Lysyl Oxidase Promotes the Formation of Vasculogenic Mimicry in Gastric Cancer through PDGF-PDGFR Pathway.

Objective: Vasculogenic mimicry (VM) generates an important supplementary form of blood supply in cancer, which many factors regulate. However, the effect of lysyl oxidase (LOX) on VM formation is unclear. In this study, gastric cancer tissues and cells were used to investigate the role of LOX in the formation of VM. Materials and Methods: The samples were collected from 49 patients with a final diagnosis of gastric cancer. According to metastasis (including lymph node metastases and distant metastases), gastric cancer samples were divided into metastasis and non-metastasis groups. Based on the degree of invasion, gastric cancer specimens were divided into T1 + T2 and T3 + T4 groups. The relative expression of LOX was detected using Western blot. The formation of VM was measured by double staining with CD34 and Periodic acid-Schiff (PAS) in gastric cancer tissue slices, and the correlation between LOX and VM was analyzed with Pearson's correlation analysis. Gastric cancer cell line BGC-803 was treated with LOX, ß-aminopropionitrile (BAPN, an inhibitor of LOX), and AG1295 or AG1296 (inhibitors of the platelet-derived growth factor receptor). The formation of VM was then measured using PAS staining. The expression of platelet-derived growth factor receptor (PDGFR)α and PDGFRß in gastric cancer cells was detected by Western blot. Results: In gastric cancer samples, the level of LOX was higher in the metastasis group than in the non-metastasis group (P < 0.05) and in the T3 + T4 group than in the T1 + T2 group (P < 0.05). VM formation was greater in the T3+T4 group than in the T1+T2 group (P < 0.05) and in the metastasis group than in the non-metastasis group (P < 0.05). The expression level of LOX was positively correlated with VM formation (P < 0.01). In gastric cancer cells, LOX concentration was positively correlated with the degree of VM, and BAPN concentration was negatively correlated with the degree of VM (P <0.05). PDGFR levels in the T3+T4 and metastasis groups were relatively higher (P <0.01) and positively correlated with LOX levels in gastric cancer specimens (P < 0.01). The relative expression of PDGFRα and PDGFRß in gastric cancer cells was up-regulated with increasing LOX and downregulated with increasing BAPN (P < 0.05). With inhibition of PDGFRα and PDGFRß using AG1295 or AG1296, VM formation in gastric cancer cells decreased (P <0.05), but the number of VM structures increased while LOX was added (P < 0.05). Conclusion: LOX partially promotes the formation of VM in gastric cancer through the PDGF-PDGFR signaling pathway.

LOX inhibition downregulates MMP-2 and MMP-9 in gastric cancer tissues and cells.

Objective: The objective of this study was to analyze the effects of lysyl oxidase (LOX) on the expression and enzyme activity of the matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) and to study its preliminary effect mechanisms. Methods: We collected fresh cancer specimens from 49 gastric cancer patients who underwent surgery. Immunohistochemistry was used to quantitate the protein expression levels of LOX and MMP-9 in gastric cancer tissues and to analyze their correlation. Also, six-week old nude mice were divided into a control group and a LOX inhibition group. SGC-7901 gastric cancer cells were inoculated subcutaneously into the backs of the two groups of these mice to construct a gastric cancer-bearing nude mouse model. In the LOX inhibition group, ß-aminopropionitrile (BAPN) was used to inhibit LOX. Western blotting was used to quantitate the relative expression levels of MMP-2 and MMP-9 in mouse tumor tissues, and gelatin zymography was used to quantitate their enzyme activity levels. In addition, BGC-823 gastric cancer cells were cultured, then 0.1 mM, 0.2 mM, and 0.3 mM BAPN and 2.5 nM, 5 nM, and 10 nM LOX were added to treat BGC-823 cells. ELISA and gelatin zymography were used to quantitate the protein concentrations and changes in enzyme activity of MMP-2 and MMP-9 in the culture supernatant. Western blotting was used to quantitate the relative expression levels of platelet derived growth factor receptor (PDGFR) in the BGC-823 gastric cancer cells after LOX inhibition and exogenous LOX addition. Results: In the tissues from the gastric cancer patients, the relative expression levels of LOX and MMP-9 were positively correlated (r = 0.326, P < 0.05). Compared with the control group, the tumor tissues from mice in the LOX inhibition group had reduced relative expression levels and enzyme activities of MMP-2 and MMP-9 (P < 0.05). After LOX were inhibited with different concentrations of BAPN in BGC-823 gastric cancer cells, the protein concentrations and enzyme activity levels of MMP-2 and MMP-9 in the culture supernatants were decreased (P < 0.05). In addition, the relative expression level of PDGFR in gastric cancer was decreased when BAPN concentrations increased, showing a negative dose-dependent manner (rPDGFR-α = -0.964, rPDGFR-ß = -0.988, P < 0.05). After exogenous LOX treating BGC-823 cells, the concentrations and enzyme activity levels of MMP-2 and MMP-9 in the cell supernatant were increased (P < 0.05). Further, the relative expression of PDGFR in gastric cancer cells was increased with the increase of exogenous LOX, showing a positive dose-dