Sayuri Omura

MicroRNA signature in diabetic wound healing: promotive role of miR-21 in fibroblast migration

A major complication of diabetes mellitus is the disruption of normal wound repair process, characterised by insufficient production of growth factors. A molecular genetic approach wherein resident cells synthesise and deliver the growth factors to the wound site would be a powerful therapeutic strategy to treat diabetic wounds. One such molecular approach could be the application of microRNAs (miRNAs). This study reports differential expression of miRNAs related to cell development and differentiation, during wound healing in diabetic mice. Comparison of skin tissue from normal and diabetic mice showed that 14 miRNAs were differentially expressed in diabetic skin; miR‐146b and miR‐21 were the most noteworthy. Expression pattern of these miRNAs was also altered during healing of diabetic wounds. A subset of miRNAs (miR‐20b, miR‐10a, miR‐10b, miR‐96, miR‐128, miR‐452 and miR‐541) exhibited similar basal levels in normal and diabetic skins, but displayed dysregulation during healing of diabetic wounds. Amongst the miRNAs studied, miR‐21 showed a distinct signature with increased expression in diabetic skin but decreased expression during diabetic wound healing. We analysed the role of miR‐21 in fibroblast migration, because migration of fibroblasts into the wound area is an important landmark facilitating secretion of growth factors and migration of other cell types into the wound, thus enhancing the healing process. Using gain‐of and loss‐of function approaches, we show that miR‐21 is involved in fibroblast migration. Our preliminary studies implicate an important role for miRNAs in the pathogenesis of diabetic wounds.

uPA dependent and independent mechanisms of wound healing by C-phycocyanin

Wound repair requires both recruitment and well co‐ordinated actions of many cell types including inflammatory cells, endothelial cells, epithelial cells and importantly fibroblast cells. Urokinase‐type plasminogen activator (uPA) system plays a vital role in wound healing phenomenon. We have previously demonstrated that C‐phycocyanin (C‐pc), a biliprotein from blue‐green algae, transcriptionally regulates uPA through cAMP‐dependent protein kinase A (PKA) pathway. To date, a role for C‐pc in wound‐healing scenario is not elucidated. This study was designed to examine the wound‐healing property of C‐pc in relation to fibroblast proliferation and migration. C‐pc increased fibroblast proliferation in a dose‐dependent manner. It also enhanced G1 phase of cell cycle and increased the expressions of cyclin‐dependent kinases 1 and 2, which facilitate cell cycle progression, in a uPA‐independent manner. In vitro wound healing and migration assays revealed the pro‐migratory properties of C‐pc. Short‐interference RNA studies demonstrated that uPA was necessary for C‐pc‐induced fibroblast migration. C‐pc also significantly elevated the expressions of chemokines (MDC, RANTES, Eotaxin, GRO α, ENA78 and TARC) and Rho‐GTPases (Cdc 42 and Rac 1) in a uPA‐dependent manner. Pre‐treatment of C‐pc‐stimulated cells with pharmacological inhibitor of PI‐3K (LY294002) annulled the expression of GTPases implying that Rac 1 and Cdc 42 were induced through PI‐3K pathway. C‐pc‐induced cellular migration towards wounded area was also negatively affected by PI‐3K inhibition. In vivo wound‐healing experiments in mice validated our finding that C‐pc accelerates wound healing. Our data provides conclusive evidence of a novel therapeutic usage for C‐pc as a wound‐healing agent. C‐pc is a food and drug administration (FDA)‐approved health supplement. We believe this compound can also be beneficial in healing of internal wounds, such as ulcers.

Curcumin Facilitates Fibrinolysis and Cellular Migration during Wound Healing by Modulating Urokinase Plasminogen Activator Expression

Urokinase plasminogen activator (uPA) plays a vital role in the early phases of wound healing by aiding fibrin dissolution and promoting the migration, proliferation, and adhesion of various cells to the wound bed. The efficacy of botanicals in healing wounds is an area of active research. Among these, curcumin, a yellow pigment abundant in turmeric rhizome, has been the center of extensive studies. This study focused on the effect of curcumin on uPA expression and its consequence on fibrin dissolution and cellular migration. Treatment of human fibroblast cells with curcumin caused an upregulation of uPA mRNA and protein. Activation of JNK and p38 MAPK signal pathways was necessary for the upregulation of uPA. Curcumin treatment resulted in an increase in fibrinolytic activity and cell migration towards the wound area. The involvement of uPA in fibrinolysis and cell migration was confirmed by zymography and siRNA studies, respectively.

