Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Methods Mol Biol ; 2206: 179-192, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754818

RESUMO

Xenograft models allow for an in vivo approach to monitor cellular functions within the context of a host microenvironment. Here we describe a protocol to generate a xenograft model of venous malformation (VM) based on the use of human umbilical vein endothelial cells (HUVEC) expressing a constitutive active form of the endothelial tyrosine kinase receptor TEK (TIE2 p.L914F) or patient-derived EC containing TIE2 and/or PIK3CA gene mutations. Hyperactive somatic TIE2 and PIK3CA mutations are a common hallmark of VM in patient lesions. The EC are injected subcutaneously on the back of athymic nude mice to generate ectatic vascular channels and recapitulate histopathological features of VM patient tissue histology. Lesion plugs with TIE2/PIK3CA-mutant EC are visibly vascularized within 7-9 days of subcutaneous injection, making this a great tool to study venous malformation.

2.
Pediatr Blood Cancer ; : e28529, 2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32634277

RESUMO

Kaposiform lymphangiomatosis (KLA) is a rare, life-threatening congenital lymphatic malformation. Diagnosis is often delayed due to complex indistinct symptoms. Blood angiopoietin-2 (ANG2) levels are elevated in KLA and may be useful as a biomarker to monitor disease status. We report a 7-year-old male child with easy bruising, inguinal swelling, and consumptive coagulopathy, diagnosed with KLA. A multimodal treatment regimen of prednisone, sirolimus, vincristine, and adjunctive zoledronate was used. Plasma ANG2 levels were highly elevated at diagnosis but decreased during treatment. The patient showed significant clinical improvement over a 38-month period and normalization of ANG2 levels correlated with resolution of the coagulopathy.

3.
J Vis Exp ; (160)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32597867

RESUMO

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7‒9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

4.
Angiogenesis ; 23(3): 425-442, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32350708

RESUMO

Capillary lymphatic venous malformations (CLVM) are complex vascular anomalies characterized by aberrant and enlarged lymphatic and blood vessels. CLVM appear during fetal development and enlarge after birth, causing life-long complications such as coagulopathy, pulmonary embolism, chronic pain, and disfigurement. Treatment includes surgical debulking, amputation, and recurrent sclerotherapy. Somatic, mosaic mutations in the 110-kD catalytic α-subunit of phosphoinositide-3-kinase (PIK3CA) gene have been previously identified in affected tissues from CLVM patients; however, the cell population harboring the mutation is still unknown. In this study, we hypothesized that endothelial cells (EC) carry the PIK3CA mutations and play a major role in the cellular origin of CLVM. We isolated EC from the lesions of seven patients with CLVM and identified PIK3CA hotspot mutations. The CLVM EC exhibited constitutive phosphorylation of the PI3K effector AKT as well as hyperproliferation and increased resistance to cell death compared to normal EC. Inhibitors of PIK3CA (BYL719) and AKT (ARQ092) attenuated the proliferation of CLVM EC in a dose-dependent manner. A xenograft model of CLVM was developed by injecting patient-derived EC into the flanks of immunocompromised mice. CLVM EC formed lesions with enlarged lymphatic and vascular channels, recapitulating the patient histology. EC subpopulations were further obtained by both immunomagnetic separation into lymphatic EC (LEC) and vascular EC (VEC) and generation of clonal populations. By sequencing these subpopulations, we determined that both LEC and VEC from the same patient express the PIK3CA mutation, exhibit increased AKT activation and can form lymphatic or vascular lesions in mouse.

5.
Sci Rep ; 9(1): 12352, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451744

RESUMO

Abnormalities in controlling key aspects of angiogenesis including vascular cell migration, lumen formation and vessel maturation are hallmarks of vascular anomalies including venous malformation (VM). Gain-of-function mutations in the tyrosine kinase receptor TIE2 can cause VM and induce a ligand-independent hyperactivation of TIE2. Despite these important findings, the TIE2-dependent mechanisms triggering enlarged vascular lesions are not well understood. Herein we studied TIE2 p.L914F, the most frequent mutation identified in VM patients. We report that endothelial cells harboring a TIE2-L914F mutation display abnormal cell migration due to a loss of front-rear polarity as demonstrated by a non-polarized Golgi apparatus. Utilizing a three-dimensional fibrin-matrix based model we show that TIE2-L914F mutant cells form enlarged lumens mimicking vascular lesions present in VM patients, independently of exogenous growth factors. Moreover, these abnormal vascular channels demonstrate a dysregulated expression pattern of apico-basal polarity markers Podocalyxin and Collagen IV. Furthermore, in this system we recapitulated another pathological feature of VM, the paucity of pericytes around ectatic veins. The presented data emphasize the value of this in vitro model as a powerful tool for the discovery of cellular and molecular signals contributing to abnormal vascular development and subsequent identification of novel therapeutic approaches.

6.
Pediatr Blood Cancer ; 66(8): e27790, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31045327

RESUMO

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a rare lymphatic anomaly with significant morbidity and mortality. KLA is characterized by diffuse multifocal lesions comprised of focal areas of "kaposiform" spindled cells accompanying malformed lymphatic channels. The goal of this study was to identify activated signaling pathways in cells isolated from three KLA patients for the purpose of testing new therapies. PROCEDURE: Cells were obtained from the lungs of one patient isolated at autopsy and the spleen of two patients removed in surgery due to disease complications. A protein kinase array was performed on the KLA cell lysates and normal lymphatic endothelial cells. RESULTS: Higher activation of key signaling pathways in the KLA cells, including PRAS40, AKT1/2/3, and ERK-1/2, was identified by protein kinase array and confirmed by Western blot analysis. This indicated a role for highly activated PI3K-AKT and MAPK-ERK-1/2 signaling pathways in KLA cells. Cell proliferation studies assessed PI3K inhibitors (LY294002; BYL719), AKT inhibitor ARQ092, mTOR inhibitor rapamycin, and MAPK inhibitor U0126. These studies demonstrated that PI3K-AKT-mTOR and MAPK signaling are important mediators of KLA cell proliferation. BYL719 and rapamycin were more effective at inhibiting KLA cell proliferation than U0126. CONCLUSIONS: Our studies using cells from KLA patient lesions demonstrate that these cells are highly proliferative and the PI3K-AKT-mTOR and MAPK pathways are promising therapeutic targets. Development and clinical trials of PI3K, AKT, and MAPK inhibitors for cancer treatment and the data in this study lend support for early clinical trials assessing the efficacy of these inhibitors in KLA patients.


Assuntos
Antineoplásicos/farmacologia , Hemangioendotelioma/patologia , Síndrome de Kasabach-Merritt/patologia , Linfangioma/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sarcoma de Kaposi/patologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adolescente , Proliferação de Células/efeitos dos fármacos , Pré-Escolar , Feminino , Seguimentos , Hemangioendotelioma/tratamento farmacológico , Hemangioendotelioma/metabolismo , Humanos , Lactente , Síndrome de Kasabach-Merritt/tratamento farmacológico , Síndrome de Kasabach-Merritt/metabolismo , Linfangioma/tratamento farmacológico , Linfangioma/metabolismo , Masculino , Inibidores de Proteínas Quinases/farmacologia , Estudos Retrospectivos , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
7.
Sci Adv ; 5(4): eaau8389, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31032403

RESUMO

Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1fl/fl;Runx3fl/fl;Nf1fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor ß (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.

8.
Arterioscler Thromb Vasc Biol ; 39(3): 496-512, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30626204

RESUMO

Objective- Venous malformations (VMs) arise from developmental defects of the vasculature and are characterized by massively enlarged and tortuous venous channels. VMs grow commensurately leading to deformity, obstruction of vital structures, bleeding, and pain. Most VMs are associated with the activating mutation L914F in the endothelial cell (EC) tyrosine kinase receptor TIE2. Therapeutic options for VM are limited and ineffective while therapy with the mammalian target of rapamycin inhibitor rapamycin shows moderate efficacy. Here, we investigated novel therapeutic targets promoting VM regression. Approach and Results- We performed an unbiased screen of Food and Drug Administration-approved drugs in human umbilical vein ECs expressing the TIE2-L914F mutation (HUVEC-TIE2-L914F). Three ABL (Abelson) kinase inhibitors prevented cell proliferation of HUVEC-TIE2-L914F. Moreover, c-ABL, common target of these inhibitors, was highly phosphorylated in HUVEC-TIE2-L914F and VM patient-derived ECs with activating TIE2 mutations. Knockdown of c-ABL/ARG in HUVEC-TIE2-L914F reduced cell proliferation and vascularity of murine VM. Combination treatment with the ABL kinase inhibitor ponatinib and rapamycin caused VM regression in a xenograft model based on injection of HUVEC-TIE2-L914F. A reduced dose of this drug combination was effective in this VM murine model with minimal side effects. The drug combination was antiproliferative, enhanced cell apoptosis and vascular channel regression both in vivo and in a 3-dimensional fibrin gel assay. Conclusions- This is the first report of a combination therapy with ponatinib and rapamycin promoting regression of VM. Mechanistically, the drug combination enhanced AKT inhibition compared with single drug treatment and reduced PLCγ (phospholipase C) and ERK (extracellular signal-regulated kinase) activity.


Assuntos
Imidazóis/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Piridazinas/uso terapêutico , Sirolimo/uso terapêutico , Malformações Vasculares/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Mutação de Sentido Incorreto , Fosfolipase C gama/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Piridazinas/administração & dosagem , Piridazinas/farmacologia , Receptor TIE-2/genética , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Malformações Vasculares/patologia
9.
Oncotarget ; 9(37): 24750-24765, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29872503

RESUMO

Angiosarcoma is an aggressive malignancy of vascular origin that occurs de novo or in the context of previous cancer therapy. Despite multi-modal aggressive treatment including surgical resection, chemotherapy, and radiation, five-year overall survival remains poor at 35%. Due to its rarity, little is known about its molecular pathology and clinical trials have been extremely difficult to conduct. Development of animal models for rare diseases like angiosarcoma is critical to improve our understanding of tumorigenesis and to test novel treatment regimens. A genetically engineered mouse model for angiosarcoma was generated by conditional deletion of Trp53, Pten, and Ptpn12 in endothelial cells. Tumors arising from these mice recapitulate the histology and molecular pathology of the human disease including hyperactivation of the PI3K/mTOR and MAPK signaling pathways. Treatment of tumor-bearing mice with mTOR or MEK inhibitors effectively inactivated signaling and resulted in reduced proliferation and elevated apoptosis leading to tumor regression. The effect of treatment on tumor growth was transient and proliferation was restored after a period of dormancy. However, combined inhibition of mTOR and MEK resulted in profound tumor regression which was sustained for the duration of treatment. These results suggest that angiosarcoma may be effectively treated by this drug combination. .

10.
Angiogenesis ; 21(4): 725-735, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29786783

RESUMO

Vascular malformations are defects caused by the abnormal growth of the vasculature. Among them, venous malformation (VM) is an anomaly characterized by slow-flow vascular lesions with abnormally shaped veins, typically in sponge-like configuration. VMs can expand over years causing disfigurement, obstruction of vital structures, thrombosis, bleeding, and pain. Treatments have been very limited and primarily based on supportive care, compression garments, sclerotherapy, and/or surgical resection. Sirolimus treatment has recently shown efficacy in some patients with complicated vascular anomalies, including VMs. Activating somatic TIE2 gene mutations have been identified in up to 60% of VMs and PIK3CA mutations have been found in another 25%. Here, we report a xenograft model of VM that reflects the patients' mutation heterogeneity. First, we established a protocol to isolate and expand in culture endothelial cells (VM-EC) from VM tissue or VM blood of nine patients. In these cells, we identified somatic mutations of TIE2, PIK3CA, or a combination of both. Both TIE2 and PIK3CA mutations induced constitutive AKT activation, while TIE2 mutations also showed high MAPK-ERK signaling. Finally, VM-EC implanted into immune-deficient mice generated lesions with ectatic blood-filled channels with scarce smooth muscle cell coverage, similar to patients' VM. This VM xenograft model could be instrumental to test the therapeutic efficacy of Sirolimus in the presence of the different TIE2 or PIK3CA mutations or to test for efficacy of additional compounds in targeting the specific mutated protein(s), thus enabling development of personalized treatment options for VM patients.


