RESUMO
KEY POINTS: Lymphatic valve defects are one of the major causes of lymph transport dysfunction; however, there are no accessible methods for quantitatively assessing valve function. This report describes a novel technique for quantifying lymphatic valve back-leak. Postnatal endothelial-specific deletion of connexin 43 (Cx43) in connexin 37 null (Cx37-/- ) mice results in rapid regression of valve leaflets and severe valve dysfunction. This method can also be used for assessing the function of venous and lymphatic valves from various species, including humans. ABSTRACT: The lymphatic system relies on robust, spontaneous contractions of collecting lymphatic vessels and one-way secondary lymphatic valves to efficiently move lymph forward. Secondary valves prevent reflux and allow for the generation of propulsive pressure during each contraction cycle. Lymphatic valve defects are one of the major causes of lymph transport dysfunction. Genetic mutations in multiple genes have been associated with the development of primary lymphoedema in humans; and many of the same mutations in mice result in valve defects that subsequently lead to chylous ascites or chylothorax. At present the only experimental technique for the quantitative assessment of lymphatic valve function utilizes the servo-null micropressure system, which is highly accurate and precise, but relatively inaccessible and difficult to use. We developed a novel, simplified alternative method for quantifying valve function and determining the degree of pressure back-leak through an intact valve in pressurized, single-valve segments of isolated lymphatic vessels. With this diameter-based method, the competence of each lymphatic valve is challenged over a physiological range of pressures (e.g. 0.5-10cmH2 O) and pressure back-leak is extrapolated from calibrated, pressure-driven changes in diameter upstream from the valve. Using mesenteric lymphatic vessels from C57BL/6J, Ub-CreERT2 ;Rasa1fx/fx , Foxc2Cre/+ , Lyve1-Cre;Cx43fx/fx , and Prox1-CreERT2 ;Cx43fx/fx ;Cx37-/- mice, we tested our method on lymphatic valves displaying a wide range of dysfunction, from fully competent to completely incompetent. Our results were validated by simultaneous direct measurement of pressure back-leak using a servo-null micropressure system. Our diameter-based technique can be used to quantify valve function in isolated lymphatic valves from a variety of species. This method also revealed that haplodeficiency in Foxc2 (Foxc2Cre/+ ) is not sufficient to cause significant valve dysfunction; however, postnatal endothelial-specific deletion of Cx43 in Cx37-/- mice results in rapid regression of valve leaflets and severe valve dysfunction.
Assuntos
Vasos Linfáticos , Linfedema , Animais , Conexina 43/genética , Conexinas , Linfedema/genética , Camundongos , Camundongos Endogâmicos C57BLRESUMO
RATIONALE: Mutations in GJC2 and GJA1, encoding Cxs (connexins) 47 and 43, respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and their electrical coupling through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels. OBJECTIVE: To determine which Cx isoforms in LMCs and lymphatic endothelial cells are required for the entrainment of lymphatic contraction waves and efficient lymph transport. METHODS AND RESULTS: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and the lymphatic endothelial cell layers. Genetic deletion of the major lymphatic endothelial cell Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle-specific Cx45 deficiency resulted in 10- to 18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45 deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load. CONCLUSIONS: Our results (1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; (2) show that major endothelial Cxs are dispensable for the entrainment of contractions; (3) reveal a lack of coupling between lymphatic endothelial cells and LMCs, in contrast to arterioles; (4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and (5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.
