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Fibroblasts play a pivotal role in wound healing. However, the molecular mechanisms determining the reparative response of fibroblasts remain unknown. Here, we identify Notch1 signaling as a molecular determinant controlling the plasticity and function of fibroblasts in modulating wound healing and angiogenesis. The Notch pathway is activated in fibroblasts of diabetic wounds but not in normal skin and non-diabetic wounds. Consistently, wound healing in the FSP-1 +/- ;ROSA LSL-N1IC+/+ mouse, in which Notch1 is activated in fibroblasts, is delayed. Increased Notch1 activity in fibroblasts suppressed their growth, migration, and differentiation into myofibroblasts. Accordingly, significantly fewer myofibroblasts and less collagen were present in granulation tissues of the FSP-1 +/- ;ROSA LSL-N1IC +/+ mice, demonstrating that high Notch1 activity inhibits fibroblast differentiation. High Notch1 activity in fibroblasts diminished their role in modulating the angiogenic response. We also identified that IL-6 is a functional Notch1 target and involved in regulating angiogenesis. These findings suggest that Notch1 signaling determines the plasticity and function of fibroblasts in wound healing and angiogenesis, unveiling intracellular Notch1 signaling in fibroblasts as potential target for therapeutic intervention in diabetic wound healing., (© 2020 Shao et al.)

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

Background: The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers, which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways. Several therapeutic anti-GFRA1 antibody-drug conjugates are under development. Demethylation (or hypomethylation) of GFRA1 CpG islands (dmGFRA1) is associated with increased gene expression and metastasis risk of gastric cancer. However, it is unknown whether dmGFRA1 affects the metastasis of other cancers, including colon cancer (CC)., Aim: To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target., Methods: CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study. The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing. Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients. Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo . The phosphorylation of AKT and ERK proteins was examined by Western blot analysis., Results: The proportion of dmGFRA1 in CC, surgical margin, and normal colon tissues by MethyLight was 68.4%, 73.4%, and 35.9% (median; nonparametric test, P = 0.001 and < 0.001), respectively. Using the median value of dmGFRA1 peak area proportion as the cutoff, the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or well-differentiated samples (92.3%% vs 55.8%, Chi-square test, P = 0.002) and significantly higher in CC samples with distant metastasis than in samples without (77.8% vs 46.0%, P = 0.021). The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC (adjusted hazard ratio = 0.49, 95% confidence interval: 0.24-0.98), especially for 89 CC patients with metastatic CC (adjusted hazard ratio = 0.41, 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration. GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways., Conclusion: GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC, especially those with metastatic CC. GFRA1 can promote the proliferation/growth of CC cells, probably by the activation of AKT and ERK pathways. GFRA1 might be a therapeutic target for CC patients, especially those with metastatic potential., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Peripheral Arterial Disease (PAD) is a progressive, atherosclerotic disease that at its end stage, Critical Limb Ischemia (CLI), results in severely diminished limb perfusion and causes leg pain at rest, non-healing ulcers, and tissue gangrene. Many patients with CLI fail current medical and surgical therapies and thus are deemed "no option" and require limb amputation. Novel therapies to attempt limb salvage in these "no option" patients are needed. Stem cell therapy is one therapeutic angiogenic avenue that has been tested over the last 20 years. To date, clinical trials have shown promise but with only modest improvement and none demonstrated a significant decrease in amputation rates in those treated with stem cell therapy. Thus, recent investigations into improving stem cell therapy have been the focus of our laboratory and many others. This review aims to describe recent advances in increasing the therapeutic potential of stem cell therapies for CLI.

