RESUMEN
OBJECTIVE: Cx40 (Connexin40) forms intercellular channels that coordinate the electric conduction in the heart and the vasomotor tone in large vessels. The protein was shown to regulate tumoral angiogenesis; however, whether Cx40 also contributes to physiological angiogenesis is still unknown. APPROACH AND RESULTS: Here, we show that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the neovascularization model of the mouse neonatal retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed endothelial cell functions as selective reexpression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular endothelial cells, by silencing its expression or by blocking gap junction channels, decreases their proliferation. Moreover, loss of Cx40 in these cells also increases their release of PDGF (platelet-derived growth factor) and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide, phenocopies the loss of Cx40 in the retinal vasculature of wild-type mice. CONCLUSIONS: Collectively, our data show that endothelial Cx40 contributes to the early stages of physiological angiogenesis in the developing retina, by regulating vessel growth and maturation. Cx40 thus represents a novel therapeutic target for treating pathological ocular angiogenesis.
Asunto(s)
Capilares/metabolismo , Conexinas/metabolismo , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Vasos Retinianos/metabolismo , Animales , Animales Recién Nacidos , Capilares/crecimiento & desarrollo , Línea Celular , Proliferación Celular , Quimiotaxis , Conexinas/deficiencia , Conexinas/genética , Regulación hacia Abajo , Uniones Comunicantes/metabolismo , Genotipo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Interferencia de ARN , Vasos Retinianos/crecimiento & desarrollo , Transducción de Señal , Transfección , Proteína alfa-5 de Unión ComunicanteRESUMEN
Store-operated Ca(2+) channels (SOCs) are voltage-independent Ca(2+) channels activated upon depletion of the endoplasmic reticulum Ca(2+) stores. Early studies suggest the contribution of such channels to Ca(2+) homeostasis in insulin-secreting pancreatic ß-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca(2+) depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca(2+) imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat ß-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca(2+) entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Canales Catiónicos TRPC/metabolismo , Acetilcolina/farmacología , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular Tumoral , Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación Missense , Proteína ORAI1 , Ratas , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/genética , Tapsigargina/farmacologíaRESUMEN
BACKGROUND: Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown. RESULTS: Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40(+/-) aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37(-/-) and Cx40(-/-) aortas but not in Cx40(+/-) aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40(-/-) and Cx40(+/-) aortas but not in Cx37(-/-) aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype. CONCLUSIONS: Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.
Asunto(s)
Aorta/fisiología , Conexinas/metabolismo , Endotelio Vascular/citología , Óxido Nítrico/metabolismo , Acetilcolina/metabolismo , Animales , Aorta/citología , Conexinas/genética , Endotelio Vascular/fisiología , Eliminación de Gen , Regulación de la Expresión Génica , Genotipo , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/agonistas , Óxido Nítrico Sintasa de Tipo III/metabolismo , Vasoconstricción , Vasodilatación , Proteína alfa-5 de Unión Comunicante , Proteína alfa-4 de Unión ComunicanteRESUMEN
BACKGROUND/AIMS: Smooth muscle tone is controlled by Ca(2+) signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40) and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca(2+) signaling of the mouse aorta. METHODS: Ca(2+) imaging was performed on intact aortic endothelium from both wild type (Cx40+/+) and Connexin40-deficient (Cx40 -/-) mice. RESULTS: Acetylcholine (ACh) induced early fast and high amplitude Ca(2+) transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca(2+) transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca(2+) waves, indicating that Cx40 contributes to the spreading of Ca(2+) signals. The propagation of those Ca(2+) responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca(2+) waves. CONCLUSIONS: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca(2+) signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.
Asunto(s)
Calcio/metabolismo , Conexinas/metabolismo , Células Endoteliales/metabolismo , Receptor Muscarínico M3/metabolismo , Acetilcolina/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antiulcerosos/farmacología , Aorta/citología , Señalización del Calcio/efectos de los fármacos , Carbenoxolona/farmacología , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Conexinas/genética , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Uniones Comunicantes/metabolismo , Ratones , Ratones Noqueados , Octanoles/farmacología , Proteína alfa-5 de Unión Comunicante , Proteína alfa-4 de Unión ComunicanteRESUMEN
Background Gap junction channels made of Connexin37 (Cx37) are expressed by aortic endothelial and smooth muscle cells of hypertensive mice, as well as by the renin-secreting cells of kidneys. Methods and Results To decipher whether Cx37 has any role in hypertension, angiotensin II (Ang II ) was infused in normotensive wild-type and Cx37-deficient mice (Cx37-/-). After 2 to 4 weeks, the resulting increase in blood pressure was lower in Cx37-/- than in wild-type mice, suggesting an alteration in the Ang II response. To investigate this possibility, mice were submitted to a 2-kidney, 1-clip procedure, a renin-dependent model of hypertension. Two weeks after this clipping, Cx37-/- mice were less hypertensive than wild-type mice and, 2 weeks later, their blood pressure had returned to control values, in spite of abnormally high plasma renin levels. In contrast, Cx37-/- and wild-type mice that received N-nitro-l-arginine-methyl-ester, a renin-independent model of hypertension, featured a similar and sustained increase in blood pressure. The data indicate that loss of Cx37 selectively altered the Ang II -dependent pathways. Consistent with this conclusion, aortas of Cx37-/- mice featured an increased basal expression of the Ang II type 2 receptors ( AT 2R), and increased transcripts levels of downstream signaling proteins, such as Cnksr1 and Ptpn6 ( SHP -1). Accordingly, the response of Cx37-/- mice aortas to an ex vivo Ang II exposure was altered, since phosphorylation levels of several proteins of the Ang II pathway ( MLC 2, ERK , and AKT ) remained unchanged. Conclusions These findings provide evidence that Cx37 selectively influences Ang II signaling, mostly via a modulation of the expression of the Ang II type 2 receptor.
Asunto(s)
Aorta/metabolismo , Presión Sanguínea/genética , Conexinas/genética , Hipertensión/genética , Receptor de Angiotensina Tipo 2/metabolismo , Renina/metabolismo , Angiotensina II/farmacología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipertensión/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasoconstrictores/farmacología , Proteína alfa-4 de Unión ComunicanteRESUMEN
AIMS: Intimal hyperplasia (IH) is an abnormal response to vessel injury characterized by the dedifferentiation, migration, and proliferation of quiescent vascular smooth muscle cells (VSMC) to form a neointima layer. Vascular connexins (Cx) are involved in the pathophysiology of various vascular diseases, and Cx43, the main Cx expressed in VSMC, has been shown to promote VSMC proliferation and IH. The aim of this study was to investigate the participation of another Cx, namely Cx37, in the formation of the neointima layer. METHODS AND RESULTS: Wild-type (WT) and Cx37-deficient (Cx37-/-) C57BL/6J mice were subjected to carotid artery ligation (CAL), a model of vessel injury and IH. The neointima developed linearly in WT until 28 days post surgery. In contrast, the neointima layer was almost absent 14 days after surgery in Cx37-/- mice, and twice as more developed after 28 days compared to WT mice. This large neointima formation correlated with a two-fold increase in cell proliferation in the media and neointima regions between 14 and 28 days in Cx37-/- mice compared to WT mice. The CAL triggered Cx43 overexpression in the media and neointima layers of ligated carotids in WT mice, and selectively up-regulated Cx37 expression in the media layer, but not in the neointima layer. The de novo expression of Cx37 in human primary VSMC reduced cell proliferation and P-Akt levels, in association with lower Cx43 levels, whereas Cx43 overexpression increased P-Akt levels. CONCLUSION: The presence of Cx37 in the media layer of injured arteries restrains VSMC proliferation and limits the development of IH, presumably by interfering with the pro-proliferative effect of Cx43 and the Akt pathway.
Asunto(s)
Traumatismos de las Arterias Carótidas/metabolismo , Estenosis Carotídea/metabolismo , Proliferación Celular , Conexinas/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Anciano , Animales , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Arterias Carótidas/cirugía , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Estenosis Carotídea/genética , Estenosis Carotídea/patología , Células Cultivadas , Conexina 43/metabolismo , Conexinas/deficiencia , Conexinas/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Hiperplasia , Ligadura , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Factores de Tiempo , Proteína alfa-4 de Unión ComunicanteRESUMEN
Endothelial connexin40 (Cx40) contributes to regulate the structure and function of vessels. We have examined whether the protein also modulates the altered growth of vessels in tumor models established in control mice (WT), mice lacking Cx40 (Cx40-/-), and mice expressing the protein solely in endothelial cells (Tie2-Cx40). Tumoral angiogenesis and growth were reduced, whereas vessel perfusion, smooth muscle cell (SMC) coverage and animal survival were increased in Cx40-/- but not Tie2-Cx40 mice, revealing a critical involvement of endothelial Cx40 in transformed tissues independently of the hypertensive status of Cx40-/- mice. As a result, Cx40-/- mice bearing tumors survived significantly longer than corresponding controls, including after a cytotoxic administration. Comparable observations were made in WT mice injected with a peptide targeting Cx40, supporting the Cx40 involvement. This involvement was further confirmed in the absence of Cx40 or by peptide-inhibition of this connexin in aorta-sprouting, matrigel plug and SMC migration assays, and associated with a decreased expression of the phosphorylated form of endothelial nitric oxide synthase. The data identify Cx40 as a potential novel target in cancer treatment.
Asunto(s)
Vasos Sanguíneos/fisiología , Conexinas/antagonistas & inhibidores , Endotelio Vascular/metabolismo , Neoplasias Pulmonares/prevención & control , Melanoma Experimental/prevención & control , Neovascularización Patológica/prevención & control , Neoplasias de la Vejiga Urinaria/prevención & control , Animales , Aorta/patología , Apoptosis , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Conexinas/metabolismo , Endotelio Vascular/patología , Femenino , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Perfusión , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/irrigación sanguínea , Neoplasias de la Vejiga Urinaria/patología , Proteína alfa-5 de Unión ComunicanteRESUMEN
Connexins (Cxs) and endothelial nitric oxide synthase (eNOS) contribute to the adaptation of endothelial and smooth muscle cells to hemodynamic changes. To decipher the in vivo interplay between these proteins, we studied Cx40-null mice, a model of renin-dependent hypertension which displays an altered endothelium-dependent relaxation of the aorta because of reduced eNOS levels. These mice, which were either untreated or subjected to the 1-kidney, 1-clip (1K1C) procedure, a model of volume-dependent hypertension, were compared with control mice submitted to either the 1K1C or the 2-kidney, 1-clip (2K1C) procedure, a model of renin-dependent hypertension. All operated mice became hypertensive and featured hypertrophy and altered Cx expression of the aorta. The combination of volume- and renin-dependent hypertension in Cx40-/- 1K1C mice raised blood pressure and cardiac weight index. Under these conditions, all aortas showed increased levels of Cx40 in endothelial cells and of both Cx37 and Cx45 in smooth muscle cells. In the wild-type 1K1C mice, the interactions between Cx40 and Cx37 with eNOS were enhanced, resulting in increased NO release. The Cx40-eNOS interaction could not be observed in mice lacking Cx40, which also featured decreased levels of eNOS. In these animals, the volume overload caused by the 1K1C procedure resulted in increased phosphorylation of eNOS and in a higher NO release. The findings provide evidence that Cx40 and Cx37 play an in vivo role in the regulation of eNOS.
Asunto(s)
Presión Sanguínea/fisiología , Conexinas/genética , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Hipertensión/genética , Óxido Nítrico Sintasa de Tipo III/genética , ARN/genética , Animales , Conexinas/biosíntesis , Modelos Animales de Enfermedad , Endotelio Vascular/fisiopatología , Hipertensión/metabolismo , Hipertensión/fisiopatología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína alfa-5 de Unión ComunicanteRESUMEN
Connexin 40 (Cx40) is expressed by the renin-producing cells (RSCs) of the kidneys and the endothelial cells of blood vessels. Cx40 null mice (Cx40(-/-)) feature a much increased renin synthesis and secretion, which results in chronic hypertension, and also display an altered endothelium-dependent relaxation of the aorta because of reduced eNOS levels and nitric oxide production. To discriminate the effect of Cx40 in renin secretion and vascular signaling, we targeted Cx40 to either the RSCs or the endothelial cells of Cx40 null mice. When compared with Cx40(-/-) controls, the animals expressing Cx40 in RSCs were less hypertensive and featured reduced renin levels, still numerous RSCs outside the wall of the afferent arterioles. In contrast, mice expressing Cx40 in the endothelial cells were as hypertensive as Cx40(-/-) mice, in spite of control levels of Cx37 and eNOS. Our data show that blood pressure is improved by restoration of Cx40 expression in RSCs but not in endothelial cells, stressing the prominent role of renin in the mouse hypertension linked to loss of Cx40.