WWTR1(TAZ)-CAMTA1 reprograms endothelial cells to drive epithelioid hemangioendothelioma.

Epithelioid hemangioendothelioma (EHE) is a poorly understood and devastating vascular cancer. Sequencing of EHE has revealed a unique gene fusion between the Hippo pathway nuclear effector TAZ (WWTR1) and the brain-enriched transcription factor CAMTA1 in ∼90% of cases. However, it remains unclear whether the TAZ-CAMTA1 gene fusion is a driver of EHE, and potential targeted therapies are unknown. Here, we show that TAZ-CAMTA1 expression in endothelial cells is sufficient to drive the formation of vascular tumors with the distinctive features of EHE, and inhibition of TAZ-CAMTA1 results in the regression of these vascular tumors. We further show that activated TAZ resembles TAZ-CAMTA1 in driving the formation of EHE-like vascular tumors, suggesting that constitutive activation of TAZ underlies the pathological features of EHE. We show that TAZ-CAMTA1 initiates an angiogenic and regenerative-like transcriptional program in endothelial cells, and disruption of the TAZ-CAMTA1-TEAD interaction or ectopic expression of a dominant negative TEAD in vivo inhibits TAZ-CAMTA1-mediated transformation. Our study provides the first genetic model of a TAZ fusion oncoprotein driving its associated human cancer, pinpointing TAZ-CAMTA1 as the key driver and a valid therapeutic target of EHE.

Lymphatics in bone arise from pre-existing lymphatics.

Bones do not normally have lymphatics. However, individuals with generalized lymphatic anomaly (GLA) or Gorham-Stout disease (GSD) develop ectopic lymphatics in bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known and the development of bone lymphatics has not been fully characterized. Here, we describe the development of bone lymphatics in mouse models of GLA and GSD. Through lineage-tracing experiments, we show that bLECs arise from pre-existing Prox1-positive LECs. We show that bone lymphatics develop in a stepwise manner where regional lymphatics grow, breach the periosteum and then invade bone. We also show that the development of bone lymphatics is impaired in mice that lack osteoclasts. Last, we show that rapamycin can suppress the growth of bone lymphatics in our models of GLA and GSD. In summary, we show that bLECs can arise from pre-existing LECs and that rapamycin can prevent the growth of bone lymphatics.

Heterozygous Mutation of Vegfr3 Reduces Renal Lymphatics without Renal Dysfunction.

BACKGROUND: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. METHODS: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy ) that abrogates its kinase ability. Vegfr3 Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3 Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue-clearing, light-sheet microscopy, and computational analyses. RESULTS: In the kidney, VEGFR3 is expressed not only in lymphatic vessels but also, in various blood capillaries. Vegfr3 Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in BP, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low-dose cisplatin (5 mg/kg), although Vegfr3 Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length but showed increased lymphatic density due to decreased cortical volume. CONCLUSIONS: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histologic changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in CKD requires assessment of changes to cortical volume.

Mir-126 is a conserved modulator of lymphatic development.

Organ homeostasis relies upon cellular and molecular processes that restore tissue structure and function in a timely fashion. Lymphatic vessels help maintain fluid equilibrium by returning interstitial fluid that evades venous uptake back to the circulation. Despite its important role in tissue homeostasis, cancer metastasis, and close developmental origins with the blood vasculature, the number of molecular players known to control lymphatic system development is relatively low. Here we show, using genetic approaches in zebrafish and mice, that the endothelial specific microRNA mir-126, previously implicated in vascular integrity, regulates lymphatic development. In zebrafish, in contrast to mir-126 morphants, double mutants (mir-126a-/-; mir-126b-/-, hereafter mir-126-/-) do not exhibit defects in vascular integrity but develop lymphatic hypoplasia; mir-126-/- animals fail to develop complete trunk and facial lymphatics, display severe edema and die as larvae. Notably, following MIR-126 inhibition, human Lymphatic Endothelial Cells (hLECs) respond poorly to VEGFA and VEGFC. In this context, we identify a concomitant reduction in Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) and Vascular Endothelial Growth Factor Receptor-3 (VEGFR3, also known as FLT4) expression upon MIR-126 inhibition. In vivo, we further show that flt4+/- zebrafish embryos exhibit lymphatic defects after mild miR-126 knockdown. Similarly, loss of Mir-126 in Flt4+/- mice results in embryonic edema and lethality. Thus, our results indicate that miR-126 modulation of Vegfr signaling is essential for lymphatic system development in fish and mammals.

Actions of the protein kinase WNK1 on endothelial cells are differentially mediated by its substrate kinases OSR1 and SPAK.

The with no lysine (K) (WNK) family of enzymes is best known for control of blood pressure through regulation of the function and membrane localization of ion cotransporters. In mice, global as well as endothelial-specific WNK1 gene disruption results in embryonic lethality due to angiogenic and cardiovascular defects. WNK1(-/-) embryos can be rescued by endothelial-specific expression of a constitutively active form of the WNK1 substrate protein kinase OSR1 (oxidative stress responsive 1). Using human umbilical vein endothelial cells (HUVECs), we explored mechanisms underlying the requirement of WNK1-OSR1 signaling for vascular development. WNK1 is required for cord formation in HUVECs, but the actions of the two major WNK1 effectors, OSR1 and its close relative SPAK (STE20/SPS1-related proline-, alanine-rich kinase), are distinct. SPAK is important for endothelial cell proliferation, whereas OSR1 is required for HUVEC chemotaxis and invasion. We also identified the zinc-finger transcription factor Slug in WNK1-mediated control of endothelial functions. Our study identifies a separation of functions for the WNK1-activated protein kinases OSR1 and SPAK in mediating proliferation, invasion, and gene expression in endothelial cells and an unanticipated link between WNK1 and Slug that is important for angiogenesis.

Hyperplasia, de novo lymphangiogenesis, and lymphatic regression in mice with tissue-specific, inducible overexpression of murine VEGF-D.

Lymphatic vessels modulate tissue fluid balance and inflammation and provide a conduit for endocrine and lipid transport. The growth of new lymphatic vessels in the adult, lymphangiogenesis, is predominantly mediated through vascular endothelial growth factor receptor-3 (VEGFR-3) signaling. We took advantage of the unique binding of murine VEGF-D specifically to VEGFR-3 and generated mice capable of inducible, tissue-specific expression of murine VEGF-D under a tightly-controlled tetracycline response element (TRE) promoter to stimulate adult tissue lymphangiogenesis. With doxycycline-activated expression, TRE-VEGF-D mouse crossed to mice with tissue-specific promoters for the lung [Clara cell secretory protein-reverse tetracycline transactivator (rtTA)] developed pulmonary lymphangiectasia. In the kidney, (kidney-specific protein-rtTA × TRE-VEGF-D) mice exhibited rapid lymphatic hyperplasia on induction of VEGF-D expression. Crossed with adipocyte-specific adiponectin-rtTA mice [Adipo-VEGF-D (VD)], chronic VEGF-D overexpression was capable of inducing de novo lymphangiogenesis in white adipose tissue and a massive expansion of brown adipose tissue lymphatics. VEGF-D expression in white adipose tissue also increased macrophage infiltration and tissue fibrosis in the tissue. Expression did not, however, measurably affect peripheral fluid transport, the blood vasculature, or basal metabolic parameters. On removal of the doxycycline stimulus, VEGF-D expression returned to normal, and the expanded adipose tissue lymphatics regressed in Adipo-VD mice. The inducible TRE-VEGF-D mouse thus provides a novel murine platform to study the adult mechanisms and therapies of an array of disease- and tissue-specific models of lymphangiogenesis.

Stepwise Ultra-pH-Sensitive Micelles Overcome a pKa Barrier for Systemic Lymph Node Delivery.

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.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

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.

Ephrin-B2 reverse signaling increases α5ß1 integrin-mediated fibronectin deposition and reduces distal lung compliance.

Alveolar growth abnormalities and severe respiratory dysfunction are often fatal. Identifying mechanisms that control epithelial proliferation and enlarged, poorly septated airspaces is essential in developing new therapies for lung disease. The membrane-bound ligand ephrin-B2 is strongly expressed in lung epithelium, and yet in contrast to its known requirement for arteriogenesis, considerably less is known regarding the function of this protein in the epithelium. We hypothesize that the vascular mediator ephrin-B2 governs alveolar growth and mechanics beyond the confines of the endothelium. We used the in vivo manipulation of ephrin-B2 reverse signaling to determine the role of this vascular mediator in the pulmonary epithelium and distal lung mechanics. We determined that the ephrin-B2 gene (EfnB2) is strongly expressed in alveolar Type 2 cells throughout development and into adulthood. The role of ephrin-B2 reverse signaling in the lung was assessed in Efnb2(LacZ/6YFΔV) mutants that coexpress the intracellular truncated ephrin-B2-ß-galactosidase fusion and an intracellular point mutant ephrin-B2 protein that is unable to become tyrosine-phosphorylated or to interact with either the SH2 or PDZ domain-containing downstream signaling proteins. In these viable mice, we observed pulmonary hypoplasia and altered pulmonary mechanics, as evidenced by a marked reduction in lung compliance. Associated with the reduction in lung compliance was a significant increase in insoluble fibronectin (FN) basement membrane matrix assembly with FN deposition, and a corresponding increase in the α5 integrin receptor required for FN fibrillogenesis. These experiments indicate that ephrin-B2 reverse signaling mediates distal alveolar formation, fibrillogenesis, and pulmonary compliance.