Atherosclerosis is the most common underlying cause of cardiovascular morbidity and mortality worldwide. c-Kit (CD117) is a member of the receptor tyrosine kinase family, which regulates differentiation, proliferation, and survival of multiple cell types. Recent studies have shown that c-Kit and its ligand stem cell factor (SCF) are present in arterial endothelial cells and smooth muscle cells (SMCs). The role of c-Kit in cardiovascular disease remains unclear. The aim of the current study is to determine the role of c-Kit in atherogenesis. For this purpose, atherosclerotic plaques were quantified in c-Kit-deficient mice (Kit Mut ) after they were fed a high-fat diet (HFD) for 16 wk. Kit Mut mice demonstrated substantially greater atherosclerosis compared with control (Kit WT ) littermates ( P < 0.01). Transplantation of c-Kit-positive bone marrow cells into Kit Mut mice failed to rescue the atherogenic phenotype, an indication that increased atherosclerosis was associated with reduced arterial c-Kit. To investigate the mechanism, SMC organization and morphology were analyzed in the aorta by histopathology and electron microscopy. SMCs were more abundant, disorganized, and vacuolated in aortas of c-Kit mutant mice compared with controls ( P < 0.05). Markers of the "contractile" SMC phenotype (calponin, SM22α) were downregulated with pharmacological and genetic c-Kit inhibition ( P < 0.05). The absence of c-Kit increased lipid accumulation and significantly reduced the expression of the ATP-binding cassette transporter G1 (ABCG1) necessary for lipid efflux in SMCs. Reconstitution of c-Kit in cultured Kit Mut SMCs resulted in increased spindle-shaped morphology, reduced proliferation, and elevated levels of contractile markers, all indicators of their restored contractile phenotype ( P < 0.05). NEW & NOTEWORTHY This study describes the novel vasculoprotective role of c-Kit against atherosclerosis and its function in the preservation of the SMC contractile phenotype.

Whitmania pigra has been used as a traditional Chinese medicine (TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the cDNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet andantithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. Thebleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1 (PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion,a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder., (Copyright © 2019 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Cancer stem cells (CSCs) play critical roles in cancer initiation, metastasis, recurrence, and drug resistance. Recent studies have revealed involvement of cancer-associated fibroblasts (CAFs) in regulating CSCs. However, the intracellular molecular mechanisms that determine the regulatory role of CAFs in modulating the plasticity of CSCs remain unknown. Here, we uncovered that intracellular Notch1 signaling in CAFs serves as a molecular switch, which modulates tumor heterogeneity and aggressiveness by inversely controlling stromal regulation of the plasticity and stemness of CSCs. Using mesenchymal stem cell-derived fibroblasts (MSC-DF) harboring reciprocal loss-of-function and gain-of-function Notch1 signaling, we found that MSC-DF Notch1-/- prompted cocultured melanoma cells to form more spheroids and acquire the phenotype (CD271 + and Nestin + ) of melanoma stem/initiating cells (MICs), whereas MSC-DF N1IC+/+ suppressed melanoma cell sphere formation and mitigated properties of MICs. MSC-DF Notch1-/- increased stemness of CD271 + MIC, which resultantly exhibited stronger aggressiveness in vitro and in vivo, by upregulating Sox2/Oct4/Nanog expression. Consistently, when cografted with melanoma cells into NOD scid gamma (NSG) mice, MSC-DF Notch1-/- increased, but MSC-DF N1IC+/+ decreased, the amounts of CD271 + MIC in melanoma tissue. The amounts of CD271 + MIC regulated by MSC-DF carrying high or low Notch1 pathway activity is well correlated with capability of melanoma metastasis, supporting that melanoma metastasis is MIC-mediated. Our data demonstrate that intracellular Notch1 signaling in CAFs is a molecular switch dictating the plasticity and stemness of MICs, thereby regulating melanoma aggressiveness, and therefore that targeting the intracellular Notch1 signaling pathway in CAFs may present a new therapeutic strategy for melanoma. Stem Cells 2019;37:865-875., (©AlphaMed Press 2019.)