Regulation of growth factors-associated cell migration by C-phycocyanin scaffold in dermal wound healing.

1. The present study examined the role of C‐phycocyanin (C‐pc) in relation to growth factors and cell migration during wound healing.

Nfκb-dependent regulation of urokinase plasminogen activator by proanthocyanidin-rich grape seed extract: effect on invasion by prostate cancer cells

Tumor invasion and metastasis present major obstacles to successful control of androgen-independent prostate cancer. Cell migration is a fundamental aspect of cancer cell metastasis. Urokinase plasminogen activator (uPA) system is implicated in cell migration and cancer metastasis and has potential to be developed as therapeutic target. In recent years, efficacy of dietary nutrients in preventing and curing cancer has gained increasing attention. One such promising candidate is proanthocyanidin-rich grape seed extract (GSE). We investigated the efficacy of GSE in regulating uPA expression and cell migration using highly metastatic androgen-independent PC3 prostate cancer cells as a model. GSE down-regulated uPA as a function of concentration. Additional studies showed that GSE inhibited DNA-binding activity of the transcription factor nuclear factor kappa B (NFκB), which in turn decreased NFκB-dependent uPA transcription. Invasion assays revealed the inhibitory effect of GSE on PC3 cell migration. These in-vitro experiments demonstrate the therapeutic property of GSE as an antimetastatic agent by targeting uPA.

Emodin upregulates urokinase plasminogen activator, plasminogen activator inhibitor-1 and promotes wound healing in human fibroblasts

Urokinase plasminogen activator (uPA) system is important for several biological processes that call for extracellular proteolysis, fibrinolysis, cell migration, proliferation and angiogenesis. The current study highlights the fibrinolytic and wound healing potential of emodin, an anthraquinone, with relevance to the uPA system. Emodin increased the fibrinolytic activity of fibroblast cells in a dose-dependent manner. Zymography linked the activity to increased uPA activity. Subsequent RT-PCR and western analyses demonstrated uPA gene upregulation. Interestingly, PAI-1, the inhibitor of uPA was also upregulated. EMSA showed the upregulation occurred independent of emodins effect on nuclear factor kappa B (NFkappaB). The effect on uPA system is supposedly via generation of reactive oxygen species (ROS) since cotreatment with ascorbic acid, an anti-oxidant, attenuated the activity. In addition to profibrinolytic potential, emodin also demonstrated wound healing activity in in vitro wound models. Presence of emodin in the medium enhanced the rate of migration of fibroblasts into the wounded region. These in vitro experiments reveal that emodin is a potent profibrinolytic and wound healing agent.

NFkappaB activation is essential for miR-21 induction by TGFβ1 in high glucose conditions.

Transforming growth factor beta1 (TGFβ1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFβ1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGFβ1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFβ1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGFβ1 in high glucose conditions. The induction by TGFβ1 was dependent on NFκB activation and subsequent ROS generation. TGFβ1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFβ1. ChIP assay revealed binding of p65 to miR-21 promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGFβ1-NFκB-miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.

Post‐transcriptional regulation of plasminogen activator inhibitor‐1 by intracellular iron in cultured human lung fibroblasts—interaction of an 81‐kDa nuclear protein with the 3′‐UTR

Summary.  The proteinase inhibitor, type‐1 plasminogen activator inhibitor (PAI‐1), is a major regulator of the plasminogen activator system involved in plasmin formation and fibrinolysis. The present study explores the effects of intracellular iron on the expression of PAI‐1 and associated cell‐surface plasmin activity in human lung fibroblasts; and reports the presence of a novel iron‐responsive protein. ELISA revealed a dose‐dependent increase in PAI‐1 antigen levels expressed in the conditioned medium of cells treated with deferoxamine, in the three cell lines studied. A concomitant increase in mRNA levels was also observed by Northern analyses. Presaturation with ferric citrate quenched the effect of deferoxamine. Experiments with transcription and translation inhibitors on TIG 3‐20 cells demonstrated that intracellular iron modulated PAI‐1 expression at the post‐transcriptional level with the requirement of de‐novo protein synthesis. Electrophoretic mobility shift assay and UV crosslinking assays revealed the presence of an ∼ 81‐kDa nuclear protein that interacted with the 3′‐UTR of PAI‐1 mRNA in an iron‐sensitive manner. Finally, we demonstrated that the increased PAI‐1 is functional in suppressing cell‐surface plasmin activity, a process that can affect wound healing and tissue remodeling.