Vascular endothelial growth factor and substrate mechanics regulate <i>in vitro</i> tubulogenesis of endothelial progenitor cells

Hanjaya‐Putra, Donny; Yee, Jane; Ceci, Doug; Truitt, Rachel; Yee, Derek; Gerecht, Sharon

doi:10.1111/j.1582-4934.2009.00981.x

Cited by 135 publications

(154 citation statements)

References 64 publications

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“…HUVECs were transfected with siGENOME SMARTpool human HIF1␣ and HIF2␣ (Dharmacon, Lafayette, CO), using the manufacturer's protocol as previously described (17). Briefly, we prepared the RNA interference (RNAi) transfection solution by mixing an antibiotic-and serum-free EGM (PromoCell) with DharmaFECT4 RNAi transfection reagent (Dharmacon).…”

Section: Methodsmentioning

confidence: 99%

“…We replaced the growth media with Calcein dye solution (2 M) and incubated the cells for 30 min at 37°C. The mean tube length and thickness and percentage of tube coverage were quantified, as we previously described (17,50). Briefly, we analyzed 18 images (ϫ10 magnification) taken at six different regions on the gel surface and three different positions in the gel depth (duplicates of n ϭ 3) using the Angiogenesis tool of MetaMorph software 6.1 (Universal Imaging, Downingtown, PA).…”

Section: Methodsmentioning

confidence: 99%

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Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

Abaci

Truitt

Tan

et al. 2011

American Journal of Physiology-Cell Physiology

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The availability of oxygen (O2) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O2 tension during 3D cultures of HUVECs. Noninvasive measurements of DO levels during culture under atmospheric conditions revealed a profound decrease that reached as low as 2% O2 at the end of 24 h. After media replacement, DO levels rose rapidly and equilibrated at ϳ15% O 2, creating a reoxygenated environment. To accurately estimate DO gradients in 3D collagen gels, we developed a 3D mathematical model and determined the Michaelis-Menten parameters, V max and K m, of HUVECs in collagen gels. We detected an increase in ROS levels throughout the culture period. Using diphenyliodonium to inhibit ROS production resulted in the complete inhibition of tube formation. Interference RNA studies further showed that hypoxiainducible factors (HIFs)-1␣ and -2␣ are not involved in the formation of 3D tubes in collagen gels. We conclude that ROS affect the tubulogenesis process through HIF␣-independent pathways, where the levels of ROS are influenced by the uncontrolled variations in DO levels. This study is the first demonstration of the critical and unexpected role of O 2 during 3D in vitro culture models of tubulogenesis in atmospheric conditions. microvasculature; reactive oxygen species; hypoxia-inducible factors AN UNDERSTANDING OF THE MECHANISMS controlling how blood vessels form from endothelial cells (ECs) is crucial for developmental biology and vascular tissue engineering. In recent decades, our understanding of the molecular regulation underlying vascular development has vastly expanded, primarily because of newly available, well-defined in vitro models. One common model is the culturing of ECs in gels made of the extracellular matrix component collagen, which has enabled us to investigate the effects of many factors on tubulogenesis (24,38,39).The availability of oxygen (O 2 ) is a critical parameter affecting how vascular cells form tubes. In vitro models have demonstrated that hypoxic conditions enhance tube formation, as do hypoxic regions formed by the O 2 consumption of the cells in threedimensional (3D) structures (3,19,32). Research has shown that the rate of O 2 consumption of cells follows the Michaelis-Menten kinetics and depends on the cell type, cell density (15), and dissolved oxygen (DO) levels (1). Variations in DO levels strongly affect angiogenesis (19, 48) through different vascular cell responses, such as viability, differentiation, migration, and remodeling of the extracellular matrix (4, 27, 40).Hypoxia-inducible factors (HIFs) and reactive oxygen species (ROS) are among the effectors responsible for cellular responses to changing DO levels. HIF1␣ and HI...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

Abaci

Truitt

Tan

et al. 2011

American Journal of Physiology-Cell Physiology

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“…55 Hanjaya-Putra et al have demonstrated that soft matrix reduces the MMP production by EPCs while increasing cell elongation and extending intracellular vacuoles to open lumen compartments through a signaling pathway involving the activation of the Rho GTPase Cdc42. 56 Not surprisingly, when normal tissue homeostasis becomes imbalanced, as can occur during tissue injury, tissue function can become impaired. Interestingly, a prominent feature of many injured tissues is altered tissue mechanics.…”

Section: Cellular Response To Ecm Stiffness During Tissue Homeostasismentioning

confidence: 99%

Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

et al. 2015

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ABSTRACT. Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression.

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“…ECFCs, from human umbilical cord blood isolated from outgrowth clones (kindly provided by Dr. Mervin C. Yoder, Indiana University School of Medicine) were expanded in endothelial growth media (EGM; PromoCell, Heidelberg, Germany, http:// www.promocell.com) and used for experiments between passages 6 and 8, as previously described [34,35].…”

Section: Human Ecfcsmentioning

confidence: 99%

Integration and Regression of Implanted Engineered Human Vascular Networks During Deep Wound Healing

Hanjaya‐Putra

Shen

Wilson

et al. 2013

Stem Cells Translational Medicine

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The ability of vascularized constructs to integrate with tissues may depend on the kinetics and stability of vascular structure development. This study assessed the functionality and durability of engineered human vasculatures from endothelial progenitors when implanted in a mouse deep burn-wound model. Human vascular networks, derived from endothelial colony-forming cells in hyaluronic acid hydrogels, were transplanted into third-degree burns. On day 3 following transplantation, macrophages rapidly degraded the hydrogel during a period of inflammation; through the transitions from inflammation to proliferation (days 5-7), the host's vasculatures infiltrated the construct, connecting with the human vessels within the wound area. The growth of mouse vessels near the wound area supported further integration with the implanted human vasculatures. During this period, the majority of the vessels (ϳ60%) in the treated wound area were human. Although no increase in the density of human vessels was detected during the proliferative phase, they temporarily increased in size. This growth peaked at day 7, the middle of the proliferation stage, and then decreased by the end of the proliferation stage. As the wound reached the remodeling period during the second week after transplantation, the vasculatures including the transplanted human vessels generally regressed, and few microvessels, wrapped by mouse smooth muscle cells and with a vessel area less than 200 m 2 (including the human ones), remained in the healed wound. Overall, this study offers useful insights for the development of vascularization strategies for wound healing and ischemic conditions, for tissue-engineered constructs, and for tissue regeneration. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:297-306

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Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells

Cited by 135 publications

References 64 publications

Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

Integration and Regression of Implanted Engineered Human Vascular Networks During Deep Wound Healing

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