Ultrashort pulse,multispectral non-linear optical microscopy(NLOM)is developed and used to image,simultaneously,a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angi...Ultrashort pulse,multispectral non-linear optical microscopy(NLOM)is developed and used to image,simultaneously,a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis.Broadband,sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen.A 16-channel multispectral detector is used to delineate the multiple non-linear optical signals,pixel by pixel,in NLOM.The ability to image multiple fluorescent protein mutants and collagen,enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.展开更多
Background:The lysosphingolipid,sphingosine-1-phosphate,is a well-described and potent pro-angiogenic factor.Receptors,as well as the sphingosine phosphorylating enzyme sphingosine kinase 1,are expressed in the placen...Background:The lysosphingolipid,sphingosine-1-phosphate,is a well-described and potent pro-angiogenic factor.Receptors,as well as the sphingosine phosphorylating enzyme sphingosine kinase 1,are expressed in the placentomes of sheep and the decidua of rodents;however,a function for this signaling pathway during pregnancy has not been established.The objective of this study was to investigate whether sphingosine-1-phosphate promoted angiogenesis within the placentomes of pregnant ewes.Ewes were given daily jugular injections of FTY720(2-amino-2[2-(−4-octylphenyl)ethyl]propate-1,3-diol hydrochloride),an S1P analog.Results:FTY720 infusion from days 30 to 60 of pregnancy did not alter maternal organ weights nor total number or mass of placentomes,but did alter placentome histoarchitecture.Interdigitation of caruncular crypts and cotyledonary villi was decreased,as was the relative area of cotyledonary tissue within placentomes.Also,the percentage of area occupied by cotyledonary villi per unit of placentome was increased,while the thickness of the caruncular capsule was decreased in ewes treated with FTY720.Further,FTY720 infusion decreased the number and density of blood vessels within caruncular tissue near the placentome capsule where the crypts emerge from the capsule.Finally,FTY720 infusion decreased asparagine and glutamine in amniotic fluid and methionine in allantoic fluid,and decreased the crown rump length of day 60 fetuses.Conclusions:While members of the sphingosine-1-phosphate signaling pathway have been characterized within the uteri and placentae of sheep and mice,the present study uses FTY720 to address the influence of S1P signaling on placental development.We present evidence that modulation of the S1P signaling pathway results in the alteration of caruncular vasculature,placentome architecture,abundance of amino acids in allantoic and amniotic fluids,and fetal growth during pregnancy in sheep.The marked morphological changes in placentome histoarchitecture,including alteration in the vasculature,may be relevant to fetal growth and survival.It is somewhat surprising that fetal length was reduced as early as day 60,because fetal growth in sheep is greatest after day 60.The subtle changes observed in the fetuses of ewes exposed to FTY720 may indicate an adaptive response of the fetuses to cope with altered placental morphology.展开更多
Vascularization is a key pre-requisite to engineered anatomical scale three dimensional(3-D)constructs to ensure their nutrient and oxygen supply upon implantation.Presently,engineered pre-vascularized 3-D tissues are...Vascularization is a key pre-requisite to engineered anatomical scale three dimensional(3-D)constructs to ensure their nutrient and oxygen supply upon implantation.Presently,engineered pre-vascularized 3-D tissues are limited to only micro-scale hydrogels,which meet neither the anatomical scale needs nor the complexity of natural extracellular matrix(ECM)environments.Anatomical scale perfusable constructs are critically needed for translational applications.To overcome this challenge,we previously developed pre-vascularized ECM sheets with long and oriented dense microvascular networks.The present study further evaluated the patency,perfusability and innate immune response toward these pre-vascularized constructs.Macrophage-co-cultured prevascularized constructs were evaluated in vitro to confirm micro-vessel patency and perturbations in macrophage metabolism.Subcutaneously implanted pre-vascularized constructs remained viable and formed a functional anastomosis with host vasculature within 3 days of implantation.This completely biological pre-vascularized construct holds great potential as a building block to engineer perfusable anatomical scale tissues.展开更多
基金funded by American Heart Association SDG(#0530020N)to KJB,National Institutes of Health(EB008366)and NSF Early Career Faculty Development Award(CAREER)to ATY.
文摘Ultrashort pulse,multispectral non-linear optical microscopy(NLOM)is developed and used to image,simultaneously,a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis.Broadband,sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen.A 16-channel multispectral detector is used to delineate the multiple non-linear optical signals,pixel by pixel,in NLOM.The ability to image multiple fluorescent protein mutants and collagen,enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.
基金National Research Initiative Competitive Grant No.2009-35203-05725(KJB and GAJ)Fellowship No.2008-35203-18830(KAD)from the USDA National Institute of Food and Agriculture.
文摘Background:The lysosphingolipid,sphingosine-1-phosphate,is a well-described and potent pro-angiogenic factor.Receptors,as well as the sphingosine phosphorylating enzyme sphingosine kinase 1,are expressed in the placentomes of sheep and the decidua of rodents;however,a function for this signaling pathway during pregnancy has not been established.The objective of this study was to investigate whether sphingosine-1-phosphate promoted angiogenesis within the placentomes of pregnant ewes.Ewes were given daily jugular injections of FTY720(2-amino-2[2-(−4-octylphenyl)ethyl]propate-1,3-diol hydrochloride),an S1P analog.Results:FTY720 infusion from days 30 to 60 of pregnancy did not alter maternal organ weights nor total number or mass of placentomes,but did alter placentome histoarchitecture.Interdigitation of caruncular crypts and cotyledonary villi was decreased,as was the relative area of cotyledonary tissue within placentomes.Also,the percentage of area occupied by cotyledonary villi per unit of placentome was increased,while the thickness of the caruncular capsule was decreased in ewes treated with FTY720.Further,FTY720 infusion decreased the number and density of blood vessels within caruncular tissue near the placentome capsule where the crypts emerge from the capsule.Finally,FTY720 infusion decreased asparagine and glutamine in amniotic fluid and methionine in allantoic fluid,and decreased the crown rump length of day 60 fetuses.Conclusions:While members of the sphingosine-1-phosphate signaling pathway have been characterized within the uteri and placentae of sheep and mice,the present study uses FTY720 to address the influence of S1P signaling on placental development.We present evidence that modulation of the S1P signaling pathway results in the alteration of caruncular vasculature,placentome architecture,abundance of amino acids in allantoic and amniotic fluids,and fetal growth during pregnancy in sheep.The marked morphological changes in placentome histoarchitecture,including alteration in the vasculature,may be relevant to fetal growth and survival.It is somewhat surprising that fetal length was reduced as early as day 60,because fetal growth in sheep is greatest after day 60.The subtle changes observed in the fetuses of ewes exposed to FTY720 may indicate an adaptive response of the fetuses to cope with altered placental morphology.
基金Texas A&M University Microscopy and Imaging Center Core Facility(RRID:SCR_022128)the Integrated Microscopy and Imaging Laboratory at Texas A&M College of Medicine(RRID:SCR_021637)for providing microscopy resources.
文摘Vascularization is a key pre-requisite to engineered anatomical scale three dimensional(3-D)constructs to ensure their nutrient and oxygen supply upon implantation.Presently,engineered pre-vascularized 3-D tissues are limited to only micro-scale hydrogels,which meet neither the anatomical scale needs nor the complexity of natural extracellular matrix(ECM)environments.Anatomical scale perfusable constructs are critically needed for translational applications.To overcome this challenge,we previously developed pre-vascularized ECM sheets with long and oriented dense microvascular networks.The present study further evaluated the patency,perfusability and innate immune response toward these pre-vascularized constructs.Macrophage-co-cultured prevascularized constructs were evaluated in vitro to confirm micro-vessel patency and perturbations in macrophage metabolism.Subcutaneously implanted pre-vascularized constructs remained viable and formed a functional anastomosis with host vasculature within 3 days of implantation.This completely biological pre-vascularized construct holds great potential as a building block to engineer perfusable anatomical scale tissues.