Background: Lack of a reliable hind limb gangrene animal model limits preclinical studies of gangrene, a severe form of critical limb ischemia. We develop a novel mouse hind limb gangrene model to facilitate translational studies., Methods: BALB/c, FVB, and C57BL/6 mice underwent femoral artery ligation (FAL) with or without administration of N G -nitro-L-arginine methyl ester (L-NAME), an endothelial nitric oxide synthase inhibitor. Gangrene was assessed using standardized ischemia scores ranging from 0 (no gangrene) to 12 (forefoot gangrene). Laser Doppler imaging (LDI) and DiI perfusion quantified hind limb reperfusion postoperatively., Results: BALB/c develops gangrene with FAL-only (n = 11/11, 100% gangrene incidence), showing mean limb ischemia score of 12 on postoperative days (PODs) 7 and 14 with LDI ranging from 0.08 to 0.12 on respective PODs. Most FVB did not develop gangrene with FAL-only (n = 3/9, 33% gangrene incidence) but with FAL and L-NAME (n = 9/9, 100% gangrene incidence). Mean limb ischemia scores for FVB undergoing FAL with L-NAME were significantly higher than for FVB receiving FAL-only. LDI score and capillary density by POD 28 were significantly lower in FVB undergoing FAL with L-NAME. C57BL/6 did not develop gangrene with FAL-only or FAL and L-NAME., Conclusions: Reproducible murine gangrene models may elucidate molecular mechanisms for gangrene development, facilitating therapeutic intervention., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo -/- mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment. Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572-83. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Pharmacological levels of ascorbate have long been suggested as a potential treatment of cancer. However, we observed that EC50 of ascorbate was at a similar level for cultured healthy melanocytes and melanoma cells, suggesting a limit of pharmacological ascorbate in treating cancer. Loss of 5-hydroxymethylcytosine (5 hmC) is an epigenetic hallmark of cancer and ascorbate promotes 5 hmC generation by serving as a cofactor for TET methylcytosine dioxygenases. Our previous work demonstrated that ascorbate treatment at physiological level (100 μM) increased 5 hmC content in melanoma cells toward the level of healthy melanocytes. Here we show that 100 µM of ascorbate induced apoptosis in A2058 melanoma cells. RNA-seq analysis revealed that expression of the Clusterin (CLU) gene, which is related to apoptosis, was downregulated by ascorbate. The suppression of CLU was verified at transcript level in different melanoma cell lines, and at protein level in A2058 cells. The anti-apoptotic cytoplasmic CLU was decreased, while the pro-apoptotic nuclear CLU was largely maintained, after ascorbate treatment. These changes in CLU subcellular localization were also associated with Bax and caspases activation, Bcl-xL sequestration, and cytochrome c release. Taken together, this study establishes an impending therapeutic role of physiological ascorbate to potentiate apoptosis in melanoma.

Type 2 diabetes mellitus (T2DM) has been considered to be a risk factor for numerous human cancers. Hyperglycemia is one of the most direct internal environmental changes for patients with T2DM. Increasing evidence reveals that a high concentration of glucose can promote tumor progression, while its role for migration and invasion of invasive ductal breast carcinoma (IDBC) cells remains unclear. In the present study, it was demonstrated that IDBC patients with T2DM suffered an increased tumor size and more frequent lymphatic and distant metastasis compared with those without T2DM (P<0.05). MCF-7 breast carcinoma cells, which were cultured in a high glucose concentration medium (25.00 mM), exhibited increased invasion (P<0.05). In addition, the expression of glucose transporters (Gluts), matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) in IDBC tissues with T2DM was significantly higher compared to those without T2DM. Downregulation of glucose transporter 1 (Glut1) by small interfering RNA may markedly suppress MCF-7 cell invasion as well as the expression of MMP2 and MMP9. These results suggest that T2DM can affect the malignant features of tumors in IDBC. The high glucose concentration in the tumor microenvironment may enhance IDBC invasion via upregulating Glut1/MMP2/MMP9 axis expression.

