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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. more...

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 (p K a 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. UPS 6.9 (subscript indicates p K a ) shows poor lymph node delivery, while UPS 5.3 delivers efficiently to lymph node sets. We investigated targeting mechanisms of UPS 5.3 , observing an accumulation among lymph node lymphatics and a dependence on lymph node-resident macrophages. To overcome the pH-threshold barrier, which limits UPS 6.9 , we rationally designed a nanoparticle coassembly of UPS 6.9 with UPS 5.3 , 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 UPS 6.9 delivery to lymph nodes, extending this platform for disease detection of lymph node metastasis. more...

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., Competing Interests: This work was supported by a grant from the NIH 1R01HL144793-01 to MTD. MTD is the Director of Research for the Lymphatic Malformation Institute. The other authors have declared that no conflict of interest exists. more...

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 KRAS G12D 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 KRAS G12D 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 KRAS G12D -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 KRAS G12D -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., Competing Interests: MD is the Director of Research for the Lymphatic Malformation Institute. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fernandes, Tresemer, Zhang, Rios, Scallan and Dellinger.) more...

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., (Copyright © 2021 by the American Society of Nephrology.) more...

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. more...

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., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.) more...

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., Competing Interests: Competing interestsM.T.D. is the Director of Research for the Lymphatic Malformation Institute. The other authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.) more...

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 Vegfr3 wt/mut mice develop significantly fewer meningeal LVs than Vegfr3 wt/wt mice. Again, meningeal lymphangiogenesis occurs in the alymphatic zone lateral to the sagittal sinus only after PT-induced stroke in Vegfr3 wt/wt mice. Interestingly, Vegfr3 wt/mut mice develop larger stroke volumes than Vegfr3 wt/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. more...

Ectopic lymphatics form in bone and promote bone destruction in diseases such as Gorham-Stout disease, generalized lymphatic anomaly, and kaposiform lymphangiomatosis. However, the role lymphatics serve in normal bone development and repair is poorly understood. The objective of this study was to characterize bone development and fracture healing in mice that have a defect in the development of the lymphatic vasculature. We found that bones in wild-type adult mice and mouse embryos did not have lymphatics. We also found that bone development was normal in Vegfr3 (Chy/Chy) embryos. These mice do not have lymphatics and die shortly after birth. To determine whether lymphatics serve a role in postnatal bone development and fracture healing, we analyzed bones from Vegfr3 (wt/Chy) mice. These mice are viable and have fewer lymphatics than wild-type mice. We found that postnatal bone development and fracture healing was normal in Vegfr3 (wt/Chy) mice. Taken together, our results suggest that lymphatics do not play a major role in normal bone development or repair., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright by International Society of Lymphology.) more...

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., (© 2018 Rodriguez-Laguna et al.) more...

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., (©2018 American Association for Cancer Research.) more...

Ever since it was discovered that endothelial cells line lymphatic vessels, investigators have been working on unraveling the mechanisms that control the growth of this distinctive endothelium and its role in normal physiology and human disease. Recent technological advances have ushered in a new era of "omics" research on the lymphatic system. Research on the genome, transcriptome, proteome, and metabolome of lymphatics has increased our understanding of the biology of the lymphatic vasculature. Here, we introduce the context-lymphatic "systemomics," then briefly review some of the latest advances in research on tumor-associated lymphatic vessels highlighting several "omic" studies that have shed light on mechanisms controlling the growth and function of tumor-associated lymphatic vessels. We conclude by returning, with unanswered questions, to the larger context of cancer and the lymphatic system as a vasculature, circulation, route of entry and transport, and control center of the immune network. more...

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., (Copyright © 2018 Elsevier Inc. All rights reserved.) more...

