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1.
Nat Commun ; 11(1): 2869, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513927

RESUMO

Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3CAH1047R mutation, resulting in constitutive activation of the p110α PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CAH1047R-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110α activation determining the LM subtype. In the postnatal vasculature, PIK3CAH1047R promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways.


Assuntos
Carcinogênese/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Vasos Linfáticos/anormalidades , Mutação/genética , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular , Criança , Células Endoteliais/metabolismo , Ativação Enzimática , Feminino , Humanos , Vasos Linfáticos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Serina-Treonina Quinases TOR/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
2.
Nat Commun ; 11(1): 3159, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572022

RESUMO

Extravasated erythrocytes in cerebrospinal fluid (CSF) critically contribute to the pathogenesis of subarachnoid hemorrhage (SAH). Meningeal lymphatics have been reported to drain macromolecules and immune cells from CSF into cervical lymph nodes (CLNs). However, whether meningeal lymphatics are involved in clearing extravasated erythrocytes in CSF after SAH remains unclear. Here we show that a markedly higher number of erythrocytes are accumulated in the lymphatics of CLNs and meningeal lymphatics after SAH. When the meningeal lymphatics are depleted in a mouse model of SAH, the degree of erythrocyte aggregation in CLNs is significantly lower, while the associated neuroinflammation and the neurologic deficits are dramatically exacerbated. In addition, during SAH lymph flow is increased but without significant lymphangiogenesis and lymphangiectasia. Taken together, this work demonstrates that the meningeal lymphatics drain extravasated erythrocytes from CSF into CLNs after SAH, while suggesting that modulating this draining may offer therapeutic approaches to alleviate SAH severity.


Assuntos
Sistema Linfático/patologia , Meninges/patologia , Hemorragia Subaracnóidea/patologia , Animais , Lesões Encefálicas , Eritrócitos , Linfonodos/fisiopatologia , Linfangiogênese , Vasos Linfáticos/fisiopatologia , Masculino , Meningite/etiologia , Meningite/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Pescoço , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(2): 157-163, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32314714

RESUMO

Objective To investigate the effect of matrine on gastric mucosal injury induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in rats and its mechanism. Methods A total of 75 Wister rats were randomly divided into a control group, a model group and three matrine-treated groups (100, 150 and 200 mg/kg). Except for the control group, the other groups were treated with MNNG to establish the models of gastric mucosal injury in the rats. After the models were successfully established, the rats in the three matrine-treated groups were administrated 100, 150, 200 mg/kg matrine, respectively, for successive 45 days. After the last administration, the body mass, daily intake of drinking water and dietary of rats were measured. And then the tissue samples were collected after the rats were sacrificed. The levels of interleukin 1ß (IL-1ß), IL-4, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) were measured by ELISA in gastric mucosa. HE staining was used to observe the pathological changes of gastric mucosa tissue. Immunohistochemical staining was performed to evaluate the expression of vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR3) in gastric mucosa. The protein levels of Bcl2, BAX, caspase-3, cytochrome C (Cyt-C), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor κB p65 (NF-κB p65) were determined by Western blotting. Results The body mass, daily intake of drinking water and dietary increased in matrine-treated rats in comparison with the model group. In addition, compared with the model group, matrine significantly reduced the expression levels of VEGF-C, VEGFR3, BAX, caspase-3, Cyt-C, TLR4, MyD88 and NF-κB p65, and increase Bcl2 protein level in the gastric mucosa tissues. Conclusion Matrine can reduce gastric mucosal damage induced by MNNG in rats, which is related to the down-regulation of VEGF-C/VEGFR3 and NF-κB/TLR4 signaling pathway.


Assuntos
Alcaloides/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Metilnitronitrosoguanidina/efeitos adversos , Quinolizinas/farmacologia , Animais , NF-kappa B/metabolismo , Ratos , Ratos Wistar , Receptor 4 Toll-Like/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
4.
Adv Exp Med Biol ; 1232: 393-399, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893436

RESUMO

Although the existence of the primo vasculature system has been shown in many species, including mice, rats, rabbits and humans, the biological role of this system, including expression of genes and proteins, has not yet been investigated. Especially the transcriptional action by mRNA, which is required for biological action, needs to be studied in primo vasculature biology. Differentially expressed genes in both isolated primo vessels and lymphatic vessels of rabbits were analyzed by RNA sequencing experiments. Primer efficiency and RNA purity of the primo vessels under lipopolysaccharides were confirmed prior to performing real-time qRT-PCR analysis following RNA extraction. We demonstrated that FLT4 was enriched in primo vessels and that several genes, including HSPH1 and EPHB2, were highly expressed in primo vessels compared with lymphatic vessels. Our data show that almost all genes, except HSPA4, were increased or sustained in isolated primo vessels compared with lymphatic vessels (FLT4 2.58 fold, HSPH1 1.83 fold, EPHB2 1.52 fold; whereas HSPA4 decreased 0.50 fold), suggesting primo vessels as a central regulator in diverse physiology. This implies that FLT4, HSPH1, and EPHB2 in high amounts may be involved in the functional activity of primo vessels. Our experimental data show that several genes are highly enriched in primo vessels in the lymphatic vessels of the rabbit.


Assuntos
Regulação da Expressão Gênica , Vasos Linfáticos , RNA-Seq , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Proteínas de Choque Térmico HSP110/genética , Vasos Linfáticos/metabolismo , RNA Mensageiro/genética , Coelhos , Receptor EphB2/genética , Análise de Sequência de RNA , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética
5.
Int J Mol Sci ; 20(24)2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31817940

RESUMO

In systemic sclerosis (SSc), the possible involvement of lymphatic microcirculation and lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on dysfunctional blood vascular system and angiogenesis. In the present in vitro study, we explore for the first time whether the SSc microenvironment may interfere with lymphangiogenesis, a complex, multi-step process in which lymphatic microvascular endothelial cells (LMVECs) sprout, migrate, and proliferate to generate new lymphatic capillaries. Normal human adult dermal LMVECs from three donors were treated with serum from SSc patients (n = 8), serum from healthy individuals (n = 8), or recombinant human vascular endothelial growth factor (VEGF)-C as a positive control for lymphangiogenesis. Cell proliferation, Boyden chamber Matrigel chemoinvasion, wound healing capacity, and lymphatic capillary morphogenesis on Geltrex were assayed. VEGF-C serum levels were measured by enzyme-linked immunosorbent assay. Gene and protein expression levels of the lymphangiogenic orchestrators VEGF receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) were determined by real-time PCR and Western blotting, respectively. Conditioning with SSc serum significantly inhibited LMVEC proliferation, Matrigel invasion, and wound healing capacity with respect to healthy serum. The ability of LMVECs to form lymphatic tubes on Geltrex was also severely compromised in the presence of SSc serum. VEGF-C levels were comparable in SSc and healthy sera. Treatment with SSc serum resulted in a significant downregulation of both VEGFR-3/Flt-4 and NRP-2 mRNA and protein levels. In SSc, the pathologic environment severely hampers every lymphangiogenesis step, likely through the reduction of pro-lymphangiogenic VEGFR-3/NRP-2 co-receptor signaling. The impairment of the lymphangiogenic process opens a new scenario underlying SSc vascular pathophysiology, which is worth investigating further.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/patologia , Linfangiogênese , Neovascularização Patológica/patologia , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/patologia , Microambiente Tumoral , Adulto , Apoptose , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Neuropilina-2/genética , Neuropilina-2/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 1023-1029, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31878999

RESUMO

Objective To explore the expression of vascular endothelial growth factor receptor 3 (VEGFR3) and its significance in lung adenocarcinoma and to examine the effect of VEGFR3 knockdown on the biological behaviors of A549 cells. Methods Immunohistochemistry was used to detect the expression of VEGFR3 in 78 pieces of lung adenocarcinoma tissue and 35 of paracancerous tissue. Relationships between VEGFR3 and clinicopathological indices were also analyzed. Correlations between lung adenocarcinoma patient survival and the expression of vascular endothelial growth factor-C (VEGF-C) or VEGFR3 were analyzed using the TCGA database. VEGFR3 expression was knocked down in A549 cells using RNA interference, and cell proliferation was assessed using a CCK-8 assay. Cell migration and invasion were detected using TranswellTM assays. The effect of siRNA-mediated knockdown of EGFR3 in A549 cells on AKT pathway activity was assessed by Western blot analysis. Results Expression of VEGFR3 was significantly higher in the lung adenocarcinoma tissue than in the adjacent tissue, and positively correlated with TNM stage and lymph node metastasis. The survival rate of patients with high VEGFR3 expression was significantly lower than that of patients with low VEGFR3 expression. Exogenous VEGF-C promoted the expression of VEGFR3, and activated the AKT signaling pathway. Silencing of VEGFR3 inhibited the proliferation, migration, and invasiveness of A549 cells, and reduced the activation of the AKT signaling pathway by VEGF-C. Conclusion High expression of VEGFR3 in the lung adenocarcinoma tissue is positively correlated with poor prognosis. Silencing VEGFR3 can block AKT pathway activity and inhibit the proliferation, migration, and invasion of A549 cells.


Assuntos
Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/patologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Invasividade Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo
7.
Nat Commun ; 10(1): 5306, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757960

RESUMO

After stroke, peripheral immune cells are activated and these systemic responses may amplify brain damage, but how the injured brain sends out signals to trigger systemic inflammation remains unclear. Here we show that a brain-to-cervical lymph node (CLN) pathway is involved. In rats subjected to focal cerebral ischemia, lymphatic endothelial cells proliferate and macrophages are rapidly activated in CLNs within 24 h, in part via VEGF-C/VEGFR3 signalling. Microarray analyses of isolated lymphatic endothelium from CLNs of ischemic mice confirm the activation of transmembrane tyrosine kinase pathways. Blockade of VEGFR3 reduces lymphatic endothelial activation, decreases pro-inflammatory macrophages, and reduces brain infarction. In vitro, VEGF-C/VEGFR3 signalling in lymphatic endothelial cells enhances inflammatory responses in co-cultured macrophages. Lastly, surgical removal of CLNs in mice significantly reduces infarction after focal cerebral ischemia. These findings suggest that modulating the brain-to-CLN pathway may offer therapeutic opportunities to ameliorate systemic inflammation and brain injury after stroke.


Assuntos
Infarto Encefálico/imunologia , Isquemia Encefálica/imunologia , Encéfalo/imunologia , Endotélio Linfático/imunologia , Linfonodos/imunologia , Macrófagos/imunologia , Fator C de Crescimento do Endotélio Vascular/imunologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologia , Animais , Encéfalo/metabolismo , Infarto Encefálico/metabolismo , Isquemia Encefálica/metabolismo , Proliferação de Células , Células Endoteliais , Endotélio Linfático/metabolismo , Inflamação , Linfonodos/metabolismo , Linfangiogênese , Camundongos , Pescoço , Ratos , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
8.
Elife ; 82019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31702553

RESUMO

The cardiac lymphatic vascular system and its potentially critical functions in heart patients have been largely underappreciated, in part due to a lack of experimentally accessible systems. We here demonstrate that cardiac lymphatic vessels develop in young adult zebrafish, using coronary arteries to guide their expansion down the ventricle. Mechanistically, we show that in cxcr4a mutants with defective coronary artery development, cardiac lymphatic vessels fail to expand onto the ventricle. In regenerating adult zebrafish hearts the lymphatic vasculature undergoes extensive lymphangiogenesis in response to a cryoinjury. A significant defect in reducing the scar size after cryoinjury is observed in zebrafish with impaired Vegfc/Vegfr3 signaling that fail to develop intact cardiac lymphatic vessels. These results suggest that the cardiac lymphatic system can influence the regenerative potential of the myocardium.


Assuntos
Coração/fisiologia , Linfangiogênese/fisiologia , Vasos Linfáticos/fisiopatologia , Miocárdio/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Vasos Coronários/metabolismo , Vasos Coronários/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Coração/crescimento & desenvolvimento , Humanos , Linfangiogênese/genética , Vasos Linfáticos/lesões , Vasos Linfáticos/metabolismo , Mutação , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Regeneração/genética , Regeneração/fisiologia , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Basic Res Cardiol ; 114(6): 43, 2019 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-31587086

RESUMO

Impairment of cardiac lymphatic vessels leads to cardiac lymphedema. Recent studies have suggested that stimulation of lymphangiogenesis may reduce cardiac lymphedema. However, effects of lymphatic endothelial progenitor cells (LEPCs) on cardiac lymphangiogenesis are poorly understood. Therefore, this study investigated effectiveness of LEPC transplantation and VEGF-C release with self-assembling peptide (SAP) on cardiac lymphangiogenesis after myocardial infarction (MI). CD34+VEGFR-3+ EPCs isolated from rat bone marrow differentiated into lymphatic endothelial cells after VEGF-C induction. VEGF-C also stimulated the cells to incorporate into the lymphatic capillary-like structures. The functionalized SAP could adhere with the cells and released VEGF-C sustainedly. In the condition of hypoxia and serum deprivation or abdominal pouch assay, the SAP hydrogel protected the cells from apoptosis and necrosis. At 4 weeks after intramyocardial transplantation of the cells and VEGF-C loaded with SAP hydrogel in rat MI models, cardiac lymphangiogenesis was increased, cardiac edema and reverse remodeling were reduced, and cardiac function was improved significantly. Delivery with SAP hydrogel favored survival of the engrafted cells. VEGF-C released from the hydrogel promoted differentiation and incorporation of the cells as well as growth of pre-existed lymphatic vessels. Cardiac lymphangiogenesis was beneficial for elimination of the inflammatory cells in the infarcted myocardium. Moreover, angiogenesis and myocardial regeneration were enhanced after reduction of lymphedema. These results demonstrate that the combined delivery of LEPCs and VEGF-C with the functionalized SAP promotes cardiac lymphangiogenesis and repair of the infarcted myocardium effectively. This study represents a novel therapy for relieving myocardial edema in cardiovascular diseases.


Assuntos
Edema Cardíaco/terapia , Células Progenitoras Endoteliais/transplante , Linfangiogênese , Fator C de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Antígenos CD34/metabolismo , Células Progenitoras Endoteliais/metabolismo , Masculino , Miocárdio/metabolismo , Neovascularização Fisiológica , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/sangue , Fator C de Crescimento do Endotélio Vascular/sangue , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
10.
Invest Ophthalmol Vis Sci ; 60(13): 4084-4096, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31574534

RESUMO

Purpose: To investigate whole transcriptional differences between proliferative diabetic retinopathy (PDR) neovascular membranes (NVMs) and retinas, and the regulatory genes participating in retinal neovascularization in PDR. Methods: We used high-throughput sequencing technology to capture the whole-genome gene expression levels of all participants, including 23 patients with PDR or branch retinal vein occlusion (BRVO), 3 normal retinal samples, and 2 retinal samples from type II diabetic (T2D) eyes by donation, followed by analyses of expression patterns using bioinformatics methods, then validation of the data by in situ hybridization and Western blotting. Results: We showed that transcriptional profiles of the NVMs were distinct from those of the retinas. Angiogenesis growth factors VEGFC, ANGPT1, ANGPT2, and EFNB2, and their receptors FLT4, TIE1, TIE2, and EPHB4, respectively, were overexpressed. Expression of VEGFA was highly upregulated in T2D retina, but low in the NVMs, while angiogenesis transcription factors, including ETS1 and ERG, were coordinately upregulated in NVMs. Conclusions: This study described a PDR neovascularization model in which pathological retina-secreted vascular endothelial growth factor A (VEGFA) enhanced the expression of a set of angiogenesis transcription factors and growth factors, to cooperatively induce the retinal neovascularization. Based on these results, novel potential therapeutic targets and biomarkers for PDR treatment and diagnosis are suggested.


Assuntos
Angiopoietina-1/metabolismo , Retinopatia Diabética/metabolismo , Efrina-B2/metabolismo , Neovascularização Retiniana/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Humanos , Receptor EphB4/metabolismo , Receptor de TIE-1/metabolismo , Receptor TIE-2/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Oclusão da Veia Retiniana/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
11.
Iran J Allergy Asthma Immunol ; 18(3): 289-299, 2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31522436

RESUMO

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologia , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos , Cromatografia de Afinidade , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Relação Estrutura-Atividade , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/química
12.
Am J Pathol ; 189(10): 2102-2114, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31369756

RESUMO

Tumor-associated lymphangiogenesis correlates with lymph node metastasis and poor outcome in several human malignancies. In addition, the presence of functional lymphatic vessels regulates the formation of tumor inflammatory and immune microenvironments. Although lymphatic structures are often found deeply integrated into the fabric of adipose tissue, the impact of lymphangiogenesis on tumor-associated adipose tissue (AT) has not yet been investigated. Using K14-VEGFR3-Ig mice that constitutively express soluble vascular endothelial growth factor receptor (VEGFR) 3-Ig in the skin, scavenging VEGF-C and VEGF-D, the role of lymphangiogenesis in the generation of an inflammatory response within tumor-associated AT was studied. Macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 were found within peritumoral adipose tissue from melanoma-bearing K14-VEGFR3-Ig mice, which were further enriched with alternatively activated macrophages based on surface marker CD301/C-type lectin domain family 10 member A expression. The blockade of lymphangiogenesis also resulted in accumulation of the cytokine IL-6, which correlated with enhanced macrophage proliferation of the alternatively activated phenotype. Furthermore, melanomas co-implanted with freshly isolated adipose tissue macrophages grew more robustly than melanomas growing alone. In human cutaneous melanomas, adipocyte-selective FABP4 transcripts closely correlated with gene signatures of CLEC10A and were associated with poor overall survival. These data suggest that the blockade of pathways regulating lymphatic vessel formation shapes an inflammatory response within tumor-associated AT by facilitating accumulation of tumor-promoting alternatively activated macrophages.


Assuntos
Tecido Adiposo/patologia , Inflamação/patologia , Linfangiogênese , Melanoma Experimental/patologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/imunologia , Animais , Feminino , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas , Microambiente Tumoral
13.
Curr Pharm Biotechnol ; 20(12): 1004-1010, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31333122

RESUMO

BACKGROUND: Vascular endothelial growth factor (VEGF)-C, -D, and VEGF receptor-3 are proteins characterized as crucial for tumor lymphangiogenesis. It is accompanied by angiogenesis during wound healing, but also in the neoplastic process. The research studies have shown that the lymphatic system plays a key role in the progression of carcinogenesis. OBJECTIVE: The aim of this study was to evaluate changes in the expression of VEGF-C, VEGF-D and VEGFR-3 in different grades of endometrial cancer (G1-G3). METHODS: The study included 45 patients diagnosed with endometrial cancer (G1=17; G2=15; G3=13) and 15 patients without neoplastic changes. The expression of VEGF-C, VEGF-D, and VEGFR-3 was assessed using microarray technique and immunohistochemistry. Statistical analysis was performed using the one-way ANOVA and Tukey's post-hoc test. RESULTS: Statistically significant changes in the expression at the transcriptome level were found only in the case of VEGF-C (G1 vs. C, fold change - FC = -1.15; G2 vs. C, FC = -2.33; G3 vs. C, FC = - 1.68). However, VEGF-D and VEGFR-3 were expressed at the protein level. Analysis of VEGF-D expression showed that the optical density of the reaction product in G1 reached 101.7, while the values in G2 and G3 were 142.7 and 184.4, respectively. For VEGF-R3, the optical density of the reaction product reached the following levels: 72 in control, 118.77 in G1, 145.8 in G2, and 170.9 in G3. CONCLUSION: An increase in VEGF-D and VEGFR-3 levels may indicate that VEGF-D-dependent processes are intensified along with the dedifferentiation of tumor cells. The lack of VEGF-C expression in endometrial cancer samples may suggest that this tumor is characterized by a different mechanism of metastasis than EMT. Our study emphasizes that when analyzing the metastatic potential of cancer, the expression of more than one factor should be taken into account.


Assuntos
Neoplasias do Endométrio/genética , Neovascularização Patológica/genética , Fator C de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Linfangiogênese/genética , Gradação de Tumores
14.
Nature ; 572(7767): 62-66, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31341278

RESUMO

Recent work has shown that meningeal lymphatic vessels (mLVs), mainly in the dorsal part of the skull, are involved in the clearance of cerebrospinal fluid (CSF), but the precise route of CSF drainage is still unknown. Here we reveal the importance of mLVs in the basal part of the skull for this process by visualizing their distinct anatomical location and characterizing their specialized morphological features, which facilitate the uptake and drainage of CSF. Unlike dorsal mLVs, basal mLVs have lymphatic valves and capillaries located adjacent to the subarachnoid space in mice. We also show that basal mLVs are hotspots for the clearance of CSF macromolecules and that both mLV integrity and CSF drainage are impaired with ageing. Our findings should increase the understanding of how mLVs contribute to the neuropathophysiological processes that are associated with ageing.


Assuntos
Líquido Cefalorraquidiano/metabolismo , Sistema Glinfático/anatomia & histologia , Sistema Glinfático/fisiologia , Vasos Linfáticos/anatomia & histologia , Vasos Linfáticos/fisiologia , Base do Crânio/anatomia & histologia , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Células Endoteliais/citologia , Células Endoteliais/patologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Sistema Glinfático/citologia , Sistema Glinfático/patologia , Proteínas de Homeodomínio/metabolismo , Vasos Linfáticos/citologia , Vasos Linfáticos/patologia , Linfedema/metabolismo , Linfedema/patologia , Imagem por Ressonância Magnética , Masculino , Camundongos , Espaço Subaracnóideo/anatomia & histologia , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
15.
PLoS One ; 14(7): e0220341, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31344105

RESUMO

The lymphatic vascular system plays important roles in the control of tissue fluid homeostasis and immune responses. While VEGF-A-induced angiogenesis promotes hair follicle (HF) growth, the potential role of lymphatic vessels (LVs) in HF cycling has remained unknown. In this study, we found that LVs are localized in close proximity to the HF bulge area throughout the postnatal and depilation-induced hair cycle in mice and that a network of LVs directly connects the individual HFs. Increased LV density in the skin of K14-VEGF-C transgenic mice was associated with prolongation of anagen HF growth. Conversely, HF entry into the catagen phase was accelerated in K14-sVEGFR3 transgenic mice that lack cutaneous LVs. Importantly, repeated intradermal injections of VEGF-C promoted hair growth in mice. Conditioned media from lymphatic endothelial cells promoted human dermal papilla cell (DPC) growth and expression of IGF-1 and alkaline phosphatase, both activators of DPCs. Our results reveal an unexpected role of LVs in coordinating and promoting HF growth and identify potential new therapeutic strategies for hair loss-associated conditions.


Assuntos
Folículo Piloso/crescimento & desenvolvimento , Linfangiogênese/fisiologia , Vasos Linfáticos/fisiologia , Pele/crescimento & desenvolvimento , Animais , Ciclo Celular/fisiologia , Proliferação de Células/genética , Células Cultivadas , Derme/citologia , Derme/crescimento & desenvolvimento , Feminino , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Remoção de Cabelo , Humanos , Linfangiogênese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regeneração/genética , Pele/citologia , Pele/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética
16.
BMC Cancer ; 19(1): 584, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31200683

RESUMO

BACKGROUND: In many malignancies including ovarian cancer, different angiogenic factors have been related to poor prognosis. However, data on their relations to each other or importance as a prognostic factor in ovarian cancer is missing. Therefore, we investigated the expressions of VEGF-A, VEGF-C, and VEGF-D, and the receptors VEGFR1, VEGFR2, and VEGFR3 in patients with malignant epithelial ovarian neoplasms. We further compared expression levels between primary tumors and related distant omental metastases. METHODS: This study included 86 patients with malignant ovarian epithelial tumors and 16 related distant metastases. Angiogenic factor expression was evaluated using immunohistochemistry (n = 102) and qRT-PCR (n = 29). RESULTS: Compared to primary high grade serous ovarian tumors, the related omental metastases showed higher expressions of VEGF-A (p = 0.022), VEGF-D (p = 0.010), and VEGFR1 (p = 0.046). In univariate survival analysis, low epithelial expression of VEGF-A in primary tumors was associated with poor prognosis (p = 0.024), and short progression-free survival was associated with high VEGF-C (p = 0.034) and low VEGFR3 (p = 0.002). The relative expressions of VEGF-D, VEGFR1, VEGFR2, and VEGFR3 mRNA determined by qRT-PCR analyses were significantly correlated with the immunohistochemically detected levels of these proteins in primary high grade serous ovarian cancer and metastases (p = 0.004, p = 0.009, p = 0.015, and p = 0.018, respectively). CONCLUSIONS: The expressions of VEGF receptors and their ligands significantly differed between malignant ovarian tumors and paired distant metastases. VEGF-A, VEGF-D, and VEGFR1 protein expressions seem to be higher in distant metastases than in the primary high grade serous ovarian cancer lesions.


Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Neoplasias Ovarianas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/secundário , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/secundário , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neovascularização Patológica/diagnóstico , Neoplasias Ovarianas/patologia , Prognóstico , Estudos Retrospectivos , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
17.
Dev Biol ; 452(2): 134-143, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31112709

RESUMO

The origin of the mammalian lymphatic vasculature has been studied for more than a century; however, details regarding organ-specific lymphatic development remain unknown. A recent study reported that cardiac lymphatic endothelial cells (LECs) stem from venous and non-venous origins in mice. Here, we identified Isl1-expressing progenitors as a potential non-venous origin of cardiac LECs. Genetic lineage tracing with Isl1-Cre reporter mice suggested a possible contribution from the Isl1-expressing pharyngeal mesoderm constituting the second heart field to lymphatic vessels around the cardiac outflow tract as well as to those in the facial skin and the lymph sac. Isl1+ lineage-specific deletion of Prox1 resulted in disrupted LYVE1+ vessel structures, indicating a Prox1-dependent mechanism in this contribution. Tracing back to earlier embryonic stages revealed the presence of VEGFR3+ and/or Prox1+ cells that overlapped with the Isl1+ pharyngeal core mesoderm. These data may provide insights into the developmental basis of heart diseases involving lymphatic vasculature and improve our understanding of organ-based lymphangiogenesis.


Assuntos
Linhagem da Célula , Coração/embriologia , Proteínas com Homeodomínio LIM/metabolismo , Linfangiogênese , Vasos Linfáticos/citologia , Vasos Linfáticos/embriologia , Fatores de Transcrição/metabolismo , Animais , Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Faringe/citologia , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Mar Drugs ; 17(4)2019 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959907

RESUMO

Lymphangiogenesis is an important biological process associated with cancer metastasis. The development of new drugs that block lymphangiogenesis represents a promising therapeutic strategy. Marine fungus-derived compound phomaketide A, isolated from the fermented broth of Phoma sp. NTOU4195, has been reported to exhibit anti-angiogenic and anti-inflammatory effects. However, its anti-lymphangiogenic activity has not been clarified to date. In this study, we showed that phomaketide A inhibited cell growth, migration, and tube formation of lymphatic endothelial cells (LECs) without an evidence of cytotoxicity. Mechanistic investigations revealed that phomaketide A reduced LECs-induced lymphangiogenesis via vascular endothelial growth factor receptor-3 (VEGFR-3), protein kinase Cδ (PKCδ), and endothelial nitric oxide synthase (eNOS) signalings. Furthermore, human proteome array analysis indicated that phomaketide A significantly enhanced the protein levels of various protease inhibitors, including cystatin A, serpin B6, tissue factor pathway inhibitor (TFPI), and tissue inhibitor matrix metalloproteinase 1 (TIMP-1). Importantly, phomaketide A impeded tumor growth and lymphangiogenesis by decreasing the expression of LYVE-1, a specific marker for lymphatic vessels, in tumor xenograft animal model. These results suggest that phomaketide A may impair lymphangiogenesis by suppressing VEGFR-3, PKCδ, and eNOS signaling cascades, while simultaneously activating protease inhibitors in human LECs. We document for the first time that phomaketide A inhibits lymphangiogenesis both in vitro and in vivo, which suggests that this natural product could potentially treat cancer metastasis.


Assuntos
Inibidores da Angiogênese/farmacologia , Antinematódeos/farmacologia , Ascomicetos/química , Linfangiogênese/efeitos dos fármacos , Policetídeos/farmacologia , Células A549 , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/uso terapêutico , Animais , Antinematódeos/isolamento & purificação , Antinematódeos/uso terapêutico , Organismos Aquáticos/química , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Metástase Linfática , Vasos Linfáticos/citologia , Masculino , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Policetídeos/isolamento & purificação , Policetídeos/uso terapêutico , Proteína Quinase C-delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Cell Death Dis ; 10(3): 219, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833548

RESUMO

Recent studies have demonstrated that chronic inflammation-induced lymphangiogenesis plays a crucial role in the progression of various renal diseases, including diabetic nephropathy. SAR131675 is a selective vascular endothelial cell growth factor receptor-3 (VEGFR-3)-tyrosine kinase inhibitor that acts as a ligand for VEGF-C and VEGF-D to inhibit lymphangiogenesis. In this study, we evaluated the effect of SAR131675 on renal lymphangiogenesis in a mouse model of type 2 diabetes. Male C57BLKS/J db/m and db/db mice were fed either a regular chow diet or a diet containing SAR131675 for 12 weeks from 8 weeks of age. In addition, we studied palmitate-induced lymphangiogenesis in human kidney-2 (HK2) cells and RAW264.7 monocytes/macrophages, which play a major role in lymphangiogenesis in the kidneys. SAR131475 ameliorated dyslipidemia, albuminuria, and lipid accumulation in the kidneys of db/db mice, with no significant changes in glucose and creatinine levels and body weight. Diabetes-induced systemic inflammation as evidenced by increased systemic monocyte chemoattractant protein-1 and tumor necrosis factor-α level was decreased by SAR131475. SAR131475 ameliorated the accumulation of triglycerides and free fatty acids and reduced inflammation in relation to decreased chemokine expression and pro-inflammatory M1 macrophage infiltration in the kidneys. Downregulation of VEGF-C and VEGFR-3 by SAR131475 inhibited lymphatic growth as demonstrated by decreased expression of LYVE-1 and podoplanin that was further accompanied by reduced tubulointerstitial fibrosis, and inflammation in relation to improvement in oxidative stress and apoptosis. Treatment with SAR131475 improved palmitate-induced increase in the expression of VEGF-C, VEGFR-3, and LYVE-1, along with improvement in cytosolic and mitochondrial oxidative stress in RAW264.7 and HK2 cells. Moreover, the enhanced expression of M1 phenotypes in RAW264.7 cells under palmitate stress was reduced by SAR131475 treatment. The results suggest that modulation of lymphatic proliferation in the kidneys is a new treatment approach for type 2 diabetic nephropathy and that SAR131675 is a promising therapy to ameliorate renal damage by reducing lipotoxicity-induced lymphangiogenesis.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Naftiridinas/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Linfangiogênese/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/uso terapêutico , Células RAW 264.7 , Triglicerídeos/metabolismo
20.
Mol Med Rep ; 19(4): 2740-2748, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816473

RESUMO

The peroxisome proliferator­activated receptor γ (PPARγ) agonist pioglitazone has been widely used in previous studies to ameliorate diabetes mellitus and regulate inflammation. However, the present study aimed to investigate the effect of pioglitazone on macrophages and determine its impact on renal fibrosis in vivo. Firstly, bone marrow­derived macrophages (BMDM) were used to detect the effects of pioglitazone on macrophages in vitro. It was demonstrated that pioglitazone promoted M2 macrophage activation and induced vascular endothelial growth factor receptor 3 (VEGFR3) upregulation in a PPARγ­dependent manner. Furthermore, pioglitazone increased macrophage proliferation and macrophage VEGFR3 expression in a murine unilateral ureteral obstruction (UUO) model; however, it had no therapeutic effect on renal fibrosis in vivo. Therefore, the results in the present study implied that presence of M2 macrophages may inhibit pioglitazone's ability to attenuate UUO­induced renal fibrosis. In addition, the results demonstrated that macrophage­associated VEGFR3 could be induced by pioglitazone, although it is still unclear what role VEGFR3+ M2 macrophages have in renal fibrosis.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , PPAR gama/metabolismo , Pioglitazona/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Fibrose , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Masculino , Camundongos
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