Vascular and Lymphatic Malformations: Perspectives From Human and Vertebrate Studies.

Vascular malformations, affecting ≈1% to 1.5% of the population, comprise a spectrum of developmental patterning defects of capillaries, arteries, veins, and/or lymphatics. The majority of vascular malformations occur sporadically; however, inherited malformations exist as a part of complex congenital diseases. The malformations, ranging from birthmarks to life-threatening conditions, are present at birth, but may reveal signs and symptoms-including pain, bleeding, disfigurement, and functional defects of vital organs-in infancy, childhood, or adulthood. Vascular malformations often exhibit recurrent patterns at affected sites due to the lack of curative treatments. This review series provides a state-of-the-art assessment of vascular malformation research at basic, clinical, genetic, and translational levels.

Lymphatic Malformations: Genetics, Mechanisms and Therapeutic Strategies.

Lymphatic vessels maintain tissue fluid homeostasis by returning to blood circulation interstitial fluid that has extravasated from the blood capillaries. They provide a trafficking route for cells of the immune system, thus critically contributing to immune surveillance. Developmental or functional defects in the lymphatic vessels, their obstruction or damage, lead to accumulation of fluid in tissues, resulting in lymphedema. Here we discuss developmental lymphatic anomalies called lymphatic malformations and complex lymphatic anomalies that manifest as localized or multifocal lesions of the lymphatic vasculature, respectively. They are rare diseases that are caused mostly by somatic mutations and can present with variable symptoms based upon the size and location of the lesions composed of fluid-filled cisterns or channels. Substantial progress has been made recently in understanding the molecular basis of their pathogenesis through the identification of their genetic causes, combined with the elucidation of the underlying mechanisms in animal disease models and patient-derived lymphatic endothelial cells. Most of the solitary somatic mutations that cause lymphatic malformations and complex lymphatic anomalies occur in genes that encode components of oncogenic growth factor signal transduction pathways. This has led to successful repurposing of some targeted cancer therapeutics to the treatment of lymphatic malformations and complex lymphatic anomalies. Apart from the mutations that act as lymphatic endothelial cell-autonomous drivers of these anomalies, current evidence points to superimposed paracrine mechanisms that critically contribute to disease pathogenesis and thus provide additional targets for therapeutic intervention. Here, we review these advances and discuss new treatment strategies that are based on the recently identified molecular pathways.

Immunohistochemical Analysis of mTOR Pathway-Related Proteins in Kaposiform Hemangioendothelioma.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors have been shown to have excellent effects in the management of kaposiform hemangioendothelioma (KHE); however, the mechanism of action is unclear. This study identified the expressions of mTOR pathway-related proteins in different vascular tumors to provide insight into the pathogenesis of KHE. METHODS: We retrospectively reviewed the pathologic specimens of 30 patients (KHE, 15; tufted angioma [TA], 5; infantile hemangioma [IH], 5; and lymphatic malformation [LM], 5). The immunohistochemical expression of mTOR-related proteins tuberous sclerosis complex 2 (TSC2), phosphatase and tensin homologue (PTEN), phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1), phosphorylated mTOR (p-mTOR), and phosphorylated ribosomal protein S6 kinase B1 (p-P70S6K) were analyzed using Image-Pro Plus software. KHE had the following pattern of expression in the spindle vascular endothelial cells: TSC2 (-); PTEN (-); p-4EBP1 (+); p-mTOR (+); and p-P70S6K (+). RESULTS: All 3 patients treated with sirolimus had good responses. The TA results were similar to KHE with no significant differences (p-4EBP1: p = 0.0687; p-mTOR: p = 0.0832). The expressions of TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K were negative or weakly positive in IH with a statistically significant difference compared to KHE (p-4EBP1: p < 0.001; p-mTOR: p < 0.001; p-P70S6K: p < 0.001). LM had no significant differences when compared to KHE. CONCLUSIONS: The absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway and may be involved in the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM was similar to KHE, unlike IH. The KHE pattern of expression [PTEN (-), TSC2 (-), p-mTOR (+), p-P70S6K (+), and p-4EBP1 (+)] suggested that sirolimus may be a good therapeutic choice.