Peripheral artery disease (PAD) is one of the major vascular complications in individuals suffering from diabetes and in the elderly that can progress to critical limb ischemia (CLI), portending significant burden in terms of patient morbidity and mortality. Over the last two decades, stem cell therapy (SCT) has risen as an attractive alternative to traditional surgical and/or endovascular revascularization to treat this disorder. The primary benefit of SCT is to induce therapeutic neovascularization and promote collateral vessel formation to increase blood flow in the ischemic limb and soft tissue. Existing evidence provides a solid rationale for ongoing in-depth studies aimed at advancing current SCT that may change the way PAD/CLI patients are treated.

Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor-regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis. We demonstrate that the Notch1 pathway's activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1-/- selectively promote, while MSC-DFN1IC+/+ preferentially suppress melanoma metastasis, but not growth, in mouse models. Consistently, conditioned media (CM) from MSC-DFNotch1-/- and MSC-DFN1IC+/+ oppositely, yet selectively regulates migration, but not growth of melanoma cells in vitro. Additionally, when co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1-/- support, while MSC-DFN1IC+/+ inhibit melanoma cells in the formation of spheroids. These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We also identified Wnt-induced secreted protein-1 (WISP-1) as a key downstream secretory mediator of Notch1 signaling to execute the influential role of MSC-DF on melanoma metastasis. These findings reveal the Notch1-WISP-1 axis as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis.

Background: RhoGTPase-activating proteins (RhoGAPs) regulate RhoGTPases in cells, but whether individual reactive oxygen species (ROS) regulate RhoGAPs is unknown. Our previous published papers have shown that deleted in liver cancer 1 (DLC1) inhibits cancer cell migration by its RhoGAP activity. The present study was designed to explore the role of H2O2 in regulation of DLC1., Materials and Methods: We treated cells with H2O2 for 24h and phenotypic changes were analyzed by MTT, RT-PCR, Western blotting, immunofluorescence staining and wound healing assays., Results: H2O2 downregulated cyclin D1 and cyclin E to inhibit proliferation, and upregulated BAX to induce apoptosis in MCF-7 cells. Compared with non-tumorigenic cells, H2O2 increased expression of DLC1 and reduced activity of RhoA in cancer cells. Stress fiber production and migration were also suppressed by H2O2 in MDA-MB-231 cells., Conclusions: Our study suggests that H2O2 inhibits proliferation through modulation of cell cycle and apoptosis-related genes, and inhibits migration by decreasing stress fibers via DLC1/RhoA signaling.

Purpose: To observe the stimulating effect of pure traditional Chinese medical mouth rinse (PTCMMR) on skin and mucosa and to evaluate the efficacy of PTCMMR on inhibiting and killing oral common bacteria., Methods: PTCMMR and normal saline (NS) were separately dripped onto 14 five-month rabbits' left and right eyes for 7 days, and were smeared on the left and right side of buttocks, abdomens, back of 10 seven-month guinea pigs (after hair removal) once a day for comparison between the two sides. Five animals were sacrificed after 3 and 7 days of smearing to make pathological sections. Bacterium suspension with Staphylococcus aureus, Escherichia coli, Candida albicans, Pseudomonas aeruginosa and Streptococcus B were dropped to nutrient solution containing different concentrations of PTCMMR or compound Borax solution to observe the growth situation of bacteria. Inhibition zone diameters were measured after 18h of incubation within 37 degrees centigrade incubator. The data was statistically analyzed using SPSS 17.0 software package., Results: There was no obvious change such as redness, dermatitis between rabbits' left eye conjunctiva and the control group. The bacteriostatic and bactericidal effect of anaerobe and aerobe with dropping of PTCMMR was stronger than that of control group., Conclusions: PTCMMR has no stimulation to skin and mucosa, while it has strong efficacy to inhibit and kill oral common bacteria.

