RESUMO
Hypoxia plays an important regulatory role in the vasculature to adjust blood flow to meet metabolic requirements. At the level of gene transcription, the responses are mediated by hypoxia-inducible factor (HIF) the stability of which is controlled by the HIF prolyl 4-hydroxylase-2 (PHD2). In the lungs hypoxia results in vasoconstriction, however, the pathophysiological relevance of PHD2 in the major arterial cell types; endothelial cells (ECs) and arterial smooth muscle cells (aSMCs) in the adult vasculature is incompletely characterized. Here, we investigated PHD2-dependent vascular homeostasis utilizing inducible deletions of PHD2 either in ECs (Phd2∆ECi) or in aSMCs (Phd2∆aSMC). Cardiovascular function and lung pathologies were studied using echocardiography, Doppler ultrasonography, intraventricular pressure measurement, histological, ultrastructural, and transcriptional methods. Cell intrinsic responses were investigated in hypoxia and in conditions mimicking hypertension-induced hemodynamic stress. Phd2∆ECi resulted in progressive pulmonary disease characterized by a thickened respiratory basement membrane (BM), alveolar fibrosis, increased pulmonary artery pressure, and adaptive hypertrophy of the right ventricle (RV). A low oxygen environment resulted in alterations in cultured ECs similar to those in Phd2∆ECi mice, involving BM components and vascular tone regulators favoring the contraction of SMCs. In contrast, Phd2∆aSMC resulted in elevated RV pressure without alterations in vascular tone regulators. Mechanistically, PHD2 inhibition in aSMCs involved actin polymerization -related tension development via activated cofilin. The results also indicated that hemodynamic stress, rather than PHD2-dependent hypoxia response alone, potentiates structural remodeling of the extracellular matrix in the pulmonary microvasculature and respiratory failure.
Assuntos
Hipertensão Pulmonar , Animais , Artérias/metabolismo , Células Endoteliais/metabolismo , Fibrose , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Camundongos , Miócitos de Músculo Liso/patologia , Prolil Hidroxilases/metabolismoRESUMO
Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2443), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5ß1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. SIGNIFICANCE: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer.See related commentary by Kamiyama and Augustin, p. 35.
Assuntos
Neoplasias Pulmonares , Melanoma , Angiopoietina-1 , Angiopoietina-2/genética , Angiopoietinas , Animais , Neoplasias Pulmonares/genética , Camundongos , Neovascularização Patológica/genética , Remodelação VascularRESUMO
Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway.
Assuntos
Tecido Adiposo/irrigação sanguínea , Obesidade/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Obesidade/complicações , Obesidade/patologiaRESUMO
The endothelial Tie1 receptor is ligand-less, but interacts with the Tie2 receptor for angiopoietins (Angpt). Angpt2 is expressed in tumor blood vessels, and its blockade inhibits tumor angiogenesis. Here we found that Tie1 deletion from the endothelium of adult mice inhibits tumor angiogenesis and growth by decreasing endothelial cell survival in tumor vessels, without affecting normal vasculature. Treatment with VEGF or VEGFR-2 blocking antibodies similarly reduced tumor angiogenesis and growth; however, no additive inhibition was obtained by targeting both Tie1 and VEGF/VEGFR-2. In contrast, treatment of Tie1-deficient mice with a soluble form of the extracellular domain of Tie2, which blocks Angpt activity, resulted in additive inhibition of tumor growth. Notably, Tie1 deletion decreased sprouting angiogenesis and increased Notch pathway activity in the postnatal retinal vasculature, while pharmacological Notch suppression in the absence of Tie1 promoted retinal hypervasularization. Moreover, substantial additive inhibition of the retinal vascular front migration was observed when Angpt2 blocking antibodies were administered to Tie1-deficient pups. Thus, Tie1 regulates tumor angiogenesis, postnatal sprouting angiogenesis, and endothelial cell survival, which are controlled by VEGF, Angpt, and Notch signals. Our results suggest that targeting Tie1 in combination with Angpt/Tie2 has the potential to improve antiangiogenic therapy.
Assuntos
Angiopoietina-1/antagonistas & inibidores , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Deleção de Genes , Receptor de TIE-1/genética , Vasos Retinianos/patologia , Inibidores da Angiogênese/química , Angiopoietina-1/metabolismo , Animais , Apoptose , Sobrevivência Celular , Homozigoto , Ligantes , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Neovascularização Patológica , Fenótipo , Receptor de TIE-1/fisiologia , Receptor TIE-2/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.
Assuntos
Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Cicatrização/fisiologia , Animais , Apoptose , Membrana Basal/fisiologia , Capilares/anatomia & histologia , Capilares/metabolismo , Capilares/patologia , Colágeno Tipo XVIII/genética , Endostatinas/genética , Células Endoteliais/fisiologia , Queratinócitos/citologia , Queratinócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica , Pele/anatomia & histologia , Pele/metabolismo , Pele/patologiaRESUMO
Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/beta-catenin activity. V3C18 (M(r) = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of beta-catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/beta-catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/beta-catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate.
Assuntos
Colágeno/fisiologia , Receptores Frizzled/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Membrana Celular/metabolismo , Colágeno/química , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. We studied the development of carcinogen-induced skin tumors in transgenic J4 mice overexpressing endostatin in their keratinocytes. Unexpectedly, we did not observe any differences in tumor incidence and multiplicity between these and control mice, nor in the rate of conversion of benign papillomas to malignant squamous cell carcinomas (SCC). We did find, however, that endostatin regulates the terminal differentiation of keratinocytes because the SCCs in the J4 mice were less aggressive and more often well differentiated than those in the control mice. We observed an inhibition of tumor angiogenesis by endostatin at an early stage in skin tumor development, but more strikingly, there was a significant reduction in lymphatic vessels in the papillomas and SCCs in association with elevated endostatin levels and also a significant inhibition of lymph node metastasis in the J4 mice. We showed that tumor-infiltrating mast cells strongly expressed vascular endothelial growth factor-C (VEGF-C), and that the accumulation of these cells was markedly decreased in the tumors of the J4 mice. Moreover, endostatin inhibited the adhesion and migration of murine MC/9 mast cells on fibronectin in vitro. Our data suggest that endostatin can inhibit tumor lymphangiogenesis by decreasing the VEGF-C levels in the tumors, apparently via inhibition of mast cell migration and adhesion, and support the view that the biological effects of endostatin are not restricted to endothelial cells because endostatin also regulates tumor-associated inflammation and differentiation, and the phenotype of epithelial tumors.
Assuntos
Endostatinas/genética , Metástase Linfática/prevenção & controle , Neovascularização Patológica/prevenção & controle , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/patologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Apoptose/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genéticaRESUMO
Endostatin, a proteolytic fragment of type XVIII collagen, has been shown to inhibit angiogenesis, tumor growth, and endothelial cell proliferation and migration. We analyzed its functions in vivo by generating transgenic mice in which it was overexpressed in the skin and lens capsule under the keratin K14 promoter. Opacity of the lens occurred at 4 months of age in the mouse line J4, with the highest level of endostatin expression. The lens epithelial cells appeared to lose contact with the capsule and began to vacuolize. In 1-year-old mice the lens epithelial cell layer had entirely degenerated, and instead, large plaques of spindle-shaped cells had formed in the anterior region of the lens. Moreover, a widening of the epidermal basement membrane (BM) zone of the skin was observed in electron microscopy. The epidermal BM was conspicuously altered in the J4 mice with high transgene expression, including clear broadening and occurrence of pearl-like protrusions in some areas, whereas the BM was more even in appearance but consistently broadened in the mouse line G20 with moderate transgene expression. In both lines the BM was continuous. Measurements indicated that the lamina densa was 78.54 +/- 53.10 nm in line J4, the large variation reflecting the protrusions of the lamina densa, and 44.24 +/- 11.52 nm in line G20, compared with 33.74 +/- 9.96 nm in wild-type adult mice. Immunoelectron microscopy of wild-type mouse skin type XVIII collagen showed a polarized orientation in the BMs, with the C-terminal endostatin region localized in the lamina densa and the N terminus in average approximately 40 nm more on the dermal side. Type XVIII collagen was dispersed in the transgenic skin, suggesting that the transgene-derived endostatin fragment displaces the full-length collagen XVIII. This may impair the anchoring of the lamina densa to the dermis and thereby lead to loosening of the BMs, resembling the previously observed situation in collagen XVIII-null mice.
Assuntos
Membrana Basal/patologia , Catarata/patologia , Endostatinas/fisiologia , Cristalino/patologia , Pele/patologia , Inibidores da Angiogênese , Animais , Membrana Basal/ultraestrutura , Primers do DNA , Humanos , Cristalino/ultraestrutura , Camundongos , Camundongos Transgênicos , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase , Pele/ultraestruturaRESUMO
Human type XVIII collagen was found to be expressed as three variants, termed NC1-303, NC1-493 and NC1-728, differing in their N-terminal non-collagenous domains (NC1). The corresponding gene was found to be approximately 105 kb in size and contain 43 exons. The short variant is derived from utilization of an upstream promoter associated with the first two exons of the gene. The two other variants are derived from a downstream promoter and alternative splicing of exon 3, resulting in 192 residues of shared sequences characterized by a putative approximately 30 residue conserved coiled-coil motif and 235 residues of sequences specific to NC1-728. The NC1-728 variant has a conserved cysteine-rich domain homologous with the ligand-binding part of the frizzled proteins. A polyclonal antibody specific to the NC1-728 variant was generated, and immunostaining of fetal tissues revealed staining in lung and skeletal muscle. Human serum contained 173- and 144-kDa alpha1(XVIII) chains corresponding to the NC1-728 and NC1-493 variants, respectively. A 200-kDa polypeptide was detected in cells transfected with a cDNA construct corresponding to the full-length NC1-728 variant, and EBNA-293 cells endogenously synthesizing low amounts of type XVIII collagen had a 45-kDa fragment in their culture medium that corresponded to most of the NC1 domain of the NC1-728 variant, suggesting processing of the N-terminal frizzled-containing domain.