The mTOR Signal Pathway Is Overactivated in Human Lymphatic Malformations.

Background: Lymphatic malformations (LMs) are congenital low-flow vascular anomalies resulting from abnormal embryogenesis. Clinical researches have shown that rapamycin, a specific inhibitor of mTOR, is effective in treating LMs. It suggests the abnormality of mTOR signal pathway in LMs. Methods and Results: From January 2009 to December 2018, 10 patients who accepted the resection of LMs were enrolled into the study. Samples of each subtype of LMs (macrocystic, microcystic, and mixed subtypes) were further investigated. Expression of molecules in mTOR signal pathway-mTORC1, p70 S6, p-p70 S6, elF4EBP1, and p-elF4EBP1-in LMs were investigated by immunohistochemical staining. Location of mTORC1, p70 S6, and elF4EBP1 in LMs were shown by immunofluorescence co-staining. Phosphorylation level of mTOR signal pathway in LMs was examined by Western blotting. Immunohistochemical staining showed the expression of mTORC1, p70 S6, p-p70 S6, eIF4EBP1, and p-eIF4EBP1 in LMs. Immunofluorescence staining further verified the co-expression of mTORC1, p70 S6, and eIF4EBP1 in the lymphatic endothelium of LMs. Western blotting analysis revealed obviously higher phosphorylation level of mTOR signal pathway in LMs than that in normal skins (P < 0.05). Conclusions: The results showed that the mTOR signal pathway was overactivated in LMs. The study provides compelling evidence for treating LMs or syndromes with lymphatic anomalies by inhibiting mTOR signaling.

The impact of sirolimus therapy on lesion size, clinical symptoms, and quality of life of patients with lymphatic anomalies.

BACKGROUND: Lymphatic anomalies (LAs) include several disorders in which abnormal lymphatic tissue invades the neck, chest, and various organs. Progressive cases may result in lethal outcomes and have proven difficult to treat. Sirolimus is showing promising results in the management of vascular anomalies. We examined the efficacy and safety of sirolimus treatment in patients with progressive LAs. METHODS: All patients with LAs treated with sirolimus from May 2015 to September 2018 were included. They received oral sirolimus once a day and the dose was adjusted so that the trough concentration remained within 5-15 ng/mL. We prospectively reviewed the response to drugs (the response rate of radiological volumetric change of the target lesion), severity scores, reported quality of life (QOL), and adverse effects at 6 months after administration. RESULTS: Twenty patients (five with cystic lymphatic malformation (LM), three with kaposiform lymphangiomatosis, three with generalized lymphatic anomaly, six with Gorham-Stout disease, and three with central conducting lymphatic anomaly) were treated with sirolimus at our institution. Fifty percent of patients (10/20) demonstrated a partial response by a radiological examination and a significant improvement in disease severity and QOL scores (P = 0.0020 and P = 0.0117, respectively). Ten patients who had no reduction in lesion size (stable disease group) showed no significant improvement in disease severity and QOL scores. Eighty percent of patients (16/20) had side effects, such as stomatitis, infection, and hyperlipidemia. CONCLUSIONS: Sirolimus impacts the reduction of the lymphatic tissue volume of LMs and could lead to improvement in clinical symptoms and QOL. TRIAL REGISTRATION: UMIN Clinical Trials Registry, UMIN000016580 . Registered 19 February 2015.

HIF-1α contributes to tube malformation of human lymphatic endothelial cells by upregulating VEGFR-3.