Assuntos
Conexinas/metabolismo , Linfa/metabolismo , Vasos Linfáticos/metabolismo , Linfedema/metabolismo , Contração Muscular , Animais , Sinalização do Cálcio , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/deficiência , Conexinas/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Predisposição Genética para Doença , Gravitação , Humanos , Técnicas In Vitro , Vasos Linfáticos/fisiopatologia , Linfedema/genética , Linfedema/fisiopatologia , Masculino , Potenciais da Membrana , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Optogenética , Fenótipo , Fatores de Tempo , Proteína alfa-4 de Junções ComunicantesRESUMO
Lymphatic valves (LVs) are cusped luminal structures that permit the movement of lymph in only one direction and are therefore critical for proper lymphatic vessel function. Congenital valve aplasia or agenesis can, in some cases, be a direct cause of lymphatic disease. Knowledge about the molecular mechanisms operating during the development and maintenance of LVs may thus aid in the establishment of novel therapeutic approaches to treat lymphatic disorders. In this study, we examined the role of Connexin43 (Cx43), a gap junction protein expressed in lymphatic endothelial cells (LECs), during valve development. Mouse embryos with a null mutation in Cx43 (Gja1) were previously shown to completely lack mesenteric LVs at embryonic day 18. However, interpreting the phenotype of Cx43-/- mice was complicated by the fact that global deletion of Cx43 causes perinatal death due to heart defects during embryogenesis. We have now generated a mouse model (Cx43∆LEC) with a lymphatic-specific ablation of Cx43 and show that the absence of Cx43 in LECs causes a delay (rather than a complete block) in LV initiation, an increase in immature valves with incomplete leaflet elongation, a reduction in the total number of valves, and altered lymphatic capillary patterning. The physiological consequences of these lymphatic changes were leaky valves, insufficient lymph transport and reflux, and a high incidence of lethal chylothorax. These results demonstrate that the expression of Cx43 is specifically required in LECs for normal development of LVs.
Assuntos
Quilotórax/metabolismo , Conexina 43/metabolismo , Deleção de Genes , Vasos Linfáticos/anormalidades , Vasos Linfáticos/embriologia , Envelhecimento/patologia , Animais , Padronização Corporal , Quilotórax/patologia , Células Endoteliais/metabolismo , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Mesentério/embriologia , Mesentério/metabolismo , Mesentério/patologia , Camundongos , Especificidade de ÓrgãosRESUMO
Venous valves (VVs) are critical for unidirectional blood flow from superficial and deep veins towards the heart. Congenital valve aplasia or agenesis may, in some cases, be a direct cause of vascular disease, motivating an understanding of the molecular mechanisms underlying the development and maintenance of VVs. Three gap junction proteins (Connexins), Cx37, Cx43, and Cx47, are specifically expressed at VVs in a highly polarized fashion. VVs are absent from adult mice lacking Cx37; however it is not known if Cx37 is required for the initial formation of valves. In addition, the requirement of Cx43 and Cx47 for VV development has not been studied. Here, we provide a detailed description of Cx37, Cx43, and Cx47 expression during mouse vein development and show by gene knockout that each Cx is necessary for normal valve development. The valve phenotypes in the knockout lines exhibit Cx-specific differences, however, including whether peripheral or central VVs are affected by gene inactivation. In addition, we show that a Cx47 null mutation impairs peripheral VV development but does not affect lymphatic valve formation, a finding of significance for understanding how some CX47 mutations cause inherited lymphedema in humans. Finally, we demonstrate a striking segregation of Foxc2 and NFATc1 transcription factor expression between the downstream and upstream faces, respectively, of developing VV leaflets and show that this segregation is closely associated with the highly polarized expression of Cx37, Cx43, and Cx47. The partition of Foxc2 and NFATc1 expression at VV leaflets makes it unlikely that these factors directly cooperate during the leaflet elongation stage of VV development.
Assuntos
Conexina 43/metabolismo , Conexinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição NFATC/metabolismo , Válvulas Venosas/metabolismo , Animais , Conexina 43/genética , Conexinas/genética , Fatores de Transcrição Forkhead/genética , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fenótipo , Fatores de Tempo , Válvulas Venosas/embriologia , Válvulas Venosas/crescimento & desenvolvimento , Proteína alfa-4 de Junções ComunicantesRESUMO
Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.
