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.

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.

Cysteine-rich cyanopeptide beta2 from Spirulina fusiformis exhibits plasmid DNA pBR322 scission prevention and cellular antioxidant activity.

Isolation of three different active peptides from C-phycocyanin (C-pc) beta chain of S. fusiformis and their biological properties are reported. Phycocyanin peptide beta fraction 2 (cyanopeptide beta 2) facilitated both antioxidant and plasmid DNA strand scission prevention activity due to higher cysteine moieties in the isolated peptide. The peptide significantly scavenged the free radicals like 1-1,-diphenyl-2-picryl hydrazyl and ferric reducing ability of plasma, increased the absorbance values in reducing power and also showed the higher trolox equivalent antioxidant capacity values in total reactive antioxidant potentials assay. Cyanopeptide beta 2 also inhibited reactive oxygen species induced DNA pBR322 damage in dose dependent manner along with free radical scavenging properties suggesting the role in the DNA integrity which is also evident by DNA binding activity of peptide. In addition, the generation of reactive oxygen species (ROS) was dose dependent (10 and 20 ng/ml) and significantly quenched by cyanopeptide beta2 in human fibroblast cell line TIG 3-20. In vitro cell scratch injury assay demonstrated the capacity of cyanopeptide beta2 in cell migration in to wounded area suggesting fibroblast proliferation and migration. The results suggest that cyanopeptide beta2 can be a free radical scavenger and effective peptide for future biomedical applications like wound healing, atherosclerosis, cell redox potential and hypoxia.

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 emodin's 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.

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 alpha, 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.

Cell cycle regulation and induction of apoptosis by beta-carotene in U937 and HL-60 leukemia cells.

In this communication, we report the efficacy of beta-carotene towards differentiation and apoptosis of leukemia cells. Dose (20 microM) and time dependence (12 h) tests of beta- carotene showed a higher magnitude of decrease (significance p < 0.05) in cell numbers and cell viability in HL-60 cells than U937 cells but not normal cell like Peripheral blood mononuclear cell (PBMC). Microscopical observation of beta-carotene treated cells showed a distinct pattern of morphological abnormalities with inclusion of apoptotic bodies in both leukemia cell lines. When cells were treated with 20 microM of beta-carotene, total genomic DNA showed a fragmentation pattern and this pattern was clear in HL-60 than U937 cells. Both the cell lines, on treatment with beta- carotene, showed a clear shift in G(1) phase of the cell cycle. In addition the study also revealed anti-oxidant properties of beta-carotene since there was reduction in relative fluorescent when treated than the control at lower concentration. Collectively this study shows the dual phenomenon of apoptosis and differentiation of leukemia cells on treatment with beta-carotene.

Pigment production in Spirulina fussiformis in different photophysical conditions.

The present investigation makes a comparative investigation of individual light source on the different commercially important pigments in Spirulina fussiformis in photobioreactor culture condition. Continuous culture system was carried out throughout the experimental condition. Initially, seed culture, corresponding to 0.2 g/L on dry weight basis was cultivated in Zarrouks medium with different colored light source in reactor. Maximum daily biomass productivity, 0.8 g/L, 0.75 g/L and 0.69 g/L in white light (WL), blue light (BL) and green light (GL), respectively, conditions was noticed. Pigment content during WL treatment showed the highest accumulation (5.5 microg/mL) of chlorophyll whereas, other pigments roughly remained constant without much change, implying WL intensity is better for chlorophyll synthesis. On the other hand, chlorophyll and phycocyanin content gradually increased up to 7 microg/mL and 2 mg/mL, respectively, at BL intensity. The response to GL was negative to all pigments studied except for phycocyanin; in this case a highest production (2.5 mg/mL) was seen during 18 days experimental period. Additionally, when yellow light (YL) treatment experiments were conducted, the rate of production gradually decreased from 6th day onward in all pigments demonstrating the photobleaching effect of YL. The average rate of pigments production did not show significant accumulation in red light (RL) light treatment except phycoerythrin which showed an increasing trend of production. It is worth to mention here that higher light intensity is better for production of phycocyanin and phycoerythrin in Spirulina.

C-phycocyanin transcriptionally regulates uPA mRNA through cAMP mediated PKA pathway in human fibroblast WI-38 cells.

We have previously demonstrated the efficacy of c-phycocyanin in up-regulation of urokinase-type plasminogen activator (uPA) in bovine endothelial cell line. However, the mechanism of action and pathway elucidation in uPA regulation is unclear. In experiments reported here, we have investigated the mechanism of action of c-phycocyanin (c-pc) induced uPA gene modulation in human fibroblast (WI-38) cell line. ELISA test confirmed that c-pc increased the uPA antigen whereas PAI-1 antigen level was unaffected. Treatment of cells with c-pc significantly (P<0.05) enhanced the uPA mRNA level in a dose (50 microg/ml) and time dependent (up to 4 h) manner. This effect of c-pc was abolished by treatment with dichloro-1-beta-D-ribofuranosyl benzamidazole (DRB) (10 microg/ml). Co-treatment of c-pc with 200 microg/ml cycloheximide (CHX), translation inhibitor, resulted in over accumulation of uPA mRNA. These results suggest that uPA induction by c-pc is transcriptionally regulated and does not require de novo protein synthesis. We also provide evidence that c-pc stimulates uPA gene through cAMP dependent pathway as adenylyl cyclase (AC) inhibitor, dideoxyadenosine (DDA) significantly inhibited the uPA mRNA expression and co-treatment with adenylyl cyclase analogue, dBcAMP recovered the effect of c-pc on gene activity. Furthermore, the present investigation provides evidence on the regulatory pathway involved in the c-pc stimulus. C-pc induced uPA expression was completely inhibited by PKA inhibitor (KT 5200), indicating the regulation is dependent on PKA pathway. Elimination of PKC pathway components by prolonged incubation with excess amount of phorbol 12-myristate 13-acetate (PMA) failed to abolish the c-pc effect on uPA expression indicating the regulation is independent of PKC pathway. Taken together, our data indicate that uPA gene regulation by c-pc is transcriptionally controlled through cAMP mediated PKA pathway.

Purification of c-phycocyanin from Spirulina fusiformis and its effect on the induction of urokinase-type plasminogen activator from calf pulmonary endothelial cells.

c-Phycocyanin (c-pc), a blue coloured, fluorescent protein was purified from blue-green alga, Spirulina fusiformis and its effect on fibrinolytic system in vascular endothelial cells was investigated. The c-pc consisted of two subunits, alpha and beta, whose molecular masses were 16 and 17 kDa, respectively. N-terminal sequences of both subunits were well conserved compared with other blue green algal phycobiliproteins. Fibrinolytic activity in the medium conditioned by calf pulmonary arterial endothelial cells was measured by the fibrin plate method. The c-pc increased the fibrinolytic activity in dose- and time-dependent manners. Fibrin zymographic studies indicated that c-pc-induced urokinase-type plasminogen activator in the cells. These in vitro results suggest that c-pc from S. fusiformis is a potent profibrinolytic protein in the vascular endothelial system.