Patients with Gorham-Stout disease (GSD) have lymphatic vessels in their bones and their bones gradually disappear. Here, we report that mice that overexpress VEGF-C in bone exhibit a phenotype that resembles GSD. To drive VEGF-C expression in bone, we generated Osx-tTA;TetO-Vegfc double-transgenic mice. In contrast to Osx-tTA mice, Osx-tTA;TetO-Vegfc mice developed lymphatics in their bones. We found that inhibition of VEGFR3, but not VEGFR2, prevented the formation of bone lymphatics in Osx-tTA;TetO-Vegfc mice. Radiological and histological analysis revealed that bones from Osx-tTA;TetO-Vegfc mice were more porous and had more osteoclasts than bones from Osx-tTA mice. Importantly, we found that bone loss in Osx-tTA;TetO-Vegfc mice could be attenuated by an osteoclast inhibitor. We also discovered that the mutant phenotype of Osx-tTA;TetO-Vegfc mice could be reversed by inhibiting the expression of VEGF-C. Taken together, our results indicate that expression of VEGF-C in bone is sufficient to induce the pathologic hallmarks of GSD in mice., Competing Interests: DH, AS, NK, YL, PD, JF, JR, MD No competing interests declared, BP During preparation of the manuscript, B Pytowski was an employee of Eli Lilly and continues to hold stock in the company. KA Reviewing editor, eLife, (© 2018, Hominick et al.) more...

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., (Copyright © 2016 the American Physiological Society.) more...

It is well established that lung tumors induce the formation of lymphatic vessels. However, the molecular mechanisms controlling tumor lymphangiogenesis in lung cancer have not been fully delineated. In the present study, we identify a panel of non-small cell lung cancer (NSCLC) cell lines that induce lymphangiogenesis and use genome-wide mRNA expression to characterize the molecular mechanisms regulating tumor lymphangiogenesis. We show that Calu-1, H1993, HCC461, HCC827, and H2122 NSCLC cell lines form tumors that induce lymphangiogenesis whereas Calu-3, H1155, H1975, and H2073 NSCLC cell lines form tumors that do not induce lymphangiogenesis. By analyzing genome-wide mRNA expression data, we identify a 17-gene expression signature that distinguishes lymphangiogenic from non-lymphangiogenic NSCLC cell lines. Importantly, VEGF-C is the only lymphatic growth factor in this expression signature and is approximately 50-fold higher in the lymphangiogenic group than in the non-lymphangiogenic group. We show that forced expression of VEGF-C by H1975 cells induces lymphangiogenesis and that knockdown of VEGF-C in H1993 cells inhibits lymphangiogenesis. Additionally, we demonstrate that the triple angiokinase inhibitor, nintedanib (small molecule that blocks all FGFRs, PDGFRs, and VEGFRs), suppresses tumor lymphangiogenesis in H1993 tumors. Together, these data suggest that VEGF-C is the dominant driver of tumor lymphangiogenesis in NSCLC and reveal a specific therapy that could potentially block tumor lymphangiogenesis in NSCLC patients. more...

Repurposing "old" drugs can facilitate rapid clinical translation but necessitates novel mechanistic insight. Warfarin, a vitamin K "antagonist" used clinically for the prevention of thrombosis for more than 50 years, has been shown to have anticancer effects. We hypothesized that the molecular mechanism underlying its antitumor activity is unrelated to its effect on coagulation, but is due to inhibition of the Axl receptor tyrosine kinase on tumor cells. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here, we show that inhibiting Gas6-dependent Axl activation with low-dose warfarin, or with other tumor-specific Axl-targeting agents, blocks the progression and spread of pancreatic cancer. Warfarin also inhibited Axl-dependent tumor cell migration, invasiveness, and proliferation while increasing apoptosis and sensitivity to chemotherapy. We conclude that Gas6-induced Axl signaling is a critical driver of pancreatic cancer progression and its inhibition with low-dose warfarin or other Axl-targeting agents may improve outcome in patients with Axl-expressing tumors., (©2015 American Association for Cancer Research.) more...

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. more...

Gorham-Stout disease (GSD) is a rare disorder characterized by the proliferation of endothelial-lined vessels in bone and the progressive destruction of bone. Although Jackson described the first case of GSD in 1838, the clinical and histological features of GSD were not defined until Gorham and Stout published their report on massive osteolysis in 1955. In the years since Gorham and Stout's groundbreaking publication, more than 300 cases of GSD have been described in the literature. These reports have revealed that the progressive resorption of bone in GSD causes severe physical deformities, disabilities, and life-threatening complications. Unfortunately, the underlying cause of GSD remains unknown and, as a result, the therapeutic options for individuals with GSD are limited. Here we review the latest advances in GSD research and present strategies to address basic and clinical research questions related to GSD., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.) more...