Assuntos
Vasos Linfáticos/embriologia , Linfedema/embriologia , Linfedema/patologia , Válvulas Venosas/embriologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Modelos Animais de Doenças , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Vasos Linfáticos/ultraestrutura , Camundongos Endogâmicos C57BL , Morfogênese , Penetrância , Fenótipo , Válvulas Venosas/ultraestruturaRESUMO
Connexins (Cxs), proteins that are vital for intercellular communication in vertebrates, have recently been shown to play a critical role in lymphatic development. However, our knowledge is currently limited regarding the functional relationships of Cxs with other proteins and signaling pathways. Cell culture studies have shown that Cx37 is necessary for coordinated activation of the transcription factor NFATc1, which cooperates with Foxc2 (another transcription factor) during lymphatic endothelial development. These data suggest that Cxs, Foxc2, and NFATc1 are part of a common developmental pathway. Here, we present a detailed characterization of Foxc2(+/-)Cx37(-/-) mice, demonstrating that lymphatic network architecture and valve formation rely on the concurrent embryonic expression and function of Foxc2 and Cx37. Foxc2(+/-)Cx37(-/-) mice have lymphedema in utero, exhibit craniofacial abnormalities, show severe dilation of intestinal lymphatics, display abnormal lacteal development, lack lymphatic valves, and typically die perinatally (outcomes not seen in Foxc2(+/-) or Cx37(-/-) mice separately). We provide a rigorous, quantitative documentation of lymphatic vascular network changes that highlight the specific structural alterations that occur in Foxc2(+/-)Cx37(-/-) mice. These data provide further evidence suggesting that Foxc2 and Cx37 are elements in a common molecular pathway directing lymphangiogenesis.
Assuntos
Conexinas/deficiência , Fatores de Transcrição Forkhead/deficiência , Deleção de Genes , Linfangiogênese , Vasos Linfáticos/anormalidades , Vasos Linfáticos/embriologia , Animais , Animais Recém-Nascidos , Padronização Corporal , Colo/patologia , Conexinas/metabolismo , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/patologia , Edema/patologia , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fatores de Transcrição Forkhead/metabolismo , Intestino Delgado/patologia , Linfangioma/patologia , Vasos Linfáticos/patologia , Linfografia , Mesentério/patologia , Camundongos Endogâmicos C57BL , Mitose , Pele/embriologia , Pele/patologia , Proteína alfa-4 de Junções ComunicantesRESUMO
Venous valves play a crucial role in blood circulation, promoting the one-way movement of blood from superficial and deep veins towards the heart. By preventing retrograde flow, venous valves spare capillaries and venules from being subjected to damaging elevations in pressure, especially during skeletal muscle contraction. Pathologically, valvular incompetence or absence of valves are common features of venous disorders such as chronic venous insufficiency and varicose veins. The underlying causes of these conditions are not well understood, but congenital venous valve aplasia or agenesis may play a role in some cases. Despite progress in the study of cardiac and lymphatic valve morphogenesis, the molecular mechanisms controlling the development and maintenance of venous valves remain poorly understood. Here, we show that in valved veins of the mouse, three gap junction proteins (Connexins, Cxs), Cx37, Cx43, and Cx47, are expressed exclusively in the valves in a highly polarized fashion, with Cx43 on the upstream side of the valve leaflet and Cx37 on the downstream side. Surprisingly, Cx43 expression is strongly induced in the non-valve venous endothelium in superficial veins following wounding of the overlying skin. Moreover, we show that in Cx37-deficient mice, venous valves are entirely absent. Thus, Cx37, a protein involved in cell-cell communication, is one of only a few proteins identified so far as critical for the development or maintenance of venous valves. Because Cxs are necessary for the development of valves in lymphatic vessels as well, our results support the notion of common molecular pathways controlling valve development in veins and lymphatic vessels.
Assuntos
Conexinas/deficiência , Válvulas Venosas/anormalidades , Válvulas Venosas/metabolismo , Animais , Conexina 43/metabolismo , Conexinas/metabolismo , Endotélio/metabolismo , Extremidades/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , Fluxo Sanguíneo Regional , Veia Safena/metabolismo , Veia Safena/patologia , Pele/irrigação sanguínea , Válvulas Venosas/patologia , Proteína alfa-4 de Junções ComunicantesRESUMO
The expression of the gap junction molecule connexin-45 (Cx45; GJC1) in lymphatic endothelium and its functional relevance were not previously known. We found that Cx45 was expressed widely in the endothelium of murine lymphatics, in both valve and non-valve regions. Cell-specific deletion of Cx45, driven by a constitutive Cre line (Lyve1-Cre) or an inducible Cre line (Prox1-CreERT2), compromised the function of lymphatic valves, as assessed by physiological tests (back leak and closure) of isolated, single-valve vessel segments. The defects were comparable to those previously reported for loss of Cx43 and, like Cx43, deletion of Cx45 resulted in shortening and/or increased asymmetry of lymphatic valve leaflets, providing an explanation for the compromised valve function. In contrast to Cx43, LEC-specific deletion of Cx45 did not alter the number of valves in mesenteric or dermal lymphatic networks, or the expression patterns of the canonical valve-associated proteins PROX1, ITGA9 or CLAUDIN5. Constitutive deletion of Cx45 from LECs resulted in increased backflow of injected tracer in popliteal networks in vivo and compromised the integrity of the LEC permeability barrier in a subset of collecting vessels. These findings provide evidence for an unexpected role of Cx45 in the development and maintenance of lymphatic valves.
RESUMO
BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.
Assuntos
Plaquetas/fisiologia , Conexinas/genética , Junções Comunicantes/fisiologia , Hemostasia/fisiologia , Trombose/fisiopatologia , Animais , Plaquetas/citologia , Plaquetas/ultraestrutura , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Sinalização do Cálcio/efeitos da radiação , Carbenoxolona/farmacologia , Comunicação Celular/fisiologia , Retração do Coágulo/fisiologia , Conexina 43/metabolismo , Conexinas/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/ultraestrutura , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Inibidores da Agregação Plaquetária/farmacologia , Proteína beta-1 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
Connexin 37 (Cx37) profoundly suppresses the proliferation of rat insulinoma (Rin) cells by unknown mechanisms. To determine whether a functional pore domain is necessary for Cx37-mediated growth suppression, we introduced a mutation that converted threonine 154 into alanine (T154A). Like other connexins mutated at the homologous site, Cx37-T154A localized to appositional membrane but failed to form functional channels and exerted a dominant-negative effect on coexpressed wild-type Cx37 or Cx43. Unlike the wild-type protein, Cx37-T154A did not suppress the proliferation of Rin cells and did not, with serum deprivation, result in cell cycle arrest. Furthermore, progression through the cell cycle was unaffected by expression of Cx37-T154A. These results indicate that a pore-forming domain that is able to form functional channels is essential for the anti-proliferative, cell-cycle arrest and serum-sensitivity effects of Cx37, and furthermore that the normally localized C-terminal domain is not sufficient for these effects of Cx37.
Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Animais , Ciclo Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Conexinas/genética , Cães , Junções Comunicantes/genética , Insulinoma/genética , Insulinoma/metabolismo , Insulinoma/patologia , Mutagênese Sítio-Dirigida , Ratos , Proteína alfa-4 de Junções ComunicantesRESUMO
Recently, we reported that recovery of tissue perfusion in the ischemic hindlimb was reduced, inflammatory response increased, and survival of distal limb tissue compromised in connexin 40 (Cx40)-deficient (Cx40(-/-)) mice. Here we evaluate whether genotype-specific differences in tissue perfusion, native vascular density, arteriogenesis, blood pressure, and chronic ANG II type 1 receptor (AT1R) activation contribute to poor recovery of ischemic hindlimb tissue in Cx40(-/-) mice. Hindlimb ischemia was induced in wild-type (WT), Cx40(-/-), and losartan-treated Cx40(-/-) mice by using surgical procedures that either maintained (mild surgery) or compromised (severe surgery) perfusion of major collateral vessels supplying the distal limb. Pre- and postsurgical hindlimb perfusion was evaluated, and tissue survival, microvascular density, and macrophage infiltration were documented during recovery. Hindlimb perfusion was compromised in presurgical Cx40(-/-) versus WT mice despite comparable native microvascular density. Hindlimb perfusion 24 h postsurgery in Cx40(-/-) and WT mice was comparable after mild surgery (collateral vessels maintained), but compromised arteriogenesis in Cx40(-/-) animals nevertheless limited subsequent recovery of tissue perfusion and compromised tissue survival. Prolonged pre- and postsurgical treatment of Cx40(-/-) mice with losartan (an AT1R antagonist) normalized blood pressure but did not improve tissue perfusion or survival, despite reduced macrophage infiltration. Thus it appears Cx40 is necessary for normal tissue perfusion and for recovery of perfusion, arteriogenesis, and tissue survival in the ischemic hindlimb. Our data suggest that Cx40(-/-) mice are at significantly greater risk for poor recovery from ischemic insult due to compromised regulation of tissue perfusion, vascular remodeling, and prolonged inflammatory response.
Assuntos
Conexinas/genética , Isquemia/patologia , Neovascularização Fisiológica , Receptor Tipo 1 de Angiotensina/metabolismo , Traumatismo por Reperfusão/patologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Membro Posterior/irrigação sanguínea , Isquemia/imunologia , Isquemia/metabolismo , Fluxometria por Laser-Doppler , Losartan/uso terapêutico , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Necrose , Receptor Tipo 1 de Angiotensina/genética , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/metabolismo , Proteína alfa-5 de Junções ComunicantesRESUMO
Intraluminal valves are required for the proper function of lymphatic collecting vessels and large lymphatic trunks like the thoracic duct. Despite recent progress in the study of lymphvasculogenesis and lymphangiogenesis, the molecular mechanisms controlling the morphogenesis of lymphatic valves remain poorly understood. Here, we report that gap junction proteins, or connexins (Cxs), are required for lymphatic valvulogenesis. Cx37 and Cx43 are expressed early in mouse lymphatic development in the jugular lymph sacs, and later in development these Cxs become enriched and differentially expressed by lymphatic endothelial cells on the upstream and downstream sides of the valves. Specific deficiencies of Cx37 and Cx43 alone or in combination result in defective valve formation in lymphatic collecting vessels, lymphedema, and chylothorax. We also show that Cx37 regulates jugular lymph sac size and that both Cx37 and Cx43 are required for normal thoracic duct development, including valve formation. Another Cx family member, Cx47, whose human analog is mutated in some families with lymphedema, is also highly enriched in a subset of endothelial cells in lymphatic valves. Mechanistically, we present data from Foxc2-/- embryos suggesting that Cx37 may be a target of regulation by Foxc2, a transcription factor that is mutated in human lymphedema-distichiasis syndrome. These results show that at least three Cxs are expressed in the developing lymphatic vasculature and, when defective, are associated with clinically manifest lymphatic disorders in mice and man.
Assuntos
Quilotórax/genética , Conexina 43/metabolismo , Conexinas/metabolismo , Linfangiogênese , Vasos Linfáticos/anormalidades , Linfedema/genética , Animais , Quilotórax/patologia , Quilotórax/fisiopatologia , Conexina 43/genética , Conexinas/genética , Fatores de Transcrição Forkhead/metabolismo , Vasos Linfáticos/metabolismo , Linfedema/patologia , Linfedema/fisiopatologia , Camundongos , Camundongos Knockout , Proteína alfa-4 de Junções ComunicantesRESUMO
BACKGROUND/AIMS: Ischemia induced by large-vessel obstruction or vascular injury induces a complex cascade of vasodilatory, remodeling and inflammatory pathways; coordination of these processes by vascular endothelium is likely to involve endothelial gap junctions. Vascular endothelium predominantly expresses two connexin (Cx) isoforms: Cx37 and Cx40. The relevance of these Cxs to postischemic limb recovery remains unclear. METHODS: In this study, we use a well-established, severe femoral-saphenous artery-vein pair resection model of unilateral hindlimb ischemia to test the relevance of Cx37 and Cx40 to postischemic tissue survival and recovery of limb perfusion. RESULTS: Cx40-deficient animals (Cx40-/-) experienced a severe reduction in limb perfusion relative to wild-type (WT) animals and exhibited profound and rapid failure of ischemic limb survival. By contrast, the deficit in limb perfusion was less severe in Cx37-ablated (Cx37-/-) animals compared to WT, corresponding with more rapid recovery of limb appearance and use. These results demonstrate that Cx40 is necessary for postischemic limb survival and reperfusion, whereas Cx37 deletion reduces the extent of ischemia in the same model. CONCLUSION: In summary, we present evidence demonstrating that Cx37 and Cx40 uniquely regulate postischemic limb perfusion, altering the severity of ischemic insult and consequent postischemic survival.
Assuntos
Conexinas/fisiologia , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Animais , Comunicação Celular , Células Endoteliais/fisiologia , Isquemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína alfa-5 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
The unique contributions of connexin (Cx)37 and Cx40, gap junction-forming proteins that are coexpressed in vascular endothelium, to the recovery of tissues from ischemic injury are unknown. We recently reported that Cx37-deficient (Cx37(-/-)) animals recovered ischemic hindlimb function more quickly and to a greater extent than wild-type (WT) or Cx40(-/-) animals, suggesting that Cx37 limits recovery in the WT animal. Here, we tested the hypothesis that enhanced angiogenesis, arteriogenesis, and vasculogenesis contribute to improved postischemic hindlimb recovery in Cx37(-/-) animals. Ischemia was induced unilaterally in the hindlimbs of WT or Cx37(-/-) mice (isoflurane anesthesia). Postsurgical limb appearance, use, and perfusion were documented during recovery, and the number (and size) of large and small vessels was determined. Native collateral number, predominantly established during embryonic development (vasculogenesis), was also determined in the pial circulation. Both microvascular density in the gastrocnemius of the ischemic limb (an angiogenic field) and the number and tortuosity of larger vessels in the gracilis vasculature (an arteriogenic field) were increased in Cx37(-/-) animals compared with WT animals. Cx37(-/-) mice also had an increased (vs. WT) number of collateral vessels in the pial circulation. These findings suggest that in Cx37(-/-) animals, improved recovery of the ischemic hindlimb involves enhanced vasculogenesis, resulting in increased numbers of collaterals in the hindlimb (and pial circulations) and more extensive collateral remodeling and angiogenesis. These results are consistent with Cx37 exerting a growth-suppressive effect in the vasculature that limits embryonic vasculogenesis as well as arteriogenic and angiogenic responses to ischemic injury in the adult animal.
Assuntos
Proteínas Angiostáticas/metabolismo , Conexinas/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Proteínas Angiostáticas/deficiência , Proteínas Angiostáticas/genética , Animais , Velocidade do Fluxo Sanguíneo , Circulação Cerebrovascular , Circulação Colateral , Conexinas/deficiência , Conexinas/genética , Modelos Animais de Doenças , Membro Posterior , Isquemia/diagnóstico , Isquemia/genética , Isquemia/fisiopatologia , Fluxometria por Laser-Doppler , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/metabolismo , Microvasos/fisiopatologia , Pia-Máter/irrigação sanguínea , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Fatores de Tempo , Microtomografia por Raio-X , Proteína alfa-4 de Junções ComunicantesRESUMO
Within the juxtaglomerular apparatus, renin-producing cells and endothelial cells of the afferent arterioles express connexin (Cx)37 and Cx40, which form abundant gap junctions among these cells. Deletion of Cx40 leads to strong hyperreninemia and ectopic localization of renin-producing cells; however, the relevance of Cx37 for the renin system in the kidney has not been investigated. We therefore studied renin expression and renin secretion in kidneys from Cx37-deficient mice, both on normal salt diet and during chronic challenge of the renin system by pretreatment of mice with a low-salt diet in combination with an angiotensin I-converting enzyme inhibitor. This treatment procedure strongly enhances renin gene expression and renin secretion. We found that renal renin mRNA abundance and plasma renin concentration did not differ between wild-type and Cx37-/- mice under normal conditions. The stimulation of renin gene expression and renin secretion by salt depletion was even more pronounced in Cx37-/- as compared to wild-type mice. The regulation of renin secretion from isolated perfused kidneys by perfusion pressure and by angiotensin II was normal in Cx37-/- mice. In addition, the localization of renin-expressing cells was also regular in Cx37-/- kidneys. Finally, the expression pattern of other vascular Cxs such as Cx40, Cx43, and Cx45 was not altered in Cx37-/- kidneys. Our findings suggest that Cx37 is not essential for normal development and function of renin-producing cells. As a consequence, it appears unlikely that Cx40 exerts its important function in renin-producing cells via Cx37/Cx40 heteromeric gap junctions.
Assuntos
Conexinas/metabolismo , Rim/citologia , Rim/metabolismo , Sistema Renina-Angiotensina/fisiologia , Renina/metabolismo , Actinas/metabolismo , Animais , Conexina 43/metabolismo , Células Endoteliais/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/análise , Renina/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína alfa-5 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
We have previously shown that increased nitric oxide (NO) production in sepsis impairs arteriolar-conducted vasoconstriction cGMP independently and that the gap junction protein connexin (Cx) 37 is required for this conducted response. In the present study, we hypothesized that NO impairs interendothelial electrical coupling in sepsis by targeting Cx37. We examined the effect of exogenous NO on coupling in monolayers of cultured microvascular endothelial cells derived from the hindlimb skeletal muscle of wild-type (WT), Cx37 null, Cx40 null, and Cx43(G60S) (nonfunctional mutant) mice. To assess coupling, we measured the spread of electrical current injected in the monolayer and calculated the monolayer intercellular resistance (inverse measure of coupling). The NO donor 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (DETA) rapidly and reversibly reduced coupling in cells from WT mice, cGMP independently. NO scavenger HbO(2) did not affect baseline coupling, but it eliminated DETA-induced reduction in coupling. Reduced coupling in response to DETA was also seen in cells from Cx40 null and Cx43(G60S) mice, but not in cells from Cx37 null mice. DETA did not alter the expression of Cx37, Cx40, and Cx43 in WT cells analyzed by immunoblotting and immunofluorescence. Furthermore, neither the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III), superoxide scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, nor preloading of WT cells with the antioxidant ascorbate affected this reduction. We conclude that NO-induced reduction of electrical coupling between microvascular endothelial cells depends on Cx37 and propose that NO in sepsis impairs arteriolar-conducted vasoconstriction by targeting Cx37 within the arteriolar wall.
Assuntos
Conexinas/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Óxido Nítrico/farmacologia , Animais , Western Blotting , Capilares/citologia , Capilares/fisiologia , Separação Celular , Conexina 43/genética , Conexina 43/fisiologia , Conexinas/genética , Eletrofisiologia , Endotélio Vascular/citologia , Imunofluorescência , Indicadores e Reagentes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doadores de Óxido Nítrico/farmacologia , Nitritos/farmacologia , Ácido Peroxinitroso/metabolismo , Proteína alfa-5 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
BACKGROUND: Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. RESULTS: We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. CONCLUSION: Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.
Assuntos
Conexinas/metabolismo , Matriz Extracelular/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Animais , Western Blotting , Comunicação Celular/fisiologia , Células Cultivadas , Conexinas/genética , Citometria de Fluxo , Imuno-Histoquímica , Laminina/metabolismo , Camundongos , Camundongos Knockout , Neurogênese/fisiologia , Oligodendroglia/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.
Assuntos
Conexina 43/análise , Conexinas/análise , Junções Comunicantes/química , Zona Pelúcida/ultraestrutura , Animais , Comunicação Celular , Feminino , Proteínas de Fluorescência Verde/análise , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/química , Folículo Ovariano/ultraestrutura , Proteínas Recombinantes/análise , Zona Pelúcida/química , Proteína alfa-4 de Junções ComunicantesRESUMO
When a short segment of arteriole is stimulated, vasomotor responses spread bidirectionally along the vessel axis purportedly via gap junctions. We used connexin40 knockout (Cx40-/-) mice to study vasomotor responses induced by 10-second trains of electrical stimulation (30 Hz, 1 ms, 30 to 50 V) in 2nd or 3rd order arterioles of the cremaster muscle. Measurements were made at the stimulation site (local) and at conducted sites (500, 1000, and 2000 microm upstream). In wild-type (Cx40+/+) animals, electrical stimulation evoked a local vasoconstriction and a conducted vasodilation that spread very rapidly along the vessel length without detectable decay. In Cx40-/- mice, the conducted dilation was converted into either vasoconstriction or a slowly developing vasodilation that decayed along the vessel length. Tetrodotoxin (TTX, 1 micromol/L) had no effect on the local vasoconstriction in either Cx40+/+ or Cx40-/- mice, but enhanced the conducted vasodilation in Cx40+/+ animals. In Cx40-/- mice, TTX abolished the conducted vasoconstriction when present and revealed a small vasodilation that decayed with distance. In the group of Cx40-/- mice in which electrical stimulation elicited a conducted vasodilation, TTX had no effect. Immunocytochemistry revealed Cx40 only in the endothelial layer of arterioles from Cx40+/+ mice and complete elimination of this connexin in the Cx40-/- animals. These results indicate that focal current stimulation causes vasoconstriction by a combination of perivascular nerve stimulation and smooth muscle activation. Moreover, electrical stimulation activates a nonneuronal, Cx40-dependent vasodilator response that spreads along the vessel length without decay.
Assuntos
Músculos Abdominais/irrigação sanguínea , Arteríolas/fisiologia , Conexinas/fisiologia , Vasodilatação/fisiologia , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/inervação , Conexinas/análise , Conexinas/genética , Estimulação Elétrica , Endotélio Vascular/química , Genótipo , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prazosina/farmacologia , Tetrodotoxina/farmacologia , Vasodilatação/efeitos dos fármacos , Proteína alfa-5 de Junções ComunicantesRESUMO
Mice lacking both connexin37 (Cx37) and connexin40 (Cx40), gap junction proteins expressed in vascular endothelium, die perinatally with pronounced vascular abnormalities. Early vasculogenesis proceeds normally, but by E18.5 Cx37(-/-)Cx40(-/-) animals display vessel dilatation and congestion as well as localized hemorrhages in skin, testis, intestines, and lungs. Abnormal vascular channels are present in the testis, often forming cavernous hemangioma-like defects. Unusually large, distended vessels are also present in the submucosa and lamina propria of the intestine. Ablation of Cx40 has a greater effect on endothelial dye-transfer than ablation of Cx37, and the effect of Cx40 ablation is age-dependent. Only in embryonic aortas lacking both Cx37 and Cx40 is there a complete loss of endothelial coupling. Surprisingly, elimination of Cx40 results in a large drop in aortic endothelial Cx37 on western blots, and deletion of Cx37 also reduces endothelial Cx40 levels. In contrast, in the medial layer, both Cx37 and Cx43 increase when Cx40 is ablated. These studies indicate that Cx37 and Cx40 are collectively critical for endothelial communication and provide evidence of an important role for gap junctions in vascular development. In addition, Cx37 and Cx40 appear to be mutually dependent on each other for normal expression in vascular endothelium.