Harishkumar Madhyastha

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.

Arsenic acid inhibits proliferation of skin fibroblasts, and increases cellular senescence through ROS mediated MST1-FOXO signaling pathway.

Arsenic exposure through drinking water is a major public health problem. It causes a number of toxic effects on skin. Arsenic has been reported to inhibit cell proliferation in in vitro conditions. However, reports about the molecular mechanisms are limited. Here, we investigated the mechanism involved in arsenic acid-mediated inhibition of cell proliferation using mouse skin fibroblast cell line. The present study found that 10 ppm arsenic acid inhibited cell proliferation, without any effect on cell death. Arsenic acid induced the generation of reactive oxygen species (ROS), resulting in oxidative stress to DNA. It also activated the mammalian Ste20-like protein kinase 1 (MST1); however the serine/threonine kinase Akt was downregulated. Forkhead box O (FOXO) transcription factors are activated through phosphorylation by MST1 under stress conditions. They are inhibited by phosphorylation by Akt through external and internal stimuli. Activation of FOXOs results in their nuclear localization, followed by an increase in transcriptional activity. Our results showed that arsenic induced the nuclear translocation of FOXO1 and FOXO3a, and altered the cell cycle, with cells accumulating at the G2/M phase. These effects caused cellular senescence. Taken together, our results indicate that arsenic acid inhibited cell proliferation through cellular senescence process regulated by MST1-FOXO signaling pathway.

Anthraquinone Glycoside Aloin Induces Osteogenic Initiation of MC3T3-E1 Cells: Involvement of MAPK Mediated Wnt and Bmp Signaling

Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.

Arsenic trioxide induces ROS activity and DNA damage, leading to G0/G1 extension in skin fibroblasts through the ATM-ATR-associated Chk pathway

Arsenic (As) toxicity is a global health problem, affecting millions of people. Exposure to arsenic, mostly via drinking water, has been associated with cancer of skin, lungs, and blood, in addition to several kinds of skin lesions. The present study focused on the effect of arsenic trioxide (As2O3) on normal skin fibroblast cells. Specifically, the effect of As2O3 on ROS generation and oxidative stress was investigated. Proteins involved in the DNA damage signaling pathway and cell cycle were also studied. As2O3 induced the generation of intracellular ROS. Immunohistochemistry analysis revealed a dose-dependent increase in the number of 8-OHdG-positive cells, an indication of oxidative stress. Cell cycle analysis by flow cytometry demonstrated that As2O3 caused a significant percentage of cells to accumulate in the G0/G1 phase with a concomitant reduction in the S phase. Increases in the activated forms of DNA damage signaling proteins, ATM and ATR, and their effector molecules, Chk2 and p53, were also observed. In addition, expression of oncogene p21 was also increased. The study shows that exposure of normal skin fibroblast cells to As2O3 could lead to cell cycle arrest through ATM/ATR and DNA damage signaling pathways. In conclusion, we report here that arsenic trioxide increases cellular oxidative stress leading to shift in cell cycle and leads to DNA damage through ATM/ATR and the CHK-dependent signaling pathway.