Background/purpose(s): We investigated whether CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are induced in patients suffering from early-stage septic shock and distinguish them from noninfectious patients with systemic inflammatory response., Methods: The study included 37 patients with early-stage septic shock, 15 patients with noninfectious systemic inflammatory response syndrome (SIRS), and 24 heath controls. We prospectively assayed the fraction of Tregs expressing high levels of CD25 and forkhead box P3 (Foxp3) as well as the plasma levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), and soluble CD25 in all the subjects studied., Results: Compared with the control groups, the plasma levels of IFN-γ [66.10 (45.23-85.08) pg/mL vs. 20.97 (17.58-26.21) pg/mL, p < 0.001] and IL-4 [100.69 (77.41-127.68) pg/mL vs. 70.40 (64.14-80.15) pg/mL, p < 0.001] as well as the IFN-γ/IL-4 ratio [0.66 (0.62-0.67) vs. 0.30 (0.27-0.33), p < 0.001] were significantly elevated in the patients with early-stage septic shock, but there was no difference between patients with sepsis and patients with SIRS. We found that the proportion of CD4(+)CD25(+)Foxp3(+) T cells was significantly increased in the patients with early-stage septic shock [(66.82 ± 21.79%) vs. (51.79 ± 21.79%) vs. (56.45 ± 10.68%), p = 0.003] in comparison with the SIRS and control groups, which could be differentiated from the patients with SIRS. The plasma levels of soluble CD25 were also increased, and positively correlated with the proportion of Tregs in patients with early-stage septic shock (Spearman correlation coefficient = 0.390, p = 0.003)., Conclusion: Our findings indicate that the proportion of CD4(+)CD25(+)Foxp3(+) T cells could be an indicator for the early diagnosis of sepsis. This proportion can also facilitate the evaluation of the patient's immune status and guide suitable immunoregulatory therapy., (Copyright © 2012. Published by Elsevier B.V.)

The Notch signaling pathway is an evolutionarily conserved, intercellular signaling cascade. The Notch proteins are single-pass receptors that are activated upon interaction with the Delta (or Delta-like) and Jagged/Serrate families of membrane-bound ligands. Association of ligand-receptor leads to proteolytic cleavages that liberate the Notch intracellular domain (NICD) from the plasma membrane. The NICD translocates to the nucleus, where it forms a complex with the DNA-binding protein CSL, displacing a histone deacetylase (HDAc)-corepressor (CoR) complex from CSL. Components of a transcriptional complex, such as MAML1 and histone acetyltransferases (HATs), are recruited to the NICD-CSL complex, leading to the transcriptional activation of Notch target genes. The Notch signaling pathway plays a critical role in cell fate decision, tissue patterning, morphogenesis, and is hence regarded as a developmental pathway. However, if this pathway goes awry, it contributes to cellular transformation and tumorigenesis. There is mounting evidence that this pathway is dysregulated in a variety of malignancies, and can behave as either an oncogene or a tumor suppressor depending upon cell context. This chapter highlights the current evidence for aberration of the Notch signaling pathway in a wide range of tumors from hematological cancers, such as leukemia and lymphoma, through to lung, skin, breast, pancreas, colon, prostate, ovarian, brain, and liver tumors. It proposes that the Notch signaling pathway may represent novel target for cancer therapeutic intervention., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Atherosclerosis and its associated complications remain the primary cause of death in humans. Aging is the main contributor to atherosclerosis, compared with any other risk factor, yet the specific manner in which age increases risk (the 'aging-risk' mechanism) remains elusive. A novel concept for atherosclerosis risk implicates a lack of endothelial progenitor cell (EPC)-dependent arterial repair in the development of the disease that is secondary to exhaustion of repair-competent EPCs. Molecular evidence derived from genetic techniques indicates atherosclerotic lesions may begin to form as arterial repair fails, rather than merely following arterial injury. Thus, chronic arterial injury may overwhelm the ability of EPCs to maintain arterial homeostasis, particularly when EPCs capable of arterial repair become exhausted. Recent studies have reported genes identified using non-biased approaches (ie, genetic linkage studies and genome-wide association studies) that are associated with susceptibility for atherosclerosis and related thromboembolic disorders; these genes may be implicated in the control of arterial wall inflammation and EPC-mediated tissue repair. Most of the genes identified by using non-biased genomic techniques are associated with inflammation, immune response and stem cells. This review focuses on new genetic data in the field of atherosclerosis and arterial homeostasis.