Hypoxia­inducible factor­1α (HIF­1α) is upregulated in various tumors and associated with lymphangiogenesis and angiogenesis during tumor development and metastasis. However, the role of HIF­1α in cystic lymphatic malformations (cLM) remains unclear. In the present study, expression of HIF­1α and vascular endothelial growth factor receptor 3 (VEGFR­3) was evaluated in 20 pairs of cLM specimens from patients who accepted curative surgery at Children's Hospital of Nanjing Medical University (Nanjing, China). Additionally, a stable HIF­1α­overexpressing human lymphatic endothelial cell (HLEC) line was established. Overexpression and silencing of HIF­1α were used to investigate the biological role in colony formation, migration and lymphatic tube formation. HIF­1α and VEGFR­3 were upregulated in cLM specimens compared with adjacent normal tissues. In addition, HIF­1α effectively induced HLEC colony formation and migration. Furthermore, lymphatic malformation of HLECs was promoted in vitro by overexpression of HIF­1α. HIF­1α overexpression upregulated VEGFR­3 during lymphangiogenesis. Additionally, expression of lymphatic endothelial markers prospero homeobox protein 1 and lymphatic vessel endothelial hyaluronan receptor 1 increased significantly during lymphatic tube malformation. The presented data demonstrated that HIF­1α overexpression in HLECs promoted colony formation, migration and tube malformation via upregulation of VEGFR­3. These findings may assist in the development of HIF­1α­targeted cLM therapeutics in the future.

Treatment of Lymphatic Malformations with the mTOR Inhibitor Sirolimus: A Systematic Review.

BACKGROUND: Extensive lymphatic malformations are low-flow vascular malformations that can cause devastating complications. Treatment of these malformations is challenging. This systematic review presents current use of sirolimus in patients with extensive lymphatic malformations. METHODS: MEDLINE and Google scholar search was conducted for studies on sirolimus treatment of lymphatic malformations up to July 2017. Search items included "lymphatic malformation," "lymphangioma," "cystic hygroma," "vascular malformation," "low-flow malformation," "sirolimus," "rapamycin," and "mTOR inhibitor." RESULTS: Twenty studies, including 71 patients receiving sirolimus, were included into this review. Forty-five patients had lymphatic malformations, eight patients venolymphatic malformations, and 19 patients capillary-lymphatico-venous malformations. Sirolimus led to a partial remission of disease in 60 patients, three patients had a progressive disease, and the outcome of eight patients was not reported. Dosing, target trough level, and duration of treatment differed between the studies. Common adverse effects were hyperlipidemia and neutropenia. CONCLUSIONS: Available literature indicated that sirolimus therapy might be effective for lymphatic malformations. However, further randomized controlled studies are required to analyze the efficacy and long-term adverse events and to clarify the potential role for sirolimus in the management of lymphatic malformations.

Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly.

Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knock-in of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.

Linkage of Metabolic Defects to Activated PIK3CA Alleles in Endothelial Cells Derived from Lymphatic Malformation.

BACKGROUND: Lymphatic endothelial cells (LECs) derived from lymphatic malformations (LMs) bear activated PIK3CA alleles yet display an inflammatory gene expression profile. A basis for the inflammatory phenotype was sought by screening for coexisting somatic mutations. METHODS AND RESULTS: Fourteen independent LEC populations bearing activated PIK3CA alleles were isolated from LM. These were characterized by the expression of growth and inflammatory genes (VEGFC, IL-6, COX-2, IL-8, HO-1, E-SEL) by qRT-PCR. Most commonly upregulated gene products were VEGFC, COX2, HO-1, and ANGPTL4. The specific inhibition of PI3K reduced VEGFC expression without resolving inflammation. Whole exome sequencing of six LM-LEC populations identified five novel somatically acquired alleles coexisting with activated PIK3CA alleles. Two affected genes regulate lipid droplet metabolism (FITM2 and ATG2A), two are gene regulators (MTA1 and TAF1L), and the fifth is an isoform of ANK3 (an endosomal/lysosomal protein). Inhibition of AMPK implicated its involvement in regulating COX-2 and HO-1 overexpression. ANGPTL4 expression was independent of AMPK and PI3K activity and reflected lipid stress demonstrated in normal LECs. AMPK activation with AICAR had a selective growth-limiting effect in a subset of LM-LEC isolates. CONCLUSIONS: Inflammatory stress displayed by LM-LECs is consistent with errors in lipid metabolism that may be linked to acquired mutations. The acquisition of PIK3CA alleles may be a permissive event that antagonizes inflammation and metabolic defect.

Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA.

