Inhibition of Notch signaling leads to increased intracellular ROS by up-regulating Nox4 expression in primary HUVECs.

The essential roles of Notch pathway in angiogenesis have been reported for years. However, how Notch pathway plays its role in regulating endothelial cells remains largely unknown. In this study we found that blockade of Notch signaling with a γ-secretase inhibitor increased reactive oxygen species (ROS) in primary human umbilical vein endothelial cells (HUVECs) under both normaxic and ischemia/reperfusion (I/R) conditions. Abruption of ROS generation with ROS scavengers or specific inhibitors of ROS production in HUVECs abolished Notch blockade-induced HUVEC proliferation, migration and adhesion, suggesting that the regulation of Notch pathway on endothelial cell behavior is at least partially dependent on its down-regulation of ROS level. We further showed that the enhanced generation of ROS after blocking Notch signal was accompanied by augmented expression of Nox4, which led to increased phosphorylation of VEGFR2 and ERK in HUVECs. In summary, our results have shown that Notch signaling regulates ROS generation by suppressing Nox4, and further modulates endothelial cell proliferation, migration and adhesion.

Insulin protects against damage to pulmonary endothelial tight junctions after thermal injury: relationship with zonula occludens-1, F-actin, and AKT activity.

Intensive insulin therapy during critical illness protects the endothelium and thereby prevents organ failure. This study tested the hypothesis that insulin directly affects the attenuation of burn injury-induced damage to pulmonary endothelial tight junction and investigated the underlying mechanisms. Sprague Dawley rats with severe burn injury were randomized to treatment with insulin dissolved in normal saline (maintenance of blood glucose at a level between 5.0 and 7.0 mmol/L) or normal saline alone (in vivo treatment). Pulmonary damage was evaluated. Rat pulmonary microvascular endothelial cells were treated with 20% burn serum or 20% burn serum + insulin (in vitro treatment). Selected cultures were pretreated with phosphatidylinositol 3-kinase/protein kinase B (AKT) inhibitor (LY294002). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine phalloidin and were examined. Cell extracts were obtained to assess zonula occludens-1, occludin, and phosphorylated AKT levels by immunoblotting. Treatment with insulin attenuated the pulmonary edema, hemorrhage, and inflammatory cell infiltration of rats with severe burn injury. Burn serum significantly enhanced monolayer permeability to albumin, whereas treatment with insulin (10(-7 ) mol/L) limited this effect. Meanwhile, insulin (10(-7 ) mol/L) reduced burn serum-induced F-actin stress fiber formation and decreased zonula occludens-1 expression. LY294002 decreased cytoplasmic AKT phosphorylation and inhibited the protection effects of insulin. Through the phosphatidylinositol 3-kinase/AKT pathway, insulin independent of glucose toxicity can attenuate increased pulmonary endothelial permeability induced by burn injury. The effect is attributed to the attenuation of the architectural disruption of protein components of the endothelial tight junction. This result is useful in inhibiting multiple organ failure after burn injury.

MiR-10a and miR-181c regulate collagen type I generation in hypertrophic scars by targeting PAI-1 and uPA.

Urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) have been proposed to play key roles in extracellular matrix (ECM) deposition in hypertrophic scars (HS). Here, we found that in HS fibroblasts (HFs) miR-181c and miR-10a were differentially-expressed and targeted uPA and PAI-1, respectively. The production of Type 1 collagen (Col1) was inhibited by miR-181c knockdown or miR-10a overexpression in HFs, and this resulted in increased levels of metalloproteinase 1 (MMP1). These results suggest that the miR-181c-uPA and miR-10a-PAI-1 regulatory pathways have an integral role in HS pathogenesis.

MicroRNA-21 regulates hTERT via PTEN in hypertrophic scar fibroblasts.

BACKGROUND: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues. CONCLUSIONS/SIGNIFICANCE: These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.

[Effects of adipose-derived stem cells on renal injury in burn mice with sepsis].