Aim: To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis., Methods: Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed., Results: GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides)., Conclusion: Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.

In the present paper the authors studied theoretically and experimentally the relationship between spectral resolution and grating density, the limitations to improve the spectral resolution by using high density grating, the use of longer focal length grating to increase spectral resolution without compromising instrument throughput and the effect of slit width on spectral resolution and sensitivity. Finally, two experiment results were provided to show why higher spectral resolution is important to ensure that critical information is not lost during a Raman measurement. Stressed silicon was produced by growing a thin crystalline layer of Si on an Si(x)Ge(1-x) substrate. It is possible to use Raman spectroscopy to probe the stress in the Si(x)Ge(1-x) and Si layers at the same time. The parameter to monitor the stress is the position of the Si-Si vibrational mode in Si(x)Ge(1-x) and Si. Such a measurement requires high spectral resolution because the peaks exhibit very subtle shifts. The authors' results clearly demonstrate that the resolution offered by a high density grating is necessary to properly monitor the very small frequency shift of this cap-layer Si-Si mode in order to properly characterize the strain structure. The Raman band around 180 cm(-1) is assigned to the radial breath mode of single wall carbon nanotube (SWCN). By measuring the frequencies excited with different laser, the diameters of the sample can be obtained. Practically, sample is always composed of SWCN with different but very close diameters and their Raman bands might overlap together and are difficult to determine the frequencies. The authors' results showed that only higher resolution with the long focal length spectrometer can give accurate number and frequencies of Raman bands, which leads to a correct analysis of the diameter distribution.

Diabetes mellitus is a common disease afflicting many people. In addition to coronary artery disease, diabetic retinopathy and renal failure, diabetic patients face abnormal wound healing and have increased lower extremity ulcers and amputations. In diabetes, wound healing is altered due to both macrovascular and microvascular processes. While the former can be addressed with surgical intervention, the latter is more difficult to correct. Neovascularization within the granulation tissue via angiogenesis and vasculogenesis is critical for wound healing. Endothelial progenitor cells (EPCs) have been implicated in vasculogenesis. Mobilization of EPCs from the bone marrow is impaired in diabetes and homing of EPCs to the wound is also abnormal. Recent studies show that hyperoxia and administration of exogenous stromal-derived factor-1α increases circulatory and wound levels of EPCs and improves wound healing in diabetic mice. These findings have great potential for translation into human counterparts as the treatment for this prevalent disease matures.

Stem cells are unspecialized precursor cells that mainly reside in the bone marrow and have important roles in the establishment of embryonic tissue. They also have critical functions during adulthood, where they replenish short-lived mature effector cells and regeneration of injured tissue. They have three main characteristics: self-renewal, differentiation and homeostatic control. In order to maintain a pool of stem cells that support the production of blood cells, stromal elements and connective tissue, stem cells must be able to constantly replenish their own number. They must also possess the ability to differentiate and give rise to a heterogeneous group of functional cells. Finally, stem cells must possess the ability to modulate and balance differentiation and self-renewal according to environmental stimuli and whole-organ needs to prevent the production of excessive number of effector cells.(1) In addition to formation of these cells, regulated movement of stem cells is critical for organogenesis, homeostasis and repair in adulthood. Stem cells require specific inputs from particular environments in order to perform their various functions. Some similar trafficking mechanisms are shared by leukocytes, adult and fetal stem cells, as well as cancer stem cells.(1,2) Achieving proper trafficking of stem cells will allow increased efficiency of targeted cell therapy and drug delivery.(2) In addition, understanding similarities and differences in homing and migration of malignant cancer stem cells will also clarify molecular events of tumor progression and metastasis.(2) This chapter focuses on the differentiation, trafficking and homing of the major types of adult bone marrow stem cells: hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) and the term"stem cell" will refer to "adult stem cells" unless otherwise specified.