Assuntos
Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Malformações Vasculares , Animais , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Malformações Vasculares/genética , Malformações Vasculares/metabolismo , Malformações Vasculares/patologia
11.
Cancer Res ; 76(21): 6396-6409, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803106

RESUMO

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH+ ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH+ ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH+ cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH+ cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.


Assuntos
Aldeído Desidrogenase/análise , Divisão Celular , Glicoproteínas de Membrana/fisiologia , Neoplasias Ovarianas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/enzimologia , Prognóstico , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia
12.
Cell Mol Life Sci ; 73(8): 1715-39, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26646071

RESUMO

The circulatory system is walled off by different cell types, including vascular mural cells and podocytes. The interaction and interplay between endothelial cells (ECs) and mural cells, such as vascular smooth muscle cells or pericytes, play a pivotal role in vascular biology. Endoglin is an RGD-containing counter-receptor for ß1 integrins and is highly expressed by ECs during angiogenesis. We find that the adhesion between vascular ECs and mural cells is enhanced by integrin activators and inhibited upon suppression of membrane endoglin or ß1-integrin, as well as by addition of soluble endoglin (SolEng), anti-integrin α5ß1 antibody or an RGD peptide. Analysis of different endoglin mutants, allowed the mapping of the endoglin RGD motif as involved in the adhesion process. In Eng (+/-) mice, a model for hereditary hemorrhagic telangectasia type 1, endoglin haploinsufficiency induces a pericyte-dependent increase in vascular permeability. Also, transgenic mice overexpressing SolEng, an animal model for preeclampsia, show podocyturia, suggesting that SolEng is responsible for podocytes detachment from glomerular capillaries. These results suggest a critical role for endoglin in integrin-mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel maturation in normal physiology as well as in pathologies such as preeclampsia or hereditary hemorrhagic telangiectasia.


Assuntos
Antígenos CD/metabolismo , Adesão Celular/fisiologia , Endotélio Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Podócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos CD/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Endoglina , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina beta1/genética , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Neovascularização Patológica/metabolismo , Pericitos/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Retina/metabolismo , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/patologia
13.
J Clin Invest ; 125(9): 3491-504, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26258417

RESUMO

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.


Assuntos
Imunossupressores/administração & dosagem , Mutação de Sentido Incorreto , Receptor TIE-2 , Transdução de Sinais , Sirolimo/administração & dosagem , Malformações Vasculares , Adolescente , Adulto , Animais , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Projetos Piloto , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Malformações Vasculares/tratamento farmacológico , Malformações Vasculares/genética , Malformações Vasculares/metabolismo , Malformações Vasculares/patologia , Veias
14.
Angiogenesis ; 18(2): 151-62, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25424831

RESUMO

Lymphatic malformations (LM) are characterized by abnormal formation of lymphatic vessels and tissue overgrowth. The lymphatic vessels present in LM lesions may become blocked and enlarged as lymphatic fluid collects, forming a mass or cyst. Lesions are typically diagnosed during childhood and are often disfiguring and life threatening. Available treatments consist of sclerotherapy, surgical removal and therapies to diminish complications. We isolated lymphatic endothelial cells (LM-LEC) from a surgically removed microcystic LM lesion. LM-LEC and normal human dermal-LEC (HD-LEC) expressed endothelial (CD31, VE-Cadherin) as well as lymphatic endothelial (Podoplanin, PROX1, LYVE1)-specific markers. Targeted gene sequencing analysis in patient-derived LM-LEC revealed the presence of two mutations in class I phosphoinositide 3-kinases (PI3K) genes. One is an inherited, premature stop codon in the PI3K regulatory subunit PIK3R3. The second is a somatic missense mutation in the PI3K catalytic subunit PIK3CA; this mutation has been found in association with overgrowth syndromes and cancer growth. LM-LEC exhibited angiogenic properties: both cellular proliferation and sprouting in collagen were significantly increased compared with HD-LEC. AKT-Thr308 was constitutively hyper-phosphorylated in LM-LEC. Treatment of LM-LEC with PI3-Kinase inhibitors Wortmannin and LY294 decreased cellular proliferation and prevented the phosphorylation of AKT-Thr308 in both HD-LEC and LM-LEC. Treatment with the mTOR inhibitor rapamycin also diminished cellular proliferation, sprouting and AKT phosphorylation, but only in LM-LEC. Our results implicate disrupted PI3K-AKT signaling in LEC isolated from a human lymphatic malformation lesion.


Assuntos
Endotélio/enzimologia , Vasos Linfáticos/anormalidades , Mutação , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Endotélio/patologia , Feminino , Humanos , Masculino , Fosforilação , Sirolimo/farmacologia
15.
Thromb Haemost ; 112(5): 1002-13, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25103869

RESUMO

Fibrotic diseases of the lung are associated with a vascular remodelling process. Fibrocytes (Fy) are a distinct population of blood-borne cells that co-express haematopoietic cell antigens and fibroblast markers, and have been shown to contribute to organ fibrosis. The purpose of this study was to determine whether fibrocytes cooperate with endothelial colony-forming cells (ECFC) to induce angiogenesis. We isolated fibrocytes from blood of patient with idiopathic pulmonary fibrosis (IPF) and characterised them by flow cytometry, quantitative reverse transcriptase PCR (RTQ-PCR), and confocal microscopy. We then investigated the angiogenic interaction between fibrocytes and cord-blood-derived ECFC, both in vitro and in an in vivo Matrigel implant model. Compared to fibroblast culture medium, fibrocyte culture medium increased ECFC proliferation and differentiation via the SDF-1/CXCR4 pathway. IPF-Fy co-implanted with human ECFC in Matrigel plugs in immunodeficient mice formed functional microvascular beds, whereas fibroblasts did not. Evaluation of implants after two weeks revealed an extensive network of erythrocyte-containing blood vessels. CXCR4 blockade significantly inhibited this blood vessel formation. The clinical relevance of these data was confirmed by strong CXCR4 expression in vessels close to fibrotic areas in biopsy specimens from patients with IPF, by comparison with control lungs. In conclusion, circulating fibrocytes might contribute to the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis.


Assuntos
Células Endoteliais/patologia , Fibroblastos/patologia , Células-Tronco Hematopoéticas/patologia , Fibrose Pulmonar Idiopática/fisiopatologia , Neovascularização Fisiológica , Receptores CXCR4/fisiologia , Proteínas Angiogênicas/biossíntese , Proteínas Angiogênicas/genética , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/genética , Colágeno , Ensaio de Unidades Formadoras de Colônias , Meios de Cultivo Condicionados/farmacologia , Combinação de Medicamentos , Sangue Fetal/citologia , Fibrose , Perfilação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/patologia , Laminina , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Nus , Proteoglicanas , Receptores CXCR4/biossíntese , Receptores CXCR4/genética
16.
Stem Cells ; 32(3): 684-93, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24022922

RESUMO

Infantile hemangioma (IH) is the most common tumor of infancy. Hemangioma stem cells (HemSC) are a mesenchymal subpopulation isolated from IH CD133+ cells. HemSC can differentiate into endothelial and pericyte/smooth muscle cells and form vascular networks when injected in immune-deficient mice. α6-Integrin subunit has been implicated in the tumorgenicity of glioblastoma stem cells and the homing properties of hematopoietic, endothelial, and mesenchymal progenitor cells. Therefore, we investigated the possible function(s) of α6-integrin in HemSC. We documented α6-integrin expression in IH tumor specimens and HemSC by RT-qPCR and flow cytometry. We examined the effect of blocking or silencing α6-integrin on the adhesive and proliferative properties of HemSC in vitro and the vasculogenic and homing properties of HemSC in vivo. Targeting α6-integrin in cultured HemSC inhibited adhesion to laminin but had no effect on proliferation. Vessel-forming ability in Matrigel implants and hepatic homing after i.v. delivery were significantly decreased in α6-integrin siRNA-transfected HemSC. In conclusion, α6-integrin is required for HemSC adherence to laminin, vessel formation in vivo, and for homing to the liver. Thus, we uncovered an important role for α6 integrin in the vasculogenic properties of HemSC. Our results suggest that α6-integrin expression on HemSC could be a new target for antihemangioma therapy.


Assuntos
Hemangioma/metabolismo , Hemangioma/patologia , Integrina alfa6/metabolismo , Neovascularização Fisiológica , Células-Tronco/metabolismo , Células-Tronco/patologia , Animais , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Pré-Escolar , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lactente , Laminina/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos
17.
Arterioscler Thromb Vasc Biol ; 33(3): 501-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23288163

RESUMO

OBJECTIVE: Infantile hemangioma (IH) is a rapidly growing vascular tumor affecting newborns. It is composed of immature endothelial cells and pericytes that proliferate into a disorganized mass of blood vessels. We isolated pericytes from IH (Hem-pericytes) to test our hypothesis that Hem-pericytes are unable to stabilize blood vessels. METHODS AND RESULTS: We injected pericytes in vivo, in combination with endothelial cells, and found that Hem-pericytes formed more microvessels compared with control retinal pericytes. We, thereby, analyzed proangiogenic properties of the Hem-pericytes. They grew fast in vitro, and were unable to stabilize endothelial cell growth and migration, and expressed high levels of vascular endothelial growth factor-A compared with retinal pericytes. Hem-pericytes from proliferating phase IH showed lower contractility in vitro, compared with Hem-pericytes from the involuting phase and retinal pericytes. Consistent with a diminished ability to stabilize endothelium, angiopoietin 1 was reduced in Hem-pericytes compared with retinal pericytes. Normal retinal pericytes in which angiopoietin 1 was silenced produced conditioned medium that stimulated endothelial cell proliferation and migration. CONCLUSIONS: We report the first successful isolation of patient-derived pericytes from IH tissue. Hem-pericytes exhibited proangiogenic properties and low levels of angiopoietin 1, consistent with a diminished ability to stabilize blood vessels in IH.


Assuntos
Angiopoietina-1/metabolismo , Hemangioma/metabolismo , Neovascularização Patológica , Pericitos/metabolismo , Angiopoietina-1/genética , Animais , Biomarcadores/metabolismo , Comunicação Celular , Movimento Celular , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Regulação para Baixo , Hemangioma/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Nus , Pericitos/patologia , Pericitos/transplante , Interferência de RNA , RNA Mensageiro/metabolismo , Vasos Retinianos/metabolismo , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Blood ; 120(14): 2925-9, 2012 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-22791286

RESUMO

Inflammatory cytokines and growth factors drive angiogenesis independently; however, their integrated role in pathologic and physiologic angiogenesis is not fully understood. Suppressor of cytokine signaling-3 (SOCS3) is an inducible negative feedback regulator of inflammation and growth factor signaling. In the present study, we show that SOCS3 curbs pathologic angiogenesis. Using a Cre/Lox system, we deleted SOCS3 in vessels and studied developmental and pathologic angiogenesis in murine models of oxygen-induced retinopathy and cancer. Conditional loss of SOCS3 leads to increased pathologic neovascularization, resulting in pronounced retinopathy and increased tumor size. In contrast, physiologic vascularization is not regulated by SOCS3. In vitro, SOCS3 knockdown increases proliferation and sprouting of endothelial cells costimulated with IGF-1 and TNFα via reduced feedback inhibition of the STAT3 and mTOR pathways. These results identify SOCS3 as a pivotal endogenous feedback inhibitor of pathologic angiogenesis and a potential therapeutic target acting at the converging crossroads of growth factor- and cytokine-induced vessel growth.