OBJECTIVES: To test the hypothesis that somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS). STUDY DESIGN: We used next generation sequencing, droplet digital polymerase chain reaction, and single molecule molecular inversion probes to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Children's Hospital who had an isolated LM (n = 17), KTS (n = 21), fibro-adipose vascular anomaly (n = 8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n = 31) from Seattle Children's Hospital. RESULTS: Most individuals from Boston Children's Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), fibro-adipose vascular anomaly (5/8), and congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (31/33) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ∼ 80% of cases. Seventy-four percent of patients with LM from Seattle Children's Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells. CONCLUSIONS: Somatic PIK3CA mutations are the most common cause of isolated LMs and disorders in which LM is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism because the abundance of mutant cells in a malformed tissue can be low.

Dynamic Toll-like receptor expression predicts outcome of sclerotherapy for lymphatic malformations with OK-432 in children.

BACKGROUND: Sclerotherapy with OK-432 is recommended as a first-line treatment for lymphatic malformations. However, 40% of patients show poor response, defined by involution to <50% of the original size. It has been suggested that the OK-432 effect is highly dependent on the Toll-like receptor (TLR) 4-dependent expression of TLR7 in antigen-presenting cells. We hypothesized that the ability for TLR expression in monocytes after treatment with the TLR4-ligand lipopolysaccharide (LPS) can be used to predict successful OK-432 treatment. METHODS: Blood was taken from children with low responder (LR, n = 6) and high responder (HR, n = 5) of previous OK-432 treatment. Monocytes were stimulated with LPS for 20 h. TLR expression was analyzed with fluorescence-activated cell sorting (mean fluorescence intensity). The level of significance was P ≤ 0.05. RESULTS: The mean age of patients in the HR group was 1.4 ± 0.9 y and in the LR group 2.8 ± 2.9 y (P = 0.31). The mean TLR4 upregulation after LPS stimulation in the HR group was significantly higher than in the LR group (factor 3.6 versus factor 1 compared with nonstimulated controls; P = 0.037). The mean TLR7 expression did not show significant differences between the groups. CONCLUSIONS: Dynamic TLR4 expression represents most probably a predictive parameter for the treatment of lymphatic malformations with OK-432 and should be further investigated.

Objective monitoring of mTOR inhibitor therapy by three-dimensional facial analysis.

With advances in therapeutics for rare, genetic and syndromic diseases, there is an increasing need for objective assessments of phenotypic endpoints. These assessments will preferentially be high precision, non-invasive, non-irradiating, and relatively inexpensive and portable. We report a case of a child with an extensive lymphatic vascular malformation of the head and neck, treated with an mammalian target of Rapamycin (mTOR) inhibitor that was assessed using 3D facial analysis. This case illustrates that this technology is prospectively a cost-effective modality for treatment monitoring, and it supports that it may also be used for novel explorations of disease biology for conditions associated with disturbances in the mTOR, and interrelated, pathways.

Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man.

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.

Acral hemosideric lymphatic malformation.

BACKGROUND: Cutaneous lymphatic malformations represent a group of heterogeneous diseases caused by developmental defects of lymphatic system. OBJECTIVE: The purpose of this study was to report the clinical, histopathological and immunohistochemical features of a distinctive lymphatic malformation. METHODS: Twelve patients with similar clinical and histopathological features were included in this study. Immunohistochemical staining of CD31, D2-40, Prox1 and Wilms tumor 1 (WT-1) were performed on all lesions. RESULTS: All cases were either congenital lesions or developed during the first 2 years of life. All presented as red to brown papules or nodules on acral sites. Histopathologically, the lesions consisted of a dermal proliferation of flat or slit-like vessels lined with a single layer of endothelial cells. Hemosiderin or extravascular red blood cells were present in all cases. The constituent vessels expressed CD31, D2-40 and Prox1 and lacked expression of WT-1. CONCLUSION: On the basis of the clinical, histopathological and immunohistochemical findings, our cases represent a unique type of lymphatic malformation that we believe is distinct from previously reported vascular proliferations. We propose the name of acral hemosideric lymphatic malformation for this entity.

Expression of androgen, estrogen, progesterone, and growth hormone receptors in vascular malformations.