Molecular configurations of CH3 OLi and CH3 CH2 OLi were structured based on the previous study that lithium atom and oxygen atom are directly joined by O-Li bond in alkoxy lithium (ROLi). Neither experimental nor theoretical Raman spectra of CH3 OLi and CH3 CH2 OLi have been reported up to now. In the present paper, DFT method at the B3LYP/ 6-31G(d,p) level was used to optimize molecular configurations of CH3 OLi and CH3 CH2 OLi, obtaining each corresponding equilibrium configuration. Vibration frequencies and Raman spectra of these two molecules were calculated based on equilibrium configuration. The vibration frequencies of obtained calculated results were analyzed by normal coordinate analysis. Besides, the Raman vibration modes of CH3 OLi and CH3 CH2 OLi were assigned according to potential energy distribution of each vibration frequency, which will provide theoretical basis for experimental workers to analyze the components of solid electrolyte interface film (SEI film) of lithium ion battery.

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.

Objective: Neovascularization, angiogenesis, and collagen constriction are essential for wound healing. We tested whether vascular endothelial growth factor-C (VEGF-C) can promote collagen constriction, capillary sprouting (angiogenesis), and invasion/migration of bone marrow-derived endothelial progenitor cells into collagen (vasculogenesis)., Methods: We used a recently characterized three-dimensional collagen matrix assay with either monolayers of human dermal microvascular endothelial cells (HMVECs) or bone marrow-derived endothelial progenitor cells (BMD EPCs), obtained from Tie-2 LacZ transgenic mice, overlaid with an acellular layer and then a cellular layer of collagen embedded with fibroblasts, that were nontransduced or transduced with either LacZ adenoviral vector (Ad5) or VEGF-C/Ad5. The ability of VEGF-C to enhance fibroblast-mediated collagen constriction was measured, and gels overlying HMVECs or BMD EPCs were co-cultured, harvested, and assayed for HMVEC migration, sprouting, and capillary-like formation; gels containing BMD EPCs were assayed for EPC invasion/migration into the collagen extracellular matrix., Results: VEGF-C significantly increased collagen constriction and formation of capillary-like structures with true lumina (P < .05) assessed by von Willebrand factor and VEGF receptor-2 immunoassaying. VEGF-C induced a significant increase in HMVEC migration, tubular polarization, and branching sprouts associated with a significant up-regulation of membrane type 1 matrix metalloproteinase (MT1-MMP) ( P < .05). Fibroblasts were necessary to support BMD-EPC invasion/migration from the monolayer into the collagen. Moreover, fibroblasts overexpressing VEGF-C significantly enhanced EPC invasion/migration ( P < .05) into the extracellular matrix by two-fold, and this effect could not be achieved with equivalent levels of exogenous VEGF-C in the absence of fibroblasts. The addition of a soluble VEGF-C competitor protein only partially inhibited these responses, reducing the EPCs by three-fold, but significant numbers of EPCs still invaded/migrated into the extracellular matrix, suggesting that other fibroblast-specific signals also contribute to the vasculogenic response., Conclusion: Fibroblast-specific expression of VEGF-C promotes collagen constriction by fibroblasts and enhances microvascular endothelial cell migration, branching, and capillary sprouting in association with up-regulating MT1-MMP expression. Fibroblasts are necessary for BMD EPC invasion/migration into collagen, and their overexpression of VEGF-C enhances this fibroblast-mediated vasculogenic effect. Collectively, these findings suggest a role for VEGF-C in multiple biologic steps required for wound healing (angiogenesis, vasculogenesis, and collagen constriction)., Clinical Relevance: Ischemic wound healing remains an unsolved problem with no previously identified molecular target for therapeutic intervention. This study demonstrates that VEGF-C overexpression by fibroblasts stimulates multiple biologic processes known to impact wound healing, such as collagen constriction, capillary sprouting, and EPC invasion and migration through extracellular matrix. Most ischemic wounds fail to heal and frequently lead to major limb amputation. Available cytokine ointments are ineffective, and revascularization is often not technically feasible. Even when these procedures are accomplished, many ischemic wounds frequently still do not heal because of multifactorial tissue level impairments in the fibroblastic and neovascularization responses at the wound base. Our findings identify an important role for two novel tissue level targets, dermis-derived fibroblasts and VEGF-C, in collagen constriction, angiogenesis, and postnatal vasculogenesis from BMD EPCs. Thus the findings are particularly relevant to the unsolved clinical problem of ischemic wound healing.

We recently showed that normal fibroblasts mediate capillary-like differentiation of human microvascular endothelial cells (HMVEC) in a 3-D angiogenesis model. Here, we show that a collaborative effect of VEGF-A and alphaVbeta3 integrin is critical in fibroblast-mediated angiogenesis because enhancement of both VEGF production by fibroblasts and beta3 integrin expression in HMVEC can rescue capillary-like endothelial differentiation under reduced serum conditions. To investigate the downstream signaling mechanisms, we compared N-Ras and Rho/Rac/Cdc42, as well as phosphatidylinositol 3-kinase (PI3-K) and Akt, for their involvement in the capillary-like network formation. The dominant-negative mutant of N-Ras (N-RasN17), but not the mutants of Rho/Rac/Cdc42, suppressed network formation. Overexpression of a constitutively active form of PI3-K rescued the network formation, which was inhibited by a dominant-negative >beta3 integrin; however, an active form of Akt failed to rescue the inhibition but induced a phenotypic change in HMVEC. Moreover, PI3-K is a downstream target of N-Ras because it could be co-immunoprecipitated with N-Ras, and its active form could rescue the inhibitory effect of N-Ras N17. Thus, our data indicate the existence of N-Ras- and PI3-K-dependent but Rho/Rac/Cdc42- and Akt-independent signaling mechanisms for the synergistic effect of VEGF-A and alphaVbeta3 on fibroblast-mediated microvascular network formation.

An in vitro model has been developed to study migration, survival, proliferation, and capillary-like differentiation of human microvascular endothelial cells (HMVECs) in an environment that avoids tumor promoters and complex matrices. HMVEC monolayers were plated, then induced to form three-dimensional, capillary-like networks by overlaying with human type I collagen followed by a second overlay of collagen with embedded fibroblasts. Detachment and migration of endothelial cells into the matrix was triggered within hours by the overlaying collagen, and the fibroblasts stimulated survival and formation of cords, vacuoles, tubes, and, after 4 to 5 days, capillary networks. The differentiation into branching capillary-like structures was dependent on direct fibroblast-endothelial cell contact and was not achieved when fibroblasts were replaced by seven types of melanoma cells, which included radial and vertical growth phase primary and metastatic stages. Vascular endothelial growth factor (VEGF), when overexpressed in fibroblasts, stimulated endothelial cell proliferation and migration, whereas angiopoietin-1 (Ang-1) had only motogenic effects. Neutralizing antibodies against VEGF and blocking antibodies for VEGF-receptor 2 (VEGFR2) significantly inhibited but not completely obliterated capillary network formation, suggesting that the VEGF signaling pathway is important but not exclusive and that other fibroblast-derived soluble factors and fibroblast-endothelial cell contact are essential for endothelial cell survival and differentiation.