Age-distribution estimation for karst groundwater: Issues of parameterization and complexity in inverse modeling by convolution

Convolution modeling is useful for investigating the temporal distribution of groundwater age based on environmental tracers. The framework of a quasi-transient convolution model that is applicable to two-domain flow in karst aquifers is presented. The model was designed to provide an acceptable level of statistical confidence in parameter estimates when only chlorofluorocarbon (CFC) and tritium (3H) data are available. We show how inverse modeling and uncertainty assessment can be used to constrain model parameterization to a level warranted by available data while allowing major aspects of the flow system to be examined. As an example, the model was applied to water from a pumped well open to the Madison aquifer in central USA with input functions of CFC-11, CFC-12, CFC-113, and 3H, and was calibrated to several samples collected during a 16-year period. A bimodal age distribution was modeled to represent quick and slow flow less than 50 years old. The effects of pumping and hydraulic head on the relative volumetric fractions of these domains were found to be influential factors for transient flow. Quick flow and slow flow were estimated to be distributed mainly within the age ranges of 0–2 and 26–41 years, respectively. The fraction of long-term flow (\u3e50 years) was estimated but was not dateable. The different tracers had different degrees of influence on parameter estimation and uncertainty assessments, where 3H was the most critical, and CFC-113 was least influential

Age-distribution estimation for karst groundwater: Issues of parameterization and complexity in inverse modeling by convolution

Convolution modeling is useful for investigating the temporal distribution of groundwater age based on environmental tracers. The framework of a quasi-transient convolution model that is applicable to two-domain flow in karst aquifers is presented. The model was designed to provide an acceptable level of statistical confidence in parameter estimates when only chlorofluorocarbon (CFC) and tritium (3H) data are available. We show how inverse modeling and uncertainty assessment can be used to constrain model parameterization to a level warranted by available data while allowing major aspects of the flow system to be examined. As an example, the model was applied to water from a pumped well open to the Madison aquifer in central USA with input functions of CFC-11, CFC-12, CFC-113, and 3H, and was calibrated to several samples collected during a 16-year period. A bimodal age distribution was modeled to represent quick and slow flow less than 50 years old. The effects of pumping and hydraulic head on the relative volumetric fractions of these domains were found to be influential factors for transient flow. Quick flow and slow flow were estimated to be distributed mainly within the age ranges of 0–2 and 26–41 years, respectively. The fraction of long-term flow (\u3e50 years) was estimated but was not dateable. The different tracers had different degrees of influence on parameter estimation and uncertainty assessments, where 3H was the most critical, and CFC-113 was least influential

Protease‐Sensitive PEG Hydrogels Regulate Vascularization In Vitro and In Vivo

Forming functional blood vessel networks in engineered or ischemic tissues is a significant scientific and clinical hurdle. Poly(ethylene glycol) (PEG)‐based hydrogels are adapted to investigate the role of mechanical properties and proteolytic susceptibility on vascularization. Four arm PEG vinyl sulfone is polymerized by Michael‐type addition with cysteine groups on a slowly degraded matrix metalloprotease (MMP) susceptible peptide, GPQG↓IWGQ, or a more rapidly cleaved peptide, VPMS↓MRGG. Co‐encapsulation of endothelial cells and supportive fibroblasts within the gels lead to vascular morphogenesis in vitro that is robust to changes in crosslinking peptide identity, but is significantly attenuated by increased crosslinking and MMP inhibition. Perfused vasculature forms from transplanted cells in vivo in all gel types; however, in contrast to the in vitro results, vascularization in vivo is not decreased in the more crosslinked gels. Collectively, these findings demonstrate the utility of this platform to support vascularization both in vitro and in vivo. Co‐encapsulation of endothelial cells and supportive fibroblasts within proteolyzable poly(ethylene glycol) (PEG)‐based hydrogels leads to vascular morphogenesis in vitro that is robust to changes in crosslinking peptide identity but is significantly attenuated by gel crosslinking and MMP inhibition. In vivo, perfused vasculature forms from transplanted cells in all gel types, and vascularization is not attenuated in more crosslinked gels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109336/1/mabi201400161.pd

Effects of Extracellular Matrix Density and Mesenchymal Stem Cells on Neovascularization In Vivo

Aberrant angiogenesis is common to a variety of diseases in which alterations in tissue mechanical properties also occur. A fundamental understanding of the interdependence of angiogenesis and tissue structural properties may enhance the development of therapeutic strategies. We previously established that increasing extracellular matrix density inhibits capillary morphogenesis in three-dimensional tissues in vitro, and that addition of human mesenchymal stem cells (MSCs) partially rescues a healthy angiogenic phenotype. This study's goal was to investigate if these effects can be recapitulated in vivo. Human umbilical vein endothelial cells, MSCs, or a mixture of both was suspended in fibrin gel precursor solutions of 5, 10, and 15-mg/mL concentrations and injected subcutaneously into SCID mice. Neovascularization was assessed in tissue constructs retrieved at 3, 7, and 21 days by quantifying vessel numbers, perfusion, thickness, maturity, and perivascular collagen deposition. The data show that changing extracellular matrix density inhibits capillary morphogenesis in vivo in a manner consistent with that observed in vitro. Delivery of both human umbilical vein endothelial cells and MSCs produced more robust and mature vessels than delivery of either cell type alone in all tissue concentrations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90460/1/ten-2Etea-2E2010-2E0275.pd

Stromal Cell Identity Influences the In Vivo Functionality of Engineered Capillary Networks Formed by Co-delivery of Endothelial Cells and Stromal Cells

A major translational challenge in the fields of therapeutic angiogenesis and tissue engineering is the ability to form functional networks of blood vessels. Cell-based strategies to promote neovascularization have been widely explored, and have led to the consensus that co-delivery of endothelial cells (ECs) (or their progenitors) with some sort of a supporting stromal cell type is the most effective approach. However, the choice of stromal cells has varied widely across studies, and their impact on the functional qualities of the capillaries produced has not been examined. In this study, we injected human umbilical vein ECs alone or with normal human lung fibroblasts (NHLFs), human bone marrow-derived mesenchymal stem cells (BMSCs), or human adipose-derived stem cells (AdSCs) in a fibrin matrix into subcutaneous pockets in SCID mice. All conditions yielded new human-derived vessels that inosculated with mouse vasculature and perfused the implant, but there were significant functional differences in the capillary networks, depending heavily on the identity of the co-delivered stromal cells. EC-alone and EC-NHLF implants yielded immature capillary beds characterized by high levels of erythrocyte pooling in the surrounding matrix. EC-BMSC and EC-AdSC implants produced more mature capillaries characterized by less extravascular leakage and the expression of mature pericyte markers. Injection of a fluorescent tracer into the circulation also showed that EC-BMSC and EC-AdSC implants formed vasculature with more tightly regulated permeability. These results suggest that the identity of the stromal cells is key to controlling the functional properties of engineered capillary networks.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140223/1/ten.tea.2012.0281.pd

A Safe and Efficient Method to Retrieve Mesenchymal Stem Cells from Three-Dimensional Fibrin Gels

Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140244/1/ten.tec.2013.0051.pd

Budd-Chiari syndrome recurring in a transplanted liver

A patient with Budd-Chiari syndrome who underwent orthotopic liver transplantation and developed recurrent disease is described. The immediate postoperative period was complicated by multiple thrombotic episodes, followed by a period of apparent remission associated with the initiation of coumadin and persantine therapy. After discontinuation of such antithrombotic therapy in order to biopsy the liver, the patient experienced another series of clinically overt vascular thromboses and ultimately died of sepsis 15 mo posttransplantation after a prolonged and complicated terminal hospital course. At autopsy, recurrent Budd-Chiari syndrome as well as thromboses in numerous other organs was demonstrated. © 1983

Assessing the ability of human endothelial cells derived from induced‐pluripotent stem cells to form functional microvasculature in vivo

Forming functional blood vessel networks is a major clinical challenge in the fields of tissue engineering and therapeutic angiogenesis. Cell‐based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation. Induced‐pluripotent stem cell‐derived endothelial cells (iPSC‐ECs) are a promising, potentially autologous, alternative cell source. However, it is unclear whether iPSC‐ECs form the same robust microvasculature in vivo documented for other EC sources. In this study, we utilized a well‐established in vivo model, in which ECs (iPSC‐EC or human umbilical vein endothelial cells [HUVEC]) were coinjected with normal human lung fibroblasts (NHLFs) and a fibrin matrix into the dorsal flank of severe combined immunodeficiency mice to assess their ability to form functional microvasculature. Qualitatively, iPSC‐ECs were capable of vessel formation and perfusion and demonstrated similar vessel morphologies to HUVECs. However, quantitatively, iPSC‐ECs exhibited a two‐fold reduction in vessel density and a three‐fold reduction in the number of perfused vessels compared with HUVECs. Further analysis revealed the presence of collagen‐IV and α‐smooth muscle actin were significantly lower around iPSC‐EC/NHLF vasculature than in HUVEC/NHLF implants, suggesting reduced vessel maturity. Collectively, these results demonstrate the need for increased iPSC‐EC maturation for clinical translation to be realized.Forming functional blood vessel networks is a major clinical challenge in the fields of tissue engineering and therapeutic angiogenesis. Cell‐based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146963/1/bit26860.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146963/2/bit26860_am.pd

Src, PKCα, and PKCδ are required for αvβ3 integrin-mediated metastatic melanoma invasion

<p>Abstract</p> <p>Background</p> <p>Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor αvβ3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.</p> <p>Results</p> <p>Two melanoma cell lines C8161.9 and M14 both express high levels of αvβ3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an αvβ3-depenent manner. Elevated levels of PKCα and PKCδ, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed αvβ3-dependent invasion. Furthermore, over expression of Src or PKCα and PKCδ was sufficient to confer αvβ3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCα expression, but not PKCδ, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCα restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCδ primarily restored stress fibers.</p> <p>Conclusion</p> <p>The misregulated expression of PKCα and PKCδ and elevated Src activity in metastatic melanoma cells is required for efficient αvβ3-mediated invasion. PKCα and Src enhance αvβ3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCα influences focal adhesion formation, while PKCδ controls stress fibers.</p