BACKGROUND: Vascular malformations frequently enlarge during adolescence, suggesting that hormones may be involved. The purpose of this study was to determine whether pubertal hormone receptors are present in vascular malformations and whether they differ from normal tissue. METHODS: Tissue specimens (arteriovenous malformation, lymphatic malformation, and venous malformation) were prospectively collected from patients undergoing resection. Immunohistochemistry was used to determine the presence of androgen, estrogen, progesterone, and growth hormone receptors. The effects of age, sex, location, and malformation type on receptor expression were analyzed. Age-, sex-, and location-matched normal tissues served as controls. RESULTS: Forty-five vascular malformation specimens were collected: arteriovenous malformation (n = 11), lymphatic malformation (n = 20), and venous malformation (n = 14). Growth hormone receptor expression was increased in arteriovenous malformation (72.7 percent), lymphatic malformation (65.0 percent), and venous malformation (57.1 percent) tissues compared with controls (25.8 percent) (p < 0.05). Growth hormone receptor was present primarily in the endothelium/perivasculature of malformations (93.1 percent), whereas in normal tissue growth hormone receptor was located only in the stroma (p < 0.0001). Neither age, nor sex, nor location influenced receptor expression (p = 0.9). No differences in androgen receptor, estrogen receptor, and progesterone receptor staining were found between malformations and control samples (p = 0.7). CONCLUSIONS: Growth hormone receptor is overexpressed and principally located in the vessels of vascular malformations. Growth hormone might contribute to the expansion of vascular malformations.

Targetoid hemosiderotic hemangiomas (hobnail hemangiomas) are vascular lymphatic malformations: a study of 12 pediatric cases.

BACKGROUND: Targetoid hemosiderotic hemangioma (THH), also called hobnail hemangioma, is a benign vascular lesion and thought to be of lymphatic origin. OBJECTIVE: We sought to perform a clinicopathologic analysis of cases diagnosed as THH in a tertiary care children's hospital. METHODS: Clinical and histopathologic data were obtained from a chart review of 12 confirmed pediatric cases of THH. To determine the presence or absence of lymphatic vessels in lesional biopsy specimens, we evaluated the expression of the lymphatic endothelial cell marker podoplanin using the D2-40 antibody. Wilms tumor-1 gene immunostaining and Ki-67 proliferation index were also performed to evaluate the proliferative nature of these lesions. RESULTS: Three children had a lesion since birth and 4 had a history of trauma before appearance of the THH. D2-40 immunostaining was positive in every case. Wilms tumor-1 gene immunostaining was negative in 9 cases, focally positive in two cases, and not performed in one case. The Ki-67 proliferation index was very low in all cases studied. LIMITATIONS: The small number of cases and restriction to a pediatric population were limitations. CONCLUSION: Our findings suggest that THH should be classified as a lymphatic vascular malformation.

Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver--re-evaluations of microlymphatic abnormalities.

BACKGROUND: From the morphological appearance, it was impossible to distinguish terminal portal venules from small lymphatic vessels in the portal tract even using histochemical microscopic techniques. Recently, D2-40 was found to be expressed at a high level in lymphatic endothelial cells (LECs). This study was undertaken to elucidate hepatic lymphatic vessels during progression of cirrhosis by examining the expression of D2-40 in LECs. METHODS: Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control) and from cirrhotic livers (LC) (Child A-LC and Child C-LC). Immunohistochemical (IHC), Western blot, and immunoelectron microscopic studies were conducted using D2-40 as markers for lymphatic vessels, as well as CD34 for capillary blood vessels. RESULTS: Imunostaining of D2-40 produced a strong reaction in lymphatic vessels only, especially in Child C-LC. It was possible to distinguish the portal venules from the small lymphatic vessels using D-40. Immunoelectron microscopy revealed strong D2-40 expression along the luminal and abluminal portions of the cell membrane of LECs in Child C-LC tissue. CONCLUSION: It is possible to distinguish portal venules from small lymphatic vessels using D2-40 as marker. D2-40- labeling in lymphatic capillary endothelial cells is related to the degree of fibrosis in cirrhotic liver.

Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice.

Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. Here we generate mice with mutations in the Pi3kca gene encoding the catalytic p110alpha isoform that block its interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110alpha is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation.