RESUMO
Disorganized vessels in the tumor vasculature lead to impaired perfusion, resulting in reduced accessibility to immune cells and chemotherapeutic drugs. In the breast tumor-stroma interplay, paracrine factors such as interleukin-6 (IL-6) often facilitate disordered angiogenesis. We show here that epigenetic mechanisms regulate the crosstalk between IL-6 and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in myoepithelial (CD10+) and endothelial (CD31+, CD105+, CD146+, and CD133-) cells isolated from malignant and nonmalignant tissues of clinically characterized human breast tumors. Tumor endothelial (Endo-T) cells in 3D cultures exhibited higher VEGFR2 expression levels, accelerated migration, invasion, and disorganized sprout formation in response to elevated IL-6 levels secreted by tumor myoepithelial (Epi-T) cells. Constitutively, compared with normal endothelial (Endo-N) cells, Endo-T cells differentially expressed DNA methyltransferase isoforms and had increased levels of IL-6 signaling intermediates such as IL-6R and signal transducer and activator of transcription 3 (STAT3). Upon IL-6 treatment, Endo-N and Endo-T cells displayed altered expression of the DNA methyltransferase 1 (DNMT1) isoform. Mechanistic studies revealed that IL-6 induced proteasomal degradation of DNMT1, but not of DNMT3A and DNMT3B and subsequently led to promoter hypomethylation and expression/activation of VEGFR2. IL-6-induced VEGFR2 up-regulation was inhibited by overexpression of DNMT1. Transfection of a dominant-negative STAT3 mutant, but not of STAT1, abrogated VEGFR2 expression. Our results indicate that in the breast tumor microenvironment, IL-6 secreted from myoepithelial cells influences DNMT1 stability, induces the expression of VEGFR2 in endothelial cells via a promoter methylation-dependent mechanism, and leads to disordered angiogenesis.
Assuntos
Neoplasias da Mama , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Interleucina-6/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-6/genética , Células MCF-7 , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Receptor-interacting protein kinase 3 (RIPK3) is a multifunctional intracellular protein that was first recognized as an important component of the necroptosis programmed cell death pathway. RIPK3 is also highly expressed in non-necroptotic murine embryonic endothelial cells (ECs) during vascular development, indicating its potential contribution to angiogenesis. To test this hypothesis, we generated mice lacking endothelial RIPK3 and found non-lethal embryonic and perinatal angiogenesis defects in multiple vascular beds. Our in vitro data indicate that RIPK3 supports angiogenesis by regulating growth factor receptor degradation in ECs. We found that RIPK3 interacted with the membrane trafficking protein myoferlin to sustain expression of vascular endothelial growth factor receptor 2 (VEGFR2) in cultured ECs following vascular endothelial growth factor A (VEGFA) stimulation. Restoration of myoferlin, which was diminished after RIPK3 knockdown, rescued decreased VEGFR2 expression and vascular sprouting in RIPK3-deficient ECs after VEGFA treatment. In addition, we found that RIPK3 modulated expression of genes involved in endothelial identity by inhibiting ERK signaling independently of growth factor receptor turnover. Altogether, our data reveal unexpected non-necroptotic roles for RIPK3 in ECs and evidence that RIPK3 promotes developmental angiogenesis in vivo.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Neovascularização Fisiológica , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Camundongos , Camundongos Transgênicos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Neovascularização Fisiológica/fisiologia , Receptores Purinérgicos P2Y2/metabolismo , Angiopoietina-2/biossíntese , Antígenos CD34/biossíntese , Células Cultivadas , Humanos , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Fosforilação/fisiologia , Agregação Plaquetária/fisiologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores CXCR4/biossíntese , Receptores Purinérgicos P2Y2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
Aggregated amyloid ß (Aß) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aß peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aß1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aß oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aß1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aß1-42 monomers, and 5 µM Aß1-42 fibrils, respectively. Brain ECs treated with Aß1-42 oligomers showed increased senescence-associated ß-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aß1-42 monomer or Aß1-42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aß1-42 oligomer-treated ECs, and these cells showed significantly increased VEGFR-1 expression and decreased VEGFR-2 levels. Overexpression of VEGFR-1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR-1 signaling events in this paradigm. More importantly, small interfering RNA-mediated knockdown of VEGFR-1 expression in brain ECs was able to prevent up-regulation of p21 protein expression and significantly reduced induction of senescence following Aß1-42 oligomer treatment. Our studies show that exposure to Aß1-42 oligomers may impair vascular functions by altering VEGFR-1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR-1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD.-Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Senescência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Encéfalo/irrigação sanguínea , Capilares/citologia , Sobrevivência Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Tachykinins such as Substance P (SP) are a group of neuropeptides that are involved in cancer development. Neurokinin-1 receptor (NK-1R) is the main tachykinin receptor mediating the effects of SP, which is overexpressed in human esophageal squamous cell carcinoma (ESCC) and other malignant tissues. However, the effects of SP/NK-1R system on the migration of esophageal cancer cells and angiogenesis is not clear yet. This study seeks to obtain data to address these research gaps. In order to assess the effects of the FDA-approved aprepitant drug, a commercially available NK-1R antagonist, on the viability of KYSE-30 ESCC cells, resazurin assay was performed. The influence of SP/NK-1R system on the migration potential of these cells was examined using scratch assay. The effects of this system on the expression levels of metastatic factors were also examined by RT-PCR and western blot analyses. The half-maximal inhibitory concentration (IC50) value for KYSE-30 cells treated with aprepitant found to be 29.88 µM. Treatment with SP significantly promoted KYSE-30 esophageal cancer cell migration, and aprepitant blocked this effect. In addition, SP significantly induced the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, vascular endothelial growth factor-A (VEGF-A), and VEGF receptor1 (VEGFR1) in the cells, whereas aprepitant inhibited the up-regulation effects caused by SP. SP plays important roles in the development of human esophageal squamous cell carcinoma by promoting cancer cell invasion and enhancing the expression of factors involved in cellular migration and angiogenesis, which can be blocked by the NK-1R antagonist, aprepitant.
Assuntos
Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Substância P/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Apoptose/efeitos dos fármacos , Aprepitanto/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Relação Dose-Resposta a Droga , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
The vascular endothelial growth factor (VEGF), a homodimeric vasoactive glycoprotein, is the key mediator of angiogenesis. Angiogenesis, the formation of new blood vessels, is responsible for a wide variety of physio/pathological processes, including cardiovascular diseases (CVD). Cardiomyocytes (CM), the main cell type present in the heart, are the source and target of VEGF-A and express its receptors, VEGFR1 and VEGFR2, on their cell surface. The relationship between VEGF-A and the heart is double-sided. On the one hand, VEGF-A activates CM, inducing morphogenesis, contractility and wound healing. On the other hand, VEGF-A is produced by CM during inflammation, mechanical stress and cytokine stimulation. Moreover, high concentrations of VEGF-A have been found in patients affected by different CVD, and are often correlated with an unfavorable prognosis and disease severity. In this review, we summarized the current knowledge about the expression and effects of VEGF-A on CM and the role of VEGF-A in CVD, which are the most important cause of disability and premature death worldwide. Based on clinical studies on angiogenesis therapy conducted to date, it is possible to think that the control of angiogenesis and VEGF-A can lead to better quality and span of life of patients with heart disease.
Assuntos
Regulação da Expressão Gênica , Cardiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Cardiopatias/patologia , Humanos , Miócitos Cardíacos/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine-arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine-arginine-rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC-like kinases-1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.
Assuntos
Comunicação Autócrina/fisiologia , Regulação da Expressão Gênica/fisiologia , Células da Granulosa/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Bovinos , Feminino , Células da Granulosa/citologiaRESUMO
BACKGROUND: Cancer pain is a pervasive clinical symptom impairing life quality. Vascular endothelial growth factor A has been well studied in tumor angiogenesis and is recognized as a therapeutic target for anti-cancer treatment. This study tested the hypothesis that vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 contribute to bone cancer pain regulation associated with spinal central sensitization. METHODS: This study was performed on female rats using a metastatic breast cancer bone pain model. Nociceptive behaviors were evaluated by mechanical allodynia, thermal hyperalgesia, spontaneous pain, and CatWalk gait analysis. Expression levels were measured by real-time quantitative polymerase chain reaction, western blot, and immunofluorescence analysis. Excitatory synaptic transmission was detected by whole-cell patch-clamp recordings. The primary outcome was the effect of pharmacologic intervention of spinal vascular endothelial growth factor A/vascular endothelial growth factor receptor 2-signaling on bone cancer pain behaviors. RESULTS: The mRNA and protein expression of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 were upregulated in tumor-bearing rats. Spinal blocking vascular endothelial growth factor A or vascular endothelial growth factor receptor 2 significantly attenuated tumor-induced mechanical allodynia (mean ± SD: vascular endothelial growth factor A, 7.6 ± 2.6 g vs. 5.3 ± 3.3 g; vascular endothelial growth factor receptor 2, 7.8 ± 3.0 g vs. 5.2 ± 3.4 g; n = 6; P < 0.0001) and thermal hyperalgesia (mean ± SD: vascular endothelial growth factor A, 9.0 ± 2.4 s vs. 7.4 ± 2.7 s; vascular endothelial growth factor receptor 2, 9.3 ± 2.5 s vs. 7.5 ± 3.1 s; n = 6; P < 0.0001), as well as spontaneous pain and abnormal gaits. Exogenous vascular endothelial growth factor A enhanced excitatory synaptic transmission in a vascular endothelial growth factor receptor 2-dependent manner, and spinal injection of exogenous vascular endothelial growth factor A was sufficient to cause pain hypersensitivity via vascular endothelial growth factor receptor 2-mediated activation of protein kinase C and Src family kinase in naïve rats. Moreover, spinal blocking vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 pathways suppressed protein kinase C-mediated N-methyl-D-aspartate receptor activation and Src family kinase-mediated proinflammatory cytokine production. CONCLUSIONS: Vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 contributes to central sensitization and bone cancer pain via activation of neuronal protein kinase C and microglial Src family kinase pathways in the spinal cord.
Assuntos
Neoplasias Ósseas/metabolismo , Dor do Câncer/metabolismo , Medição da Dor/métodos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Neoplasias Ósseas/patologia , Dor do Câncer/patologia , Feminino , Injeções Espinhais , Medição da Dor/efeitos dos fármacos , Quinazolinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong antiangiogenic effect both on directly targeted cells and on naive cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.
Assuntos
Íntrons , Poliadenilação , Receptores Proteína Tirosina Quinases/biossíntese , Humanos , Neovascularização Fisiológica/fisiologia , Poli A/química , Poli A/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Splicing de RNA , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/fisiologia , Ribonucleoproteína Nuclear Pequena U1/fisiologia , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
This experiment investigated the impact of serum from patients with type 2 diabetes mellitus on the angiogenic behavior of human mesenchymal stem cells in vitro. Changes in the level of Ang-1, Ang-2, cell migration, and trans-differentiation into pericytes and endothelial lineage were monitored after 7 days. The interaction of mesenchymal stem cells with endothelial cells were evaluated using surface plasmon resonance technique. Paracrine restorative effect of diabetic stem cells was tested on pancreatic ß cells. Compared to data from FBS and normal serum, diabetic serum reduced the stem cell survival and chemotaxis toward VEGF and SDF-1α (P < 0.05). Diabetic condition were found to decline cell migration rate and the activity of MMP-2 and -9 (P < 0.05). The down-regulation of VEGFR-2 and CXCR-4 was observed with an increase in the level of miR-1-3p and miR-15b-5p at the same time. The paracrine angiogenic potential of diabetic stem cells was disturbed via the changes in the dynamic of Ang-1, Ang-2, and VEGF. Surface plasmon resonance analysis showed that diabetes could induce an aberrant increase in the interaction of stem cells with endothelial cells. After treatment with diabetic serum, the expression of VE-cadherin and NG2 and ability for uptake of Dil-Ac-LDL were reduced (P < 0.01). Conditioned media prepared from diabetic stem cells were unable to decrease fatty acid accumulation in ß-cells (P < 0.05). The level of insulin secreted by ß-cells was not affected after exposure to supernatant from diabetic or non-diabetic mesenchymal stem cells. Data suggest diabetes could decrease angiogenic and restorative effect of stem cells in vitro. J. Cell. Biochem. 119: 524-535, 2018. © 2017 Wiley Periodicals, Inc.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Soro , Idoso , Angiopoietina-1/biossíntese , Angiopoietina-2/biossíntese , Humanos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Pessoa de Meia-Idade , Receptores CXCR4/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
The identification of informative biomarkers that could predict the treatment response is particularly important in the triple-negative (TN) breast cancer, which is characterized by biological diversity. The aim of this study was to investigate the impact of vascular endothelial growth factor receptor (VEGFR2) expression and its gene polymorphisms on pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) in Russian patients with TN breast cancer. We performed a retrospective analysis of 70 women with operable TN breast cancer, who underwent NCT with 5-fluorouracil, adriamycin, and cyclophosphamide (FAC) or cyclophosphamide, adriamycin, and capecitabine (CAX) between 2007 and 2013. VEGFR2 expression was evaluated before NCT by immunohistochemistry. TaqMan SNP assays were used for genotyping KDR - 604T>C (rs2071559) and KDR 1192G>A (rs2305948) polymorphisms. The pCR was used as an end-point in the treatment efficacy analysis. In the univariate analysis, the pCR rate was strongly associated with young age (P = 0.004), high Ki67 expression (P = 0.012), lymph node negativity (P = 0.023) as well as with positive VEGFR2 expression (P = 0.019) and the CAX regimen (P = 0.005). In the multivariate analysis, only patient's age (P = 0.005) and pre-NCT VEGFR2 expression (P = 0.048) remained significant predictors of pCR. The pCR rate was higher in the CAX-treated patients than that in the FAC-treated patients (P = 0.005). Our results revealed that - 604TT genotype of rs2071559 and age < 50 years were correlated with a pCR in the CAX-treated patients. VEGFR2 expression in pre-NCT tumors and KDR gene polymorphism can be considered as additional predictive molecular markers of pCR in Russian TN breast cancer patients treated with NCT.
Assuntos
Regulação Neoplásica da Expressão Gênica , Terapia Neoadjuvante , Proteínas de Neoplasias , Polimorfismo Genético , Neoplasias de Mama Triplo Negativas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Valor Preditivo dos Testes , Estudos Retrospectivos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/terapia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Studies have shown that sFlt-1 overproduction stimulated by excess VEGF of deciduous origin in trophoblasts can cause preeclampsia. However, the mechanism underlying how VEGF regulates sFtl-1 expression in trophoblasts remains unknown. To address this issue, JEG3 and HTR-8/SV neo (HTR8) trophoblast cell lines were used to investigate the signaling pathways involved in the regulation of sFlt-1 production via VEGF overexpression in vitro. JEG3 (VEGF-GFP-JEG3, V-J) and HTR8 (VEGF-GFP-HTR8, V-H) cells overexpressing VEGF165 were established by infecting the JEG3 and HTR8 cell lines with lentivirus expressing VEGF165. Both the mRNA and protein levels of VEGF and sFlt-1 were dramatically up-regulated in the V-J and V-H cells compared to the JEG3 and HTR8 cells, and they were significantly decreased after treatment with an Flt-1 receptor inhibitor (MK-2461), a KDR receptor inhibitor (XL-184), or an Flt-1 and KDR receptor inhibitor (ABT-869). The mRNA levels of sFlt-1, Flt-1, and KDR were increased in V-H cells after treatment, and the VEGF-A mRNA levels were also elevated. The migration and invasion abilities of JEG3 and HTR8 cells were decreased after VEGF overexpression, and this reduction could be reversed with VEGF receptor inhibitor treatment. In addition, after the different treatments, the cell migration rates of V-J cells were significantly increased compared with the control treatment. Taken together, these results indicate that sFlt-1 up-regulation by VEGF may be mediated by the VEGF/Flt-1 and/or VEGF/KDR signaling pathways. However, elucidating which pathway plays this key role requires further investigation.
Assuntos
Regulação da Expressão Gênica , Transdução de Sinais , Trofoblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Linhagem Celular Tumoral , Humanos , Inibidores de Proteínas Quinases/farmacologia , Trofoblastos/citologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Molecular pathways that regulate alveolar development and adult repair represent potential therapeutic targets for emphysema. Signalling via retinoic acid (RA), derived from vitamin A, is required for mammalian alveologenesis, and exogenous RA can induce alveolar regeneration in rodents. Little is known about RA signalling in the human lung and its potential role in lung disease. OBJECTIVES: To examine regulation of human alveolar epithelial and endothelial repair by RA, and characterise RA signalling in human emphysema. METHODS: The role of RA signalling in alveolar epithelial repair was investigated with a scratch assay using an alveolar cell line (A549) and primary human alveolar type 2 (AT2) cells from resected lung, and the role in angiogenesis using a tube formation assay with human lung microvascular endothelial cells (HLMVEC). Localisation of RA synthetic (RALDH-1) and degrading (cytochrome P450 subfamily 26 A1 (CYP26A1)) enzymes in human lung was determined by immunofluorescence. Regulation of RA pathway components was investigated in emphysematous and control human lung tissue by quantitative real-time PCR and Western analysis. RESULTS: RA stimulated HLMVEC angiogenesis in vitro; this was partially reproduced with a RAR-α agonist. RA induced mRNA expression of vascular endothelial growth factor A (VEGFA) and VEGFR2. RA did not modulate AT2 repair. CYP26A1 protein was identified in human lung microvasculature, whereas RALDH-1 partially co-localised with vimentin-positive fibroblasts. CYP26A1 mRNA and protein were increased in emphysema. CONCLUSIONS: RA regulates lung microvascular angiogenesis; the endothelium produces CYP26A1 which is increased in emphysema, possibly leading to reduced RA availability. These data highlight a role for RA in maintenance of the human pulmonary microvascular endothelium.
Assuntos
Pulmão/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Enfisema Pulmonar/fisiopatologia , Regeneração/fisiologia , Tretinoína/farmacologia , Idoso , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/fisiologia , Linhagem Celular , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/patologia , RNA Mensageiro/genética , Receptores do Ácido Retinoico/metabolismo , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
The SMAD proteins are responsible for transducing signals from activated transforming growth factor-beta. This is the first study assessing the expression of SMAD-1/8, SMAD-2/3, SMAD-4, and SMAD-7 in chronic lymphocytic leukemia cells with regard to their clinical significance and potential prognostic value. Overexpression of SMAD-1/8 was observed in 160 chronic lymphocytic leukemia patients compared to 42 healthy volunteers (p = 0.023) and was associated with a more progressive course of the disease (p = 0.016). Moreover, the high expression of SMAD-1/8 correlated with other, well-established prognostic factors, including clinical stage (p = 0.010) and lymphocyte doubling time (p = 0.021). The expression of SMAD-4 was lower in chronic lymphocytic leukemia patients compared with the control group (p = 0.003). Importantly, lower SMAD-4 levels correlated with longer progression-free survival (p = 0.009), progressive course of the disease (p = 0.002), advanced clinical stage (p = 0.0004), elevated beta-2-microglobulin and lactate dehydrogenase levels (p < 0.05), shorter lymphocyte doubling time (p = 0.009), and CD38 antigen expression (p = 0.039). In addition, lower SMAD-4 expression correlated with lower apoptotic index (p = 0.0007) and lower expression of receptors for vascular endothelial growth factors VEGFR-1 and VEGFR-2. A significant association was found between the low expression of inhibitory protein SMAD-7 and both zeta-chain-associated protein kinase 70-negative cells (p = 0.04) and lower apoptotic index (p = 0.004). No differences were observed in SMAD-2/3 expression. In conclusion, our results demonstrate a significant correlation between greater SMAD-1/8 and lower SMAD-4 expression in chronic lymphocytic leukemia cells, as well as more progressive outcome and poor prognosis. These data provide supporting evidence that the expression of SMAD proteins plays an important role in disease development and may be considered as a novel, biologic prognostic factor in this disease.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Proteína Smad1/biossíntese , Proteína Smad2/biossíntese , Proteína Smad4/biossíntese , Proteína Smad7/biossíntese , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Intervalo Livre de Doença , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteína Smad1/genética , Proteína Smad2/genética , Proteína Smad4/genética , Proteína Smad7/genética , Fator de Crescimento Transformador beta/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Proteína-Tirosina Quinase ZAP-70/biossínteseRESUMO
The limited availability of qualified endothelial progenitor cells (EPCs) is a major challenge for regenerative medicine. In the present study, we isolated human EPCs from human umbilical vein endothelial cells (HUVECs) by using magnetic micro-beads coated with an antibody against human CD34. Flow cytometric assay showed that majority of these cells expressed VEGFR2 (KDR), CD34 and CD133, three molecular markers for early EPCs. It was also found that a bioreactor micro-carrier cell culture system (bio-MCCS) was superior to dish culture for in vitro expansion of EPCs. It expanded more EPCs which were in the early stage, as shown by the expression of characteristic molecular markers and had better angiogenic potential, as shown by matrix-gel based in vitro angiogenesis assay. These results suggest that HUVECs might be a novel promising resource of EPCs for regenerative medicine and that a bio-MCCS cell culture system might be broadly used for in vitro expansion of EPCs.
Assuntos
Diferenciação Celular/genética , Células Progenitoras Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Medicina Regenerativa , Antígeno AC133/biossíntese , Antígenos CD34/biossíntese , Reatores Biológicos , Proliferação de Células/genética , Citometria de Fluxo , Humanos , Técnicas In Vitro , Veias Umbilicais/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney-cancer metastasis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Gestational diabetes mellitus (GDM) is one of the most common medical complications of pregnancy, and has important health implications for mother and child. Changes in the fetoplacental vessels may predict those in the vasculature of the developing fetus, as these have been implicated in the pathogenesis of human GDM. This study aimed to determine the differences in the localization and expression level of VEGFA and VEGFR2 between placentas of women with GDM and placentas of normal pregnancies, which is the first step in elucidating the possible roles of VEGFA and VEGFR2 in the altered uteroplacental function resulting from maternal hyperglycaemia and ultimately in the manifestation of GDM. METHODS: The expressions of VEGFA and VEGFR2 mRNA and protein in 20 samples from each group were analyzed by real-time PCR, immunohistochemistry and Western blot. The placental blood barrier and angiogenesis were observed by the transmission electron microscopy (TEM) in10 GDM samples and ten controls. RESULTS: The expression levels of VEGFA and VEGFR2 mRNA and protein were significantly decreased in the GDM group (P < 0.05 or 0.01). Immunohistochemical analysis showed the reduced expression of VEGFA and VEGFR2 protein in GDM-affected placental tissues, and the degenerative alterations of the terminal villi vascular. CONCLUSION: The expressions of VEGFA and VEGFR-2 mRNAs and protein were reduced in GDM-affected placental tissues, suggesting that maternal GDM affects the pathophysiological function of placentas.
Assuntos
Diabetes Gestacional/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Placenta/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Adulto , Diabetes Gestacional/diagnóstico , Diabetes Gestacional/genética , Feminino , Humanos , Gravidez , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adulto JovemRESUMO
Thrombospondin-1 (TSP1) inhibits angiogenesis, in part, by interacting with the ubiquitous cell-surface receptor CD47. In endothelial cells, CD47 interacts directly with vascular endothelial growth factor receptor (VEGFR)-2, and TSP1 inhibits VEGFR2 phosphorylation and signaling by disrupting this association. We show that CD47 similarly associates with and regulates VEGFR2 in T cells. TSP1 inhibits phosphorylation of VEGFR2 and its downstream target Src in wild type but not in CD47-deficient human Jurkat and primary murine T cells. VEGFR2 signaling inhibits proliferation and TCR signaling in wild type T cells. However, ligation of CD47 by TSP1 or loss of CD47 expression reverses some inhibitory effects of VEGF on proliferation and T cell activation. We further found that VEGF and VEGFR2 expression are upregulated in CD47-deficient murine CD4(+) and human Jurkat T cells, and the resulting autocrine VEGFR2 signaling enhances proliferation and some TCR responses in the absence of CD47. Thus, CD47 signaling modulates the ability of VEGF to regulate proliferation and TCR signaling, and autocrine production of VEGF by T cells contributes to this regulation. This provides a mechanism to understand the context-dependent effects of TSP1 and VEGF on T cell activation, and reveals an important role for CD47 signaling in regulating T cell production of the major angiogenic factor VEGF.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígeno CD47/imunologia , Tolerância Imunológica , Trombospondina 1/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antígeno CD47/biossíntese , Antígeno CD47/genética , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/imunologia , Fosforilação , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: Mesenchymal stem cells (MSC) have shown therapeutic potential to engraft and either differentiate into or support differentiation of vascular endothelial cells (EC), smooth muscle cells and cardiomyocytes in animal models of ischemic heart disease. Following intracoronary or transendocardial delivery of MSCs, however, only a small fraction of cells engraft and the majority of those persist as an immature cell phenotype. The goal of the current study was to decipher the molecular pathways and mechanisms that control MSC differentiation into ECs. Vascular endothelial growth factor (VEGF-165) treatment is known to enhance in vitro differentiation of MSCs into ECs. We tested the possible involvement of the Sry-type HMG box (Sox) family of transcription factors in this process. METHOD AND RESULTS: MSCs were isolated from the bone marrow of Yucatan microswine and underwent a 10 day differentiation protocol. VEGF-165 (50ng/ml) treatment of MSCs in vitro induced a significant increase in the protein expression of VEGFR-2, Sox9 and Sox18, in addition to the EC markers PECAM-1, VE-cadherin and vWF, as determined by Western blot or flow cytometry. siRNA-mediated knockdown of Sox18, as opposed to Sox9, in MSCs prevented VEGF-165-mediated induction of EC markers and capillary tube formation. Inhibition of VEGFR-2 signaling (SC-202850) reduced Sox18 and reduced VEGF-165-induced differentiation of MSCs to ECs. CONCLUSION: Here we demonstrate that VEGF-165 mediates MSC differentiation into ECs via VEGFR-2-dependent induction of Sox18, which ultimately coordinates the transcriptional upregulation of specific markers of the EC phenotype.
Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , Fatores de Transcrição SOXF/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Fatores de Transcrição SOXF/genética , Transdução de Sinais , Suínos , Porco Miniatura , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: The brain vascular system arises from the perineural vascular plexus (PNVP) which sprouts radially into the neuroepithelium and subsequently branches off laterally to form a secondary plexus in the subventricular zone (SVZ), the subventricular vascular plexus (SVP). The process of SVP formation remains to be fully elucidated. We investigated the role of Foxc1 in early stage vascular formation in the ventral telencephalon. RESULTS: The Foxc1 loss of function mutant mouse, Foxc1(ch/ch) , showed enlarged telencephalon and hemorrhaging in the ventral telencephalon by embryonic day 11.0. The mutant demonstrated blood vessel dilation and aggregation of endothelial cells in the SVZ after the invasion of endothelial cells through the radial path, which lead to failure of SVP formation. During this early stage of vascular development, Foxc1 was expressed in endothelial cells and pericytes, as well as in cranial mesenchyme surrounding the neural tube. Correspondingly, abnormal deposition pattern of basement membrane proteins around the vessels and increased strong Vegfr2 staining dots were found in the aggregation sites. CONCLUSIONS: These observations reveal an essential role for Foxc1 in the early stage of vascular formation in the telencephalon.