OBJECTIVE: To investigate the effect of adipose-derived stem cells (ADSC) on renal injury in mice with burn injury and sepsis and its underlying mechanism. METHODS: (1) Adipose tissue was collected from both inguinal regions of 5 C57BL/6J mice to isolate, culture and purify ADSC through enzyme digestion, density gradient centrifugation, and adherence method. Cells of the third passage were used in the experiment. The morphologic change in cells was observed and the growth curve of cells was determined. The expression of cell surface antigen phenotype was analyzed by flow cytometry, and the cells were identified by adipogenic and osteogenic differentiation. (2) Another 37 C57BL/6J mice were divided into normal control group (n = 5), saline group (n = 16), and group ADSC (n = 16) according to the random number table. The mice in saline group and group ADSC were injected with Pseudomonas aeruginosa after being subjected to 15% TBSA full-thickness burn on the back to reproduce septic burn model. Then the mice were injected with saline and ADSC through tail vein respectively. At post burn hour (PBH) 12, 24, 48, and 72, the pathological change in kidney tissue was observed, the levels of blood urea nitrogen and serum creatinine were determined, and the levels of TNF-α, IL-12, IL-10, and cyclooxygenase-2 (COX2) mRNA were determined with real-time fluorescence quantitative PCR in both groups. Above-mentioned indexes were also examined in the normal control group (without burn). Data were processed with multifactor analysis of variance and LSD- t test. RESULTS: (1) Cells in the third passage were orderly arranged with the shape similar to fibroblasts. The percentages of CD90(+), CD105(+), CD34(-), and CD45(-) cells were all above 90%. The cells could differentiate into osteoblasts and adipocytes. The cells were identified to be ADSC. (2) From PBH 12 to PBH 72, the neutrophil infiltration gradually increased, and the structure of kidney tubules and glomeruli were deranged in saline group. The pathological change in kidney tissue in group ADSC was less serious than that of normal control group at each time point. From PBH 12 to PBH 72, the levels of blood urea nitrogen and serum creatinine in saline group were significantly higher than those of normal control group and group ADSC (P values all below 0.01). Compared with those of the normal control group, the levels of TNF-α and IL-12 mRNA were higher in group ADSC and saline group at PBH 24 (P values all below 0.05). At PBH 24, the level of TNF-α mRNA in group ADSC (1.58 ± 0.19) was lower than that of saline group (3.36 ± 0.30, P < 0.05). At PBH 24, the levels of IL-10 and COX2 mRNA in group ADSC (2.89 ± 0.47, 4.90 ± 0.59) were higher than those in normal control group (1.00 ± 0.15, 1.00 ± 0.27) and saline group (1.32 ± 0.38, 1.57 ± 0.38, P values all below 0.05). CONCLUSIONS: ADSC can decrease the levels of blood urea nitrogen and serum creatinine, promote the production of anti-inflammatory cytokines IL-10 and COX2, and reduce the release of the pro-inflammatory cytokines TNF-α and IL-12 to offer protective effects against renal injury in burn mice with sepsis.

Protection against TGF-ß1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring.

Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-ß1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.

A potential skin substitute constructed with hEGF gene modified HaCaT cells for treatment of burn wounds in a rat model.

This study aimed to investigate the feasibility of using an immortal keratinocyte cell line, HaCaT cells, to effectively deliver epidermal growth factor (EGF) in a skin substitute to treat burn wounds. The skin equivalent was constructed with human EGF (hEGF) gene modified HaCaT cells obtained through stable gene transfection; these were applied to full thickness burn wounds in a rat model. The results showed that the hEGF gene modified HaCaT cells produced more than 390ng/l of bioactive hEGF in the culture supernatant. K19 and integrin-ß1 as keratinocyte differentiation markers were elevated in the hEGF gene modified HaCaT cells which were shown to be non-tumorigenic. The skin equivalent constructed with hEGF gene modified HaCaT cells demonstrated improved epidermal morphogenesis with a thick and compact epidermis. Wound healing was accelerated noticeably when applied with this skin substitute seeded with hEGF gene modified HaCaT cells in vivo. The results suggest that HaCaT cells modified with hEGF gene might be promising seed cells for construction of genetically modified skin substitute which can effectively secrete hEGF to accelerate wound repair and regeneration.

Overexpression of Notch ligand Dll1 in B16 melanoma cells leads to reduced tumor growth due to attenuated vascularization.

Notch signaling plays an important role in vascular development and tumor angiogenesis. It has been shown that disruption of Dll4-triggered Notch signal activation effectively inhibits tumor growth, but this treatment also results in the formation of vascular neoplasms. In this study, we investigate the effects of over-expressing Notch ligand Dll1 in B16 melanoma cells on tumor cell proliferation and tumor growth in vitro and in vivo. Our results showed that over-expression of Dll1 could activate Notch signaling in tumor cells, and promote tumor cell proliferation in vitro. In contrast, growth of Dll1-over-expressing tumors in vivo was reduced, due to abnormal tumor vessel formation. Impaired tumor vasculature enhanced hypoxia and necrosis in tumor tissues, leading to retarded tumor growth. These results suggest that activation of Notch signaling may serve as an anti-angiogenesis strategy in the treatment of malignant tumors.

[Biological effects of paracrine from insulin stimulated adipose-derived stem cells (ADSC) on human vascular endothelial cells].

OBJECTIVE: To study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells. METHODS: (1) ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group (I, cultured with serum-free DMEM containing 1 x 10(-7) mol/L insulin) and control group (C, cultured with serum-free DMEM) according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium (ADSC-CM) was collected for determination of levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by ELISA. (2) Human umbilical vein endothelial cells (HUVEC) were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group (AI), ADSC-CM without insulin stimulation group (AC), insulin group (I, with same concentration as above), blank control group (BC) according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method (with expression of absorbance value). Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and HUVEC migration with scratch adhesion test at post scratch hour (PSH) 12, 24, 36, 48. Data were processed with t test. RESULTS: (1) Compared with those in C group [(287 +/- 47), (577 +/- 84) pg/mL, respectively], the secretion levels of VEGF and HGF in I group [(643 +/- 64), (930 +/- 68) pg/mL, respectively] were significantly increased (with t value respectively 18.869, 18.475, P values all below 0.05). (2) The absorbance value of HUVEC in AI and AC groups was 0.847 +/- 0.042, 0.798 +/- 0.022, respectively, which were higher than that in I and BC groups [0.665 +/- 0.028 (with t value respectively 4.579, 3.732), 0.674 +/- 0.031 (with t value respectively 3.761, 4.073), P values all below 0.01], and that in AI group was higher than that in AC group (t = 2.576, P < 0.05). The apoptosis rates of HUVEC in AI and AC groups [(5.8 +/- 1.9)%, (9.0 +/- 2.0)%, respectively] were obviously lower as compared with that in I and BC groups [(30.4 +/- 6.0)% (with t value respectively 12.891, 10.417), (31.4 +/- 7.4)% (with t value respectively 11.474, 9.783), P values all below 0.05 ], and that in AC group was higher than that in AI group (t = 8.548, P < 0.05). The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group (with t value respectively 4.076, 4.573, P values all below 0.05). CONCLUSIONS: Paracrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.

Smad interacting protein 1 as a regulator of skin fibrosis in pathological scars.

Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by the excessive and abnormal deposition of collagen-based extracellular matrix (ECM) components. However, the molecular basis of keloid and hypertrophic scar formation has not been fully elucidated. Here, we demonstrated that down-regulation of the transcription factor Smad interacting protein 1 (SIP1) could be relevant to keloid and hypertrophic scar formation. The results of the present study show that the level of SIP1 mRNA is significantly decreased in pathological scar tissues and in normal skin and pathological scar fibroblasts treated with transforming growth factor ß1 (TGF-ß1). In contrast, the expression of SIP1 mRNA is not decreased in normotrophic scar samples. The SIP1 mRNA level inversely correlates with the mRNA level of type I collagen (COL1A2) and directly correlates with the mRNA level of matrix metalloproteinase-1 (MMP1). Overexpression of SIP1 in keloid and hypertrophic scar fibroblasts represses TGF-ß1-stimulated COL1A2 expression and induces MMP1 expression. Alternatively, knockdown of SIP1 in normal skin fibroblasts enhance TGF-ß1-induced COL1A2 levels. These findings suggest that SIP1 could be a regulator of skin fibrosis, and depletion of SIP1 in pathological scar tissues could result in an up-regulation of collagen and down-regulation of matrix metalloproteinase, leading to an abnormal accumulation of ECM along with fibrosis and pathological scar formation.

[Effect of heat injured keratinocytes supernatant on biological behavior of fibroblasts].

OBJECTIVE: To observe the effect of the supernatant of heat injured keratinocytes (KC) on biological behavior of the dermal fibroblasts (Fb). METHODS: Human dermal Fb were isolated and cultured. A model of heat injured KC (HaCaT) was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1:1 volume ratio to make normal KC conditioned medium (NKCM) and heat injury KC conditioned medium (HKCM) respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. (1) The proliferation of Fb was detected with MTT method at post culture hour (PCH) 12, 24, 36, 48. (2) The apoptosis of Fb was determined by flow cytometry at PCH 12 (Fb were heat injured in advance; Fb without heat treatment was used as control). (3) At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test. RESULTS: (1) The proliferation of Fb: the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 was respectively higher than that of Fb cultured with non-serum DMEM (with t value respectively 1.89, 2.35, 2.02, 1.94, and P values all below 0.01). There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with NKCM at PCH 12, 24, and 48 (at PCH 12, t = 1.83, P < 0.01; at PCH 24, t = 2.91, P < 0.05; at PCH 48, t = 1.83, P < 0.05). (2) Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment (t = 3.31, P < 0.05; t = 1.47, P < 0.01). (3) The expression of alpha-SMA (red fluorescence) in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was obviously increased in Fb cultured with HKCM. (4) The relative expression amount of alpha-SMA mRNA in Fb cultured with HKCM was 1.32 +/- 0.06, which was higher than that both in Fb cultured with NKCM (1.14 +/- 0.07, t = 2.51, P < 0.05) and in Fb cultured with non-serum DMEM (1.00 +/- 0.09, t = 1.77, P < 0.05). CONCLUSIONS: The supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.

[Effects of insulin on the growth factor secreting function of adipose-derived stem cells].

OBJECTIVE: To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs). METHODS: ADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry. RESULTS: The secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01). CONCLUSIONS: Insulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.

[The protective effect of intensive insulin treatment on the myocardium in severely scalded rats].

OBJECTIVE: To study the protective effect of intensive insulin treatment on the myocardium of severely scalded rats, and to primarily explore its mechanism. METHODS: Eighteen SD rats were divided into three groups, with 6 rats in each group. The rats in burn and intensive insulin group were inflicted with 30% TBSA full-thickness injury on the back. Isotonic saline containing 0.12 U/ml insulin solution, and 100 g/L glucose solution were infused into the rats in the intensive insulin group to keep plasma glucose at the level of 4.0 - 6.6 mmol/L (the total fluid amount was 2 ml x kg(-1) x 8h(-1)). In sham burn group,fluid was given according to physiological demand. The same amount of isotonic saline was infused into the rats in burn group. The venous blood was obtained for the detection of plasma glucose contents, and the left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were recorded via aortic ventricle cannula before scald and at 1, 2, 3, 4, 5, 6 post-scald hours (PSH). The tissue of the left ventricle was harvested at 6 PSH for the detection of troponin T expression in myocardiocytes. RESULTS: Plasma glucose level was increased to (7.6 +/- 1.7) mmol/L - (8.4 +/- 4.7) mmol/L in burn group during 1-6 PSH, which was significantly higher than that in intensive insulin group (4.5 +/- 0.9) mmol/L - (5.2 +/- 1.3) mmol/L, P < 0.01). Compared with the intensive insulin group, LVSP was markedly decreased in the burn group (60 +/- 11 mm Hg vs 72 +/- 8 mm Hg, P < 0.05) at 1 PSH,whereas LVEDP was increased significantly (21.3 +/- 11.3 mmHg vs 11.7 +/- 5.2 mmHg, P < 0.05). Intensive insulin treatment could significantly inhibit the loss of troponin T protein in myofilaments of myocardium. CONCLUSION: Intensive insulin treatment possesses a protective effect on myocardia function after severe burns, and it may be related to its preventive effect on the loss of contractile protein in cardiocytes.