound, and consisted of control (saline, n = 6), gemcitabine 25 mg/kg twice weekly plus erlotinib 100 μg daily (standard of care, n = 6) as described in Kirane et al. (29), or mcr84 plus apricoxib (n = 10) and continued for 3 weeks. Tumor weight was normalized to the control and data from the treatment groups were compared. Data are displayed as mean {plus minus} SEM. *P < 0.05 vs. control, by ANOVA with Dunn's MCT. Supplementary Figure 2. Human pancreatic cancer cell lines, Colo357 (a) and AsPc-1 (b) were plated either under normal conditions or under conditions of forced EMT (treated with 20 ng/mL TGFβ on collagen I -coated plates for 72 hours). PGE2 levels were measured by ELISA after 500 nM apricoxib treatment. (c) Colo357 and AsPC-1 cells were plated under normal conditions or conditions of forced EMT (50 ng/ml TGFβ on collagen I -coated plates for 24 hours). Lysates were probed for the indicated targets by Western blotting. The induced EMT conditions promoted loss of E-Cadherin, gain of Vimentin expression. In Colo357, the expression of N-Cadherin was also upregulated by collagen I and TGFβ stimulation. β-actin was used as a loading control. Supplementary Figure 3. Microvessel density is not decreased by COX-2 inhibition. Supplementary Figure 4. KIC pancreatic tissues from the treated mice were subjected to immunohistochemistry Supplementary Figure 5. Anti-VEGF therapy results in reduced microvessel density, hypoxia induced TGFβ expression, epithelial plasticity and enhanced collagen deposition Table S1: List of antibodies

Supplementary Figure 1 PDF file - 311K, This Figure shows that the KrasG12D;Ink4a/Arf -/- mice show increased tumor burden, increased Ki-67, Cyclin D1 expression and decreased survival compared to KrasG12D;Ink4a/Arf +/+ mice

Supplementary Methods PDF file - 123K, This file contains the Supplementary materials and methods linked to the manuscript. In addition the references associated with the materials are reported

PDF file, 156K, Species and target specificity of IMC-3G3 and 1E10.

Supplementary Figure 5 PDF file - 199K, This Figure shows that FAK is a critical target of RHOA in mutant KRAS;INK4a/ARF deficient NSCLC cells

Supplementary Figure 6 PDF file - 141K, This Figure shows that FAK is co-expressed with RHOA in lung adenocarcinomas. In addition, we provide the validation of RHOA-GTP antibody on human specimens by IHC

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

Objective:. Gorham-Stout disease (GSD) is a rare lymphatic anomaly that can be caused by somatic activating mutations in KRAS. This discovery has led investigators to suggest that MEK inhibitors could be a novel treatment for GSD. However, the effect of MEK inhibitors on bone disease in animal models of GSD has not been investigated. We recently reported that Osx-tTA;TetO-Vegfc mice exhibit a phenotype that resembles GSD. Osx-tTA;TetO-Vegfc mice overexpress vascular endothelial growth factor-C (VEGF-C) in bone, which stimulates the development of lymphatic vessels in bone and the gradual loss of cortical bone. The objective of this study was to characterize the effect of trametinib, an FDA-approved MEK1/2 inhibitor, on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. Methods:. Immunoblotting was performed to assess the effect of trametinib on VEGF-C-induced phosphorylation of ERK1/2, AKT, and S6 in primary human lymphatic endothelial cells. Prevention and intervention experiments were performed to determine the effect of trametinib on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. Results:. We found that trametinib blocked VEGF-C-induced phosphorylation of ERK1/2 in primary human lymphatic endothelial cells. We also found that trametinib prevented VEGF-C-induced lymphatic invasion of bone and cortical bone loss in Osx-tTA;TetO-Vegfc mice. Additionally, trametinib slowed the progression of disease in Osx-tTA;TetO-Vegfc mice with established disease. However, it did not reverse disease in Osx-tTA;TetO-Vegfc mice. Conclusion:. Our results show trametinib impacts bone disease in Osx-tTA;TetO-Vegfc mice. These findings further support the testing of MEK inhibitors in patients with GSD and other RAS pathway-driven complex lymphatic anomalies with bone involvement.

Supplementary Tables S1-S2. Metastatic events in preclinical models of PDA (S1); Warfarin enhances the activity of gemcitabine in an Axl-dependent manner (S2).

Supplementary Figure 4 PDF file - 169K, This Figure shows that RHOA activation is essential for the survival of NSCLC expressing mutant KRAS in association with INK4a/ARF or p53 deficiency