Assuntos
Carcinoma Pulmonar de Lewis/prevenção & controle , Hipóxia/patologia , Melanoma Experimental/prevenção & controle , Neovascularização Patológica/prevenção & controle , Síndromes Paraneoplásicas Oculares/prevenção & controle , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Animais , Western Blotting , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/patologia , Proliferação de Células , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Integrases/metabolismo , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/etiologia , Síndromes Paraneoplásicas Oculares/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
19.
J Invest Dermatol ; 131(12): 2467-76, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21938011

RESUMO

Infantile hemangioma (IH) is a common childhood vascular tumor. Although benign, some hemangiomas cause deformation and destruction of features or endanger life. The current treatments, corticosteroid or propranolol, are administered for several months and can have adverse effects on the infant. We designed a high-throughput screen to identify the Food and Drug Administration-approved drugs that could be used to treat this tumor. Rapamycin, an mTOR (mammalian target of Rapamycin) inhibitor, was identified, based on its ability to inhibit proliferation of a hemangioma-derived stem cell population, human vasculogenic cells, which we had previously discovered. In vitro and in vivo studies show that Rapamycin reduces the self-renewal capacity of the hemangioma stem cells, diminishes differentiation potential, and inhibits the vasculogenic activity of these cells in vivo. Longitudinal in vivo imaging of blood flow through vessels formed with hemangioma stem cells shows that Rapamycin also leads to regression of hemangioma blood vessels, consistent with its known anti-angiogenic activity. Finally, we demonstrate that Rapamycin-induced loss of stemness can work in concert with corticosteroid, the current standard therapy for problematic hemangioma, to block hemangioma formation in vivo. Our studies reveal that Rapamycin targets the self-renewal and vascular differentiation potential in patient-derived hemangioma stem cells, and suggests a novel therapeutic strategy to prevent formation of this disfiguring and endangering childhood tumor.


Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Hemangioma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Sirolimo/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Dexametasona/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Lactente
20.
Am J Pathol ; 179(5): 2266-77, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21945324

RESUMO

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a member of the VEGFR family, and binds to VEGF-A, VEGF-B, and placental growth factor. VEGFR-1 contributes to tumor growth and metastasis, but its role in the pathological formation of blood vessels is still poorly understood. Herein, we used infantile hemangioma (IH), the most common tumor of infancy, as a means to study VEGFR-1 activation in pathological vasculogenesis. IH arises from stem cells (HemSCs) that can form the three most prominent cell types in the tumor: endothelial cells, pericytes, and adipocytes. HemSCs can recapitulate the IH life cycle when injected in immuncompromised mice, and are targeted by corticosteroids, the traditional treatment for IH. We report here that VEGF-A or VEGF-B induces VEGFR-1-mediated ERK1/2 phosphorylation in HemSCs and promotes differentiation of HemSCs to endothelial cells. Studies of VEGFR-2 phosphorylation status and down-regulation of neuropilin-1 in the HemSCs demonstrate that VEGFR-2 and NRP1 are not needed for VEGF-A- or VEGF-B-induced ERK1/2 activation. U0216-mediated blockade of ERK1/2 phosphorylation or shRNA-mediated suppression of VEGFR-1 prevents HemSC-to-EC differentiation. Furthermore, the down-regulation of VEGFR-1 in the HemSCs results in decreased formation of blood vessels in vivo and reduced ERK1/2 activation. Thus, our study reveals a critical role for VEGFR-1 in the HemSC-to-EC differentiation that underpins pathological vasculogenesis in IH.


Assuntos
Células Endoteliais/citologia , Hemangioma/irrigação sanguínea , Neovascularização Patológica/fisiopatologia , Neoplasias Cutâneas/irrigação sanguínea , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Proliferação de Células , Transformação Celular Neoplásica/patologia , Inibidores Enzimáticos/farmacologia , Hemangioma/patologia , Humanos , Lactente , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Nus , Microvasos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Neuropilina-1/metabolismo , Fosforilação , Neoplasias Cutâneas/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA