1 Department of Physiology and Biophysics and 2 Bioengineering Laboratory, Department of Chemical and Biological Engineering, State University of New York, Buffalo, New York
Submitted 20 May 2004
; ...accepted in final form 7 October 2004
We engineered implantable small-diameter blood vessels based on ovine smooth muscle and endothelial cells embedded in fibrin gels. Cylindrical tissue constructs remodeled the fibrin matrix and exhibited considerable reactivity in response to receptor- and nonreceptor-mediated vasoconstrictors and dilators. Aprotinin, a protease inhibitor of fibrinolysis, was added at varying concentrations and affected the development and functionality of tissue-engineered blood vessels (TEVs) in a concentration-dependent manner. Interestingly, at moderate concentrations, aprotinin increased mechanical strength but decreased vascular reactivity, indicating a possible relationship between matrix degradation/remodeling, vasoreactivity, and mechanical properties. TEVs developed considerable mechanical strength to withstand interpositional implantation in jugular veins of lambs. Implanted TEVs integrated well with the native vessel and demonstrated patency and similar blood flow rates as the native vessels. At 15 wk postimplantation, TEVs exhibited remarkable matrix remodeling with production of collagen and elastin fibers and orientation of smooth muscle cells perpendicular to the direction of blood flow. Implanted vessels gained significant mechanical strength and reactivity that were comparable to those of native veins. Our work demonstrates that fibrin-based TEVs hold significant promise for treatment of vascular disease and as a biological model for studying vascular development and pathophysiology.
matrix degradation/remodeling; vascular disease; vascular reactivity; vascular tissue engineering; smooth muscle; endothelial cells
Address for reprint requests and other correspondence: S. T. Andreadis, Bioengineering Laboratory, 908 Furnas Hall, Dept. of Chemical and Biological Engineering, State Univ. of New York, Buffalo, NY 14260 (E-mail: sandread{at}eng.buffalo.edu )
Ionizing radiation, commonly used for head and neck cancer treatment, typically damages the salivary glands, resulting in hyposalivation. The development of treatments to restore this lost function ...is crucial for improving the quality of life for patients suffering from this condition. To address this clinical need, we have developed an innovative hydrogel by chemically conjugating laminin-1 peptides (A99 and YIGSR) and growth factors, FGF-7 and FGF-10, to fibrin hydrogels. Our results demonstrate that FGF-7/10 and laminin-1 peptides fortified fibrin hydrogel enhanced laminin-1 peptides fibrin hydrogel (Ep-FH) promotes salivary gland regeneration and functionality by improving epithelial tissue organization, establishing a healthy network of blood vessels and nerves, while reducing fibrosis in a head and neck irradiated mouse model. These results indicate that fibrin hydrogel-based implantable scaffolds containing pro-regenerative signals promote sustained secretory function of irradiated salivary glands, offering a potential alternative treatment for hyposalivation in head and neck cancer patients undergoing radiation treatment. These unique findings emphasize the potential of fibrin hydrogel-based implantable scaffolds enriched with pro-regenerative signals in sustaining the secretory function of irradiated salivary glands and offer a promising alternative treatment for addressing hyposalivation in head and neck cancer patients undergoing radiation therapy. STATEMENT OF SIGNIFICANCE: Radiation therapies used to treat head and neck cancers often result in damaged salivary gland, leading to severe dryness of the oral cavity. In this study, we engineered FGF-7 and FGF-10 and immobilized them into L
-FH. The resulting hydrogel, Ep-FH, restored irradiated salivary gland functionality by enhancing epithelial tissue organization, promoting the development of a healthy network of blood vessels and nerves as well as reduction of fibrosis.
Cell migration through confining three dimensional (3D) topographies can lead to loss of nuclear envelope integrity, DNA damage, and genomic instability. Despite these detrimental phenomena, cells ...transiently exposed to confinement do not usually die. Whether this is also true for cells subjected to long‐term confinement remains unclear at present. To investigate this, photopatterning and microfluidics are employed to fabricate a high‐throughput device that circumvents limitations of previous cell confinement models and enables prolonged culture of single cells in microchannels with physiologically relevant length scales. The results of this study show that continuous exposure to tight confinement can trigger frequent nuclear envelope rupture events, which in turn promote P53 activation and cell apoptosis. Migrating cells eventually adapt to confinement and evade cell death by downregulating YAP activity. Reduced YAP activity, which is the consequence of confinement‐induced YAP1/2 translocation to the cytoplasm, suppresses the incidence of nuclear envelope rupture and abolishes P53‐mediated cell death. Cumulatively, this work establishes advanced, high‐throughput biomimetic models for better understanding cell behavior in health and disease, and underscores the critical role of topographical cues and mechanotransduction pathways in the regulation of cell life and death.
This study employs novel microfluidic devices to demonstrate how migrating cells respond and adapt to long‐term confinement. Constant exposure to confined microenvironments that deform the nucleus can reduce cell viability. However, YAP translocation to the cytoplasm helps cells adapt to confinement by reducing the frequency of nuclear envelope rupture events and preventing P53‐mediated cell death.
The c-Jun amino-terminal kinase (JNK) is an important player in inflammation, proliferation, and apoptosis. More recently, JNK was found to regulate cell migration by phosphorylating paxillin. Here, ...we report a novel role of JNK in cell adhesion. Specifically, we provide evidence that JNK binds to E-cadherin/β-catenin complex and phosphorylates β-catenin at serine 37 and threonine 41, the sites also phosphorylated by GSK-3β. Inhibition of JNK kinase activity using dominant-negative constructs reduces phosphorylation of β-catenin and promotes localization of E-cadherin/β-catenin complex to cell-cell contact sites. Conversely, activation of JNK induces β-catenin phosphorylation and disruption of cell contacts, which are prevented by JNK siRNA. We propose that JNK binds to β-catenin and regulates formation of adherens junctions, ultimately controlling cell-to-cell adhesion.--Lee, M.-H., Koria, P., Qu, J., Andreadis, S. T. JNK phosphorylates β-catenin and regulates adherens junctions.
Mitochondrial dysfunction, a hallmark of aging, has been associated with the onset of aging phenotypes and age-related diseases. Here, we report that impaired mitochondrial function is associated ...with increased glutamine catabolism in senescent human mesenchymal stem cells (MSCs) and myofibroblasts derived from patients suffering from Hutchinson-Gilford progeria syndrome. Increased glutaminase (GLS1) activity accompanied by loss of urea transporter SLC14A1 induces urea accumulation, mitochondrial dysfunction, and DNA damage. Conversely, blocking GLS1 activity restores mitochondrial function and leads to amelioration of aging hallmarks. Interestingly, GLS1 expression is regulated through the JNK pathway, as demonstrated by chemical and genetic inhibition. In agreement with our in vitro findings, tissues isolated from aged or progeria mice display increased urea accumulation and GLS1 activity, concomitant with declined mitochondrial function. Inhibition of glutaminolysis in progeria mice improves mitochondrial respiratory chain activity, suggesting that targeting glutaminolysis may be a promising strategy for restoring age-associated loss of mitochondrial function.
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•Overactivated glutaminolysis leads to increased urea production in senescent cells•Loss of urea transporter leads to urea accumulation causing mitochondrial dysfunction•JNK pathway positively regulates GLS1 expression•Inhibiting GLS1 improves mitochondrial function and ameliorates aging hallmarks
Choudhury et al. report that senescent cells exhibit enhanced glutaminolysis and loss of urea transporter, increasing urea production and accumulation. Increased intracellular urea severely impairs mitochondrial function and exacerbates the aging phenotype. Inhibiting glutaminolysis by blocking GLS1 significantly improves mitochondrial function in senescent cells and progeria mice.
During epithelial-mesenchymal transition (EMT) in cancer progression, tumor cells switch cadherin profile from E-cadherin to cadherin-11 (CDH11), which is accompanied by increased invasiveness and ...metastatic activity. However, the mechanism through which CDH11 may affect tumor growth and metastasis remains elusive. Here, we report that CDH11 was highly expressed in multiple human tumors and was localized on the membrane, in the cytoplasm and, surprisingly, also in the nucleus. Interestingly, β-catenin remained bound to carboxy-terminal fragments (CTFs) of CDH11, the products of CDH11 cleavage, and co-localized with CTFs in the nucleus in the majority of breast cancer samples. Binding of β-catenin to CTFs preserved β-catenin activity, whereas inhibiting CDH11 cleavage led to β-catenin phosphorylation and diminished Wnt signaling, similar to CDH11 knockout. Our data elucidates a previously unknown role of CDH11, which serves to stabilize β-catenin in the cytoplasm and facilitate its translocation to the nucleus, resulting in activation of Wnt signaling, with subsequent increased proliferation, migration and invasion potential.
Large size cell-laden hydrogel models hold great promise for tissue repair and organ transplantation, but their fabrication using 3D bioprinting is limited by the slow printing speed that can affect ...the part quality and the biological activity of the encapsulated cells. Here a fast hydrogel stereolithography printing (FLOAT) method is presented that allows the creation of a centimeter-sized, multiscale solid hydrogel model within minutes. Through precisely controlling the photopolymerization condition, low suction force-driven, high-velocity flow of the hydrogel prepolymer is established that supports the continuous replenishment of the prepolymer solution below the curing part and the nonstop part growth. The rapid printing of centimeter-sized hydrogel models using FLOAT is shown to significantly reduce the part deformation and cellular injury caused by the prolonged exposure to the environmental stresses in conventional 3D printing methods. Embedded vessel networks fabricated through multiscale printing allows media perfusion needed to maintain the high cellular viability and metabolic functions in the deep core of the large-sized models. The endothelialization of this vessel network allows the establishment of barrier functions. Together, these studies demonstrate a rapid 3D hydrogel printing method and represent a first step toward the fabrication of large-sized engineered tissue models.
Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in ...cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase α and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated β-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks.
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•Senescent MSCs exhibit reduced PYCR1 and PYCR2 expression•Proline induces Parkin-mediated mitophagy leading to improved mitochondrial function•Proline ameliorates several aging hallmarks, including SA-β-Gal and DNA damage•Proline improves myogenic differentiation capacity of senescent MSCs
Choudhury et al. report that senescent cells exhibit diminished proline biosynthesis and benefit from proline supplementation. Proline promotes mitophagy by activating AMPKα and upregulating Parkin, improving mitochondrial function, and ameliorating aging hallmarks. Parkin is necessary to mediate the effects of proline and reverse aging hallmarks, but it is not sufficient.
ABSTRACT
Collagen type III (COL3) is one of the 3 major collagens in the body, and loss of expression or mutations in the COL3 gene have been associated with the onset of vascular diseases such the ...Ehlers‐Danlos syndrome. Previous work reported a significant reduction of COL3 in tissues such as skin and vessels with aging. In agreement, we found that COL3 was significantly reduced in senescent human mesenchymal stem cells and myofibroblasts derived from patients with Hutchinson‐Gilford progeria syndrome, a premature aging syndrome. Most notably, we discovered that ectopic expression of the embryonic transcription factor Nanog homeobox (NANOG) restored COL3 expression by restoring the activity of the TGF‐β pathway that was impaired in senescent cells. RNA sequencing analysis showed that genes associated with the activation of the TGF‐β pathway were up‐regulated, whereas negative regulators of the pathway were down‐regulated upon NANOG expression. Chromatin immunoprecipitation sequencing and immunoprecipitation experiments revealed that NANOG bound to the mothers against decapentaplegic (SMAD)2 and SMAD3 promoters, in agreement with increased expression and phosphorylation levels of both proteins. Using chemical inhibition, short hairpin RNA knockdown, and gain of function approaches, we established that both SMAD2 and SMAD3 were necessary to mediate the effects of NANOG, but SMAD3 overexpression was also sufficient for COL3 production. In summary, NANOG restored production of COL3, which was impaired by cellular aging, suggesting novel strategies to restore the impaired extracellular matrix production and biomechanical function of aged tissues, with potential implications for regenerative medicine and anti‐aging treatments.—Rong, N., Mistriotis, P., Wang, X., Tseropoulos, G., Rajabian, N., Zhang, Y., Wang, J., Liu, S., Andreadis, S. T. Restoring extracellular matrix synthesis in senescent stem cells. FASEB J. 33, 10954–10965 (2019). www.fasebj.org
This review focuses on the stem cell sources with the potential to be used in vascular tissue engineering and to promote vascular regeneration. The first clinical studies using tissue-engineered ...vascular grafts are already under way, supporting the potential of this technology in the treatment of cardiovascular and other diseases. Despite progress in engineering biomaterials with the appropriate mechanical properties and biological cues as well as bioreactors for generating the correct tissue microenvironment, the source of cells that make up the vascular tissues remains a major challenge for tissue engineers and physicians. Mature cells from the tissue of origin may be difficult to obtain and suffer from limited proliferative capacity, which may further decline as a function of donor age. On the other hand, multipotent and pluripotent stem cells have great potential to provide large numbers of autologous cells with a great differentiation capacity. Here, we discuss the adult multipotent as well as embryonic and induced pluripotent stem cells, their differentiation potential toward vascular lineages, and their use in engineering functional and implantable vascular tissues. We also discuss the associated challenges that need to be addressed in order to facilitate the transition of this technology from the bench to the bedside.
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