Sung HJ, Chandra P, Treiser MD, Liu E, Iovine CP, Moghe PV, Kohn J. Synthetic polymeric substrates as potent pro-oxidant versus anti-oxidant regulators of cytoskeletal remodeling and cell apoptosis. (2009) J Cell Physiol 218: 549-57
Show Abstract · Added July 2, 2010The role of reactive oxygen species (ROS)-mediated cell signal transduction pathways emanating from engineered cell substrates remains unclear. To elucidate the role, polymers derived from the amino acid L-tyrosine were used as synthetic matrix substrates. Variations in their chemical properties were created by co-polymerizing hydrophobic L-tyrosine derivatives with uncharged hydrophilic poly(ethylene glycol) (PEG, Mw = 1,000 Da), and negatively charged desaminotyrosyl-tyrosine (DT). These substrates were characterized for their intrinsic ability to generate ROS, as well as their ability to elicit Saos-2 cell responses in terms of intracellular ROS production, actin remodeling, and apoptosis. PEG-containing substrates induced both exogenous and intracellular ROS production, whereas the charged substrates reduced production of both types, indicating a coupling of exogenous ROS generation and intracellular ROS production. Furthermore, PEG-mediated ROS induction caused nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase and an increase in caspase-3 activity, confirming a link with apoptosis. PEG-rich pro-oxidant substrates caused cytoskeletal actin remodeling through beta-actin cleavage by caspase-3 into fractins. The fractins co-localized to the mitochondria and reduced the mitochondrial membrane potential. The remnant cytosolic beta-actin was polymerized and condensed, events consistent with apoptotic cell shrinkage. The cytoskeletal remodeling was integral to the further augmentation of intracellular ROS production. Conversely, the anti-oxidant DT-containing charged substrates suppressed the entire cascade of apoptotic progression. We demonstrate that ROS activity serves an important role in "outside-in" signaling for cells grown on substrates: the ROS activity couples exogenous stress, driven by substrate composition, to changes in intracellular signaling. This signaling causes cell apoptosis, which is mediated by actin remodeling. | Publication | 19016472 (PMID)
10.1002/jcp.21629 (DOI) |
Sung HJ, Sakala Labazzo KM, Bolikal D, Weiner MJ, Zimnisky R, Kohn J. Angiogenic competency of biodegradable hydrogels fabricated from polyethylene glycol-crosslinked tyrosine-derived polycarbonates. (2008) Eur Cell Mater 15: 77-87
Show Abstract · Added July 2, 2010Synthetic biomaterials can be used as instructive biological milieus to guide cellular behaviour and function. To further realize this application, we synthesized a series of structurally similar hydrogels and tested their ability to modulate angiogenesis. Hydrogels were synthesized from poly(DTE-co-x% DT carbonate) crosslinked by y% poly(ethylene glycol) (PEG). Hydrogel desaminotyrosyl tyrosine (DT) contents (x%) ranged from 10-100%, and crosslink densities (y% PEG-crosslinker) ranged from 5-80%. The hydrogels were fashioned into porous scaffolds with highly interconnected macro- and micro-pore (>100 and 10 mm in diameter, respectively) architecture using poly(DTE-co-10%DT carbonate% crosslinked with 8% PEG. Under physiological conditions (in vitro), the hydrogels degraded into three major products: desaminotyrosyl-tyrosine ethyl ester (DTE), desaminotyrosyl tyrosine (DT), and poly(ethylene glycol)-di-DT-hydrazide (PEG-di-DT hydrazide). Increasing either DT content or crosslink density brought quickened degradation. Because DT and DTE, two of the three major degradation products, have not demonstrated any noticeable cytotoxicity or angiogenic effect in previous studies, we measured the cytotoxicity of PEG-di-DT hydrazide, the third major degradation product. We found that PEG-di-DT hydrazide only displayed significant cytotoxicity at the high concentration of 100 mg/mL. Interestingly, PEG-di-DT hydrazide and its further degradation product PEG-dihydrazide stimulated in vitro endothelial cell migration and tubulogenesis, which is comparable to results found with FGF-beta treatment. Subcutaneous implantation of the PEG-crosslinked poly(DTE-co-10%DT carbonate) scaffolds into the backs of rats elicited greater tissue growth over time and superior vascularization than poly(DTE carbonate) implantation. These results show that this new class of biomaterials has a strong potential to modulate angiogenesis. | Publication | 18438755 (PMID) |
Sung HJ, Yee A, Eskin SG, McIntire LV. Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types. (2007) Am J Physiol Cell Physiol 293: C87-94
Show Abstract · Added July 2, 2010The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H(2)O(2) production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H(2)O(2) levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC. | Publication | 17314265 (PMID)
10.1152/ajpcell.00585.2006 (DOI) |
Sung HJ, Eskin SG, Sakurai Y, Yee A, Kataoka N, McIntire LV. Oxidative stress produced with cell migration increases synthetic phenotype of vascular smooth muscle cells. (2005) Ann Biomed Eng 33: 1546-54
Show Abstract · Added July 2, 2010Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H2O2 generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tgp22phox VSMC in an in vitro wound scratch model. Enhanced H2O2 production in Tgp22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H2O2 than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle alpha-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tgp22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased alpha-actin. | Publication | 16341922 (PMID)
10.1007/s10439-005-7545-2 (DOI) |
Sung HJ, Johnson CE, Lessner SM, Magid R, Drury DN, Galis ZS. Matrix metalloproteinase 9 facilitates collagen remodeling and angiogenesis for vascular constructs. (2005) Tissue Eng 11: 267-76
Show Abstract · Added July 2, 2010Degradation of the extracellular matrix, facilitated by matrix metalloproteinases (MMPs), can lead to mechanical failure of vascular constructs, suggesting that MMP inhibition could improve survival of constructs. Therefore, we investigated the role of MMP-9 in collagen remodeling in vitro, focusing on the three major steps of production, degradation, and organization. Because an adequate blood supply is essential for survival of tissue-engineered constructs, we also evaluated the influence of MMP-9 deficiency on angiogenesis in vivo by implantation of thin biodegradable polymer scaffolds. Using aortic smooth muscle cells (SMCs) from wild-type and genetically deficient (9KO) mice, we examined the role of MMP-9 in collagen mRNA expression and protein accumulation, both with and without ascorbic acid treatment. We measured collagen assembly in a fibrillogenesis assay. We investigated in vivo angiogenesis and cell invasion, using fluorescence microangiography and histology. MMP-9 deficiency did not affect collagen mRNA production or polymer scaffold degradation, but collagen accumulation was greater in cultures of 9KO SMCs than in wild-type SMCs. Both MMP-9 deficiency and chemical inhibition impaired collagen degradation. Ascorbic acid treatment enhanced collagen production by 9KO SMCs compared with wild-type SMCs at 3 days, but by 7 days this effect was reversed. MMP-9 improved fibrillogenesis of collagen, significantly more on ascorbic acid treatment. MMP-9 deficiency dramatically decreased inflammatory cell invasion, but also capillary formation within biodegradable polymer scaffolds in vivo. Our data suggest that MMP inhibition, by impairing collagen organization and angiogenesis, might have detrimental effects on the survival of vascular constructs. | Publication | 15738681 (PMID)
10.1089/ten.2005.11.267 (DOI) |
Sung HJ, Su J, Berglund JD, Russ BV, Meredith JC, Galis ZS. The use of temperature-composition combinatorial libraries to study the effects of biodegradable polymer blend surfaces on vascular cells. (2005) Biomaterials 26: 4557-67
Show Abstract · Added July 2, 2010Controlling cellular and physiological responses such as adhesion, proliferation and migration is a highly desirable feature of engineered scaffolds. One important application would be the design of tissue engineered vascular grafts that regulate cell adhesion and growth. We utilized temperature-composition combinatorial polymer libraries to investigate the effects of surfaces of blended poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(epsilon-caprolactone) (PCL) on murine vascular smooth muscle cells (SMC). In this manner, SMCs were exposed to approximately 1000 distinguishable surfaces in a single experiment, allowing the discovery of optimal polymer compositions and processing conditions. SMC adhesion, aggregation, proliferation, and protein production were highest in regions with mid- to high-PCL concentrations and high annealing temperatures. These regions exhibited increased surface roughness, increased microscale PLGA-rich matrix stiffness, and significant change of bulk PCL-rich crystallinity relative to other library regions. This study revealed a previously unknown processing temperature and blending composition for two well-known polymers that optimized SMC interactions. | Publication | 15722125 (PMID)
10.1016/j.biomaterials.2004.11.034 (DOI) |
Sung HJ, Meredith C, Johnson C, Galis ZS. The effect of scaffold degradation rate on three-dimensional cell growth and angiogenesis. (2004) Biomaterials 25: 5735-42
Show Abstract · Added July 2, 2010Even though degradation products of biodegradable polymers are known to be largely non-cytotoxic, little detailed information is available regarding the degradation rate-dependent acidic byproduct effect of the scaffold. In vitro and in vivo scaffold degradation rate could be differentiated using a fast degrading polymer (e.g., poly D, L-lactic-glycolic acid co-polymer, PLGA, 50:50) and a slow degrading polymer (e.g., poly epsilon-caprolactone, PCL). We applied a new method to develop uniform 10 microm thickness of high porous scaffolds using a computer-controlled knife coater with a motion stage and exploiting phase transition properties of a combination of salts and water in salt-leaching method. We then verified in vitro the effect of fast degradation by assessing the viability of primary mouse aortic smooth muscle cell cultured in the three-dimensional scaffolds. We found that cell viability was inversely related to degradation rate and was dependent on the depth from the seeding (upper) surface toward the lower surface. The pH measurement of culture medium using fluorescence probes showed time-dependent decrease in pH in the PLGA scaffolds, corresponding to PLGA degradation, and closely related to cell viability. In vivo analysis of scaffolds implanted subcutaneously into the back of mice, showed significant differences in inflammation and cell invasion into PLGA vs. PCL. Importantly, these were correlated with the degree of the functional angiogenesis within the scaffolds. Again, PLGA scaffolds demonstrated less cell mobilization and less angiogenesis, further supporting the negative effect of the acidic environment created by the degradation of biocompatible polymers. | Publication | 15147819 (PMID)
10.1016/j.biomaterials.2004.01.066 (DOI) |
Khatri JJ, Johnson C, Magid R, Lessner SM, Laude KM, Dikalov SI, Harrison DG, Sung HJ, Rong Y, Galis ZS. Vascular oxidant stress enhances progression and angiogenesis of experimental atheroma. (2004) Circulation 109: 520-5
Show Abstract · Added July 2, 2010BACKGROUND: Although multiple pathological processes have been associated with oxidative stress, the causative relation between oxidative stress and arterial lesion progression remains unclear. METHODS AND RESULTS: To test the effect of creating arterial wall oxidative stress, we compared progression of mouse carotid lesions induced by flow cessation in the wild-type (WT) versus transgenic mice (Tg(p22vsmc)), in which overexpression of p22phox, a critical component of NAD(P)H oxidase was targeted to smooth muscle cell (SMC). Compared with WT mice, arterial lesions grew significantly larger in Tg(p22vsmc) (P<0.001) and demonstrated elevated hydrogen peroxide (H2O2) and vascular endothelial growth factor (VEGF) levels at all time points examined (P<0.001, n=4 animals per time point), probably related to increased expression of hypoxia inducible factor (HIF)-1alpha via SMC oxidative stress in the Tg(p22vsmc) arteries, both basally (203+/-12% versus WT, P<0.001, n=3) and after lesion formation. Interestingly, Tg(p22vsmc) lesions were complicated by extensive neointimal angiogenesis. In vitro experiments confirmed SMCs isolated from Tg(p22vsmc) to be the source for increased H2O2, VEGF, and HIF-1alpha and their capacity to induce angiogenic cord-like structures when cocultured with endothelial cells. The antioxidant ebselen inhibited SMC activities in vitro and intralesion angiogenesis and lesion progression in vivo. CONCLUSIONS: We have demonstrated a novel pathway by which oxidative stress can trigger in vivo an angiogenic switch associated with experimental plaque progression and angiogenesis. This pathway may be related to human atheroma progression and destabilization through intraplaque hemorrhage. | Publication | 14744973 (PMID)
10.1161/01.CIR.0000109698.70638.2B (DOI) |
Johnson C, Sung HJ, Lessner SM, Fini ME, Galis ZS. Matrix metalloproteinase-9 is required for adequate angiogenic revascularization of ischemic tissues: potential role in capillary branching. (2004) Circ Res 94: 262-8
Show Abstract · Added July 2, 2010Angiogenesis, an essential component of a variety of physiological and pathological processes, offers attractive opportunities for therapeutic regulation. We hypothesized that matrix metalloproteinase-9 genetic deficiency (MMP-9-/-) will impair angiogenesis triggered by tissue ischemia, induced experimentally by femoral artery ligation in mice. To investigate the role of MMP-9, we performed a series of biochemical and histological analyses, including zymography, simultaneous detection of perfused capillaries, MMP-9 promoter activity, MMP-9 protein, and macrophages in MMP-9-/- and wild-type (WT) mice. We found that ischemia resulted in doubling of capillary density in WT and no change in the MMP-9-/- ischemic tissues, which translated into increased (39%) perfusion capacity only in the WT at 14 days after ligation. We also confirmed that capillaries in the MMP-9-/- presented significantly (P<0.05) less points of capillary intersections, interpreted by us as decreased branching. The combined conclusions from simultaneous localizations of MMP-9 expression, capillaries, and macrophages suggested that macrophage MMP-9 participates in capillary branching. Transplantation of WT bone marrow into the MMP-9-/-, restored capillary branching, further supporting the contribution of bone marrow-derived macrophages in supplying the necessary MMP-9. Our study indicates that angiogenesis triggered by tissue ischemia requires MMP-9, which may be involved in capillary branching, a potential novel role for this MMP that could be exploited to control angiogenesis. | Publication | 14670843 (PMID)
10.1161/01.RES.0000111527.42357.62 (DOI) |
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