Inhibition of multiple pathogenic pathways by histone deacetylase inhibitor SAHA in a corneal alkali-burn injury model.

Neovascularization (NV) in the cornea is a major cause of vision impairment and corneal blindness. Hemangiogenesis and lymphangiogenesis induced by inflammation underlie the pathogenesis of corneal NV. The current mainstay treatment, corticosteroid, treats the inflammation associated with corneal NV, but is not satisfactory due to such side effects as cataract and the increase in intraocular pressure. It is imperative to develop a novel therapy that specifically targets the hemangiogenesis, lymphangiogenesis, and inflammation pathways underlying corneal NV. Histone deacetylase inhibitors (HDACi) have been in clinical trials for cancer and other diseases. In particular, HDACi suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The functional mechanism of SAHA in cancer and especially in corneal NV remains unclear. Here, we show that topical application of SAHA inhibits neovascularization in an alkali-burn corneal injury model. Mechanistically, SAHA inhibits corneal NV by repressing hemangiogenesis, inflammation pathways, and previously overlooked lymphangiogenesis. Topical SAHA is well tolerated on the ocular surface. In addition, the potency of SAHA in corneal NV appears to be comparable to the current steroid therapy. SAHA may possess promising therapeutic potential in alkali-burn corneal injury and other inflammatory neovascularization disorders.

Trametinib inhibits lymphatic vessel invasion of bone in a mouse model of Gorham-Stout disease.

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 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 (LECs). 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 LECs. 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.

Hyperactive KRAS/MAPK signaling disrupts normal lymphatic vessel architecture and function.

Complex lymphatic anomalies (CLAs) are sporadically occurring diseases caused by the maldevelopment of lymphatic vessels. We and others recently reported that somatic activating mutations in KRAS can cause CLAs. However, the mechanisms by which activating KRAS mutations cause CLAs are poorly understood. Here, we show that KRASG12D expression in lymphatic endothelial cells (LECs) during embryonic development impairs the formation of lymphovenous valves and causes the enlargement of lymphatic vessels. We demonstrate that KRASG12D expression in primary human LECs induces cell spindling, proliferation, and migration. It also increases AKT and ERK1/2 phosphorylation and decreases the expression of genes that regulate the maturation of lymphatic vessels. We show that MEK1/2 inhibition with the FDA-approved drug trametinib suppresses KRASG12D-induced morphological changes, proliferation, and migration. Trametinib also decreases ERK1/2 phosphorylation and increases the expression of genes that regulate the maturation of lymphatic vessels. We also show that trametinib and Cre-mediated expression of a dominant-negative form of MEK1 (Map2k1 K97M) suppresses KRASG12D-induced lymphatic vessel hyperplasia in embryos. Last, we demonstrate that conditional knockout of wild-type Kras in LECs does not affect the formation or function of lymphatic vessels. Together, our data indicate that KRAS/MAPK signaling must be tightly regulated during embryonic development for the proper development of lymphatic vessels and further support the testing of MEK1/2 inhibitors for treating CLAs.

Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax.

Intraluminal valves are required for the proper function of lymphatic collecting vessels and large lymphatic trunks like the thoracic duct. Despite recent progress in the study of lymphvasculogenesis and lymphangiogenesis, the molecular mechanisms controlling the morphogenesis of lymphatic valves remain poorly understood. Here, we report that gap junction proteins, or connexins (Cxs), are required for lymphatic valvulogenesis. Cx37 and Cx43 are expressed early in mouse lymphatic development in the jugular lymph sacs, and later in development these Cxs become enriched and differentially expressed by lymphatic endothelial cells on the upstream and downstream sides of the valves. Specific deficiencies of Cx37 and Cx43 alone or in combination result in defective valve formation in lymphatic collecting vessels, lymphedema, and chylothorax. We also show that Cx37 regulates jugular lymph sac size and that both Cx37 and Cx43 are required for normal thoracic duct development, including valve formation. Another Cx family member, Cx47, whose human analog is mutated in some families with lymphedema, is also highly enriched in a subset of endothelial cells in lymphatic valves. Mechanistically, we present data from Foxc2-/- embryos suggesting that Cx37 may be a target of regulation by Foxc2, a transcription factor that is mutated in human lymphedema-distichiasis syndrome. These results show that at least three Cxs are expressed in the developing lymphatic vasculature and, when defective, are associated with clinically manifest lymphatic disorders in mice and man.

Lymphangiogenesis and hemangiogenesis: potential targets for therapy.

This review updates historical background from century-old observations on embryonic lymphatic system development through current understanding of the molecular basis of lymphvasculogenesis/lymphangiogenesis ("molecular lymphology"), highlighting similarities and differences with analogous blood vasculature processes. Topics covered include molecular mechanisms in lymphatic development, structural adaptations of the lymphatic vasculature to particulate and cellular transport and trafficking, lymphogenous route of clinical cancer spread, preservation of delineated lymphatic pathways during cancer operations, and anti-lymphangiogenesis in cancer therapy.

KRAS-driven model of Gorham-Stout disease effectively treated with trametinib.

Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatics in bone, gradually lose bone, and can have life-threatening complications, such as chylothorax. The etiology of GSD is poorly understood, and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a patient with GSD and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.

The emergence of targetable MEKanisms in sporadic lymphatic disorders.

Sporadic lymphatic diseases are orphans among orphans in the medical community, a diverse collection of disorders at the intersection of cardiac, gastrointestinal, pulmonary, dermatologic, and oncologic disease that receives only passing attention in medical school and that no subspecialty in medicine fully embraces as its own. They often present in a confusing and illusive manner, with a fractured bone, expectoration of blood or a branching airway cast, a swollen limb or a collection of chylous material; protean manifestations that can challenge even the most expert diagnostician. Yet many of these acquired disorders have been discovered to have a targetable genetic basis, and as the case report of Foster et al (2020) demonstrates, the sedulous clinician-patient dyad can be rewarded with an almost miraculous result when the molecular pathogenesis of the disease is pursued and an exquisitely targeted therapy is administered.

Impaired meningeal lymphatic vessel development worsens stroke outcome.

The discovery of meningeal lymphatic vessels (LVs) has sparked interest in identifying their role in diseases of the central nervous system. Similar to peripheral LVs, meningeal LVs depend on vascular endothelial growth factor receptor-3 (VEGFR3) signaling for development. Here we characterize the effect of stroke on meningeal LVs, and the impact of meningeal lymphatic hypoplasia on post-stroke outcomes. We show that photothrombosis (PT), but not transient middle cerebral artery occlusion (tMCAo), induces meningeal lymphangiogenesis in young male C57Bl/J6 mice. We also show that Vegfr3wt/mut mice develop significantly fewer meningeal LVs than Vegfr3wt/wt mice. Again, meningeal lymphangiogenesis occurs in the alymphatic zone lateral to the sagittal sinus only after PT-induced stroke in Vegfr3wt/wt mice. Interestingly, Vegfr3wt/mut mice develop larger stroke volumes than Vegfr3wt/wt mice after tMCAo, but not after PT. Our results reveal differences between PT and tMCAo models of stroke and underscore the need to consider method of stroke induction when investigating the role of meningeal lymphatics. Taken together, our data indicate that ischemic injury can induce the growth of meningeal LVs and that the absence of these LVs can impact post-stroke outcomes.

Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice.

Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2(-/-) mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2(-/-) mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2(-/-) pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knock-out Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2(-/-) mice and further defines the sequential steps in lymphatic vessel remodeling.

Cyclooxygenase-2 Inhibition Potentiates the Efficacy of Vascular Endothelial Growth Factor Blockade and Promotes an Immune Stimulatory Microenvironment in Preclinical Models of Pancreatic Cancer.

Resistance to standard therapy remains a major challenge in the treatment of pancreatic ductal adenocarcinoma (PDA). Although anti-VEGF therapy delays PDA progression, therapy-induced hypoxia results in a less differentiated mesenchymal-like tumor cell phenotype, which reinforces the need for effective companion therapies. COX-2 inhibition has been shown to promote tumor cell differentiation and improve standard therapy response in PDA. Here, we evaluate the efficacy of COX-2 inhibition and VEGF blockade in preclinical models of PDA. In vivo, the combination therapy was more effective in limiting tumor growth and metastasis than single-agent therapy. Combination therapy also reversed anti-VEGF-induced epithelial-mesenchymal transition and collagen deposition and altered the immune landscape by increasing tumor-associated CD8+ T cells while reducing FoxP3+ T cells and FasL expression on the tumor endothelium. IMPLICATIONS: Together, these findings demonstrate that COX-2 inhibition enhances the efficacy of anti-VEGF therapy by reducing hypoxia-induced epithelial plasticity and promoting an immune landscape that might facilitate immune activation.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/2/348/F1.large.jpg.

Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.