There is growing evidence that antiangiogenic therapy stimulates cancer cell invasion and metastasis. However, the underlying molecular mechanisms responsible for these changes have not been fully defined. Here, we report that anti-VEGF therapy promotes local invasion and metastasis by inducing collagen signaling in cancer cells. We show that chronic VEGF inhibition in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDA) induces hypoxia, a less differentiated mesenchymal-like tumor cell phenotype, TGF-β expression, and collagen deposition and signaling. In addition, we show that collagen signaling is critical for protumorigenic activity of TGF-β in vitro. To further model the impact of collagen signaling in tumors, we evaluated PDA in mice lacking Sparc, a protein that reduces collagen binding to cell surface receptors. Importantly, we show that loss of Sparc increases collagen signaling and tumor progression. Together, these findings suggest that collagen actively promotes PDA spread and that enhanced disease progression associated with anti-VEGF therapy can arise from elevated extracellular matrix-mediated signaling., (©2013 AACR.) more...

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed by lymphatic endothelial cells and has been shown to stimulate lymphangiogenesis in adult mice. However, the role VEGFR2 serves in the development of the lymphatic vascular system has not been defined. Here we use the Cre-lox system to show that the proper development of the lymphatic vasculature requires VEGFR2 expression by lymphatic endothelium. We show that Lyve-1(wt/Cre);Vegfr2(flox/flox) mice possess significantly fewer dermal lymphatic vessels than Vegfr2(flox/flox) mice. Although Lyve-1(wt/Cre);Vegfr2(flox/flox) mice exhibit lymphatic hypoplasia, the lymphatic network is functional and contains all of the key features of a normal lymphatic network (initial lymphatic vessels and valved collecting vessels surrounded by smooth muscle cells (SMCs)). We also show that Lyve-1(Cre) mice display robust Cre activity in macrophages and in blood vessels in the yolk sac, liver and lung. This activity dramatically impairs the development of blood vessels in these tissues in Lyve-1(wt/Cre);Vegfr2(flox/flox) embryos, most of which die after embryonic day14.5. Lastly, we show that inactivation of Vegfr2 in the myeloid lineage does not affect the development of the lymphatic vasculature. Therefore, the abnormal lymphatic phenotype of Lyve-1(wt/Cre);Vegfr2(flox/flox) mice is due to the deletion of Vegfr2 in the lymphatic vasculature not macrophages. Together, this work demonstrates that VEGFR2 directly promotes the expansion of the lymphatic network and further defines the molecular mechanisms controlling the development of the lymphatic vascular system. more...

Unlabelled: Non-small cell lung cancer (NSCLC) often expresses mutant KRAS together with tumor-associated mutations of the CDKN2A locus, which are associated with aggressive, therapy-resistant tumors. Here, we unravel specific requirements for the maintenance of NSCLC that carries this genotype. We establish that the extracellular signal-regulated kinase (ERK)/RHOA/focal adhesion kinase (FAK) network is deregulated in high-grade lung tumors. Suppression of RHOA or FAK induces cell death selectively in mutant KRAS;INK4A/ARF-deficient lung cancer cells. Furthermore, pharmacologic inhibition of FAK caused tumor regression specifically in the high-grade lung cancer that developed in mutant Kras;Cdkn2a-null mice. These findings provide a rationale for the rapid implementation of genotype-specific targeted therapies using FAK inhibitors in patients with cancer., Significance: Targeted therapies are effective for only a small fraction of patients with cancer. We report that FAK inhibitors exert potent antitumor effects in NSCLCs that express mutant KRAS in association with INK4A/ARF deficiency. These results reveal a novel genotype-specific vulnerability of cancer cells that can be exploited for therapeutic purposes., (©2013 AACR.) more...

In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα monoclonal antibodies (mAb) on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were conducted using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression., (©2012 AACR.) more...

Recent discoveries in molecular lymphology, developmental biology, and tumor biology in the context of long-standing concepts and observations on development, growth, and neoplasia implicate overlapping pathways, processes, and clinical manifestations in developmental disorders and cancer metastasis. Highlighted in this review are some of what is known (and speculated) about the genes, proteins, and signaling pathways and processes involved in lymphatic/blood vascular development in comparison to those involved in cancer progression and spread. Clues and conundra from clinical disorders that mix these processes and mute them, including embryonic rests, multicentric nests of displaced cells, uncontrolled/invasive "benign" proliferation and lymphogenous/hematogenous "spread", represent a fine line between normal development and growth, dysplasia, benign and malignant neoplasia, and "metastasis". Improved understanding of these normal and pathologic processes and their underlying pathomechanisms, e.g., stem cell origin and bidirectional epithelial-mesenchymal transition, could lead to more successful approaches in classification, treatment, and even prevention of cancer and a whole host of other diseases. more...

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., (Copyright © 2011 Elsevier Inc. All rights reserved.) more...

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., (Copyright © 2011 Wiley-Liss, Inc.) more...

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels. more...

Vascular endothelial growth factor (VEGF) is a primary stimulant of angiogenesis and is a macrophage chemotactic protein. Inhibition of VEGF is beneficial in combination with chemotherapy for some breast cancer patients. However, the mechanism by which inhibition of VEGF affects tumor growth seems to involve more than its effect on endothelial cells. In general, increased immune cell infiltration into breast tumors confers a worse prognosis. We have shown previously that 2C3, a mouse monoclonal antibody that prevents VEGF from binding to VEGF receptor 2 (VEGFR2), decreases tumor growth, angiogenesis, and macrophage infiltration into pancreatic tumors and therefore hypothesized that r84, a fully human IgG that phenocopies 2C3, would similarly affect breast tumor growth and immune cell infiltration. In this study, we show that anti-VEGF therapy with bevacizumab, 2C3, or r84 inhibits the growth of established orthotopic MDA-MB-231 breast tumors in severe combined immunodeficiency (SCID) mice, reduces tumor microvessel density, limits the infiltration of tumor-associated macrophages, but is associated with elevated numbers of tumor-associated neutrophils. In addition, we found that treatment with r84 reduced the number of CD11b(+)Gr1(+) double-positive cells in the tumor compared with tumors from control-treated animals. These results show that selective inhibition of VEGFR2 with an anti-VEGF antibody is sufficient for effective blockade of the protumorigenic activity of VEGF in breast cancer xenografts. These findings further define the complex molecular interactions in the tumor microenvironment and provide a translational tool that may be relevant to the treatment of breast cancer. more...

Lymphedema-distichiasis (OMIM 153400) is a dominantly inherited disorder typically presenting with lymphedema at puberty and distichiasis at birth. The condition has been decisively linked to mutations in the forkhead transcription factor FOXC2 which have been primarily frameshift mutations truncating the protein. We report here a novel missense mutation along with a literature review summarizing reported mutations. more...

Recent advances in molecular lymphology and lymphatic phenotyping techniques in small animals offer new opportunities to delineate mutant mouse models. Chy-3 mutant mice were originally named for their chylous ascites, but the underlying lymphatic disorder was not defined. We now re-examined these mice and applied advanced genotyping and lymphatic phenotyping techniques to pinpoint the specific lymphatic defect in this mouse model. We demonstrated that Chy-3 mice carry a large chromosomal deletion that includes Vegfc and narrowed this region by monitoring the heterozygosity of genetic markers. We found that Chy-3 mice not only exhibited chylous ascites but also lymphedema of the hind paws and, in approximately half of the males, lymphedema of the penis. Visual lymphangiography and immunofluorescence staining showed a hypoplastic dermal lymphatic network, whereas the blood vasculature appeared unaffected. This hypoplastic lymphatic network was functional, and all adult Chy-3 mice exhibited a lateral lymphatic pathway directly connecting the inguinal to the axillary lymph node. The dermal superficial to deep lymphatic connections in upper limbs and in all cervical regions were intact and functionally drained the upper body. Lymphatic tracer was not transported from the dermal to the deep truncal lymphatic system in the lower limbs, even though the deep lymphatic vessels and nodes were present and patent. These findings further delineate the lymphatic phenotype of Chy-3 mice, identify a collateral lymph drainage pathway previously undescribed in other genetic models of lymphedema, and demonstrate a predilection for lymphatic abnormalities of the lower limbs., ((c) 2007 Wiley-Liss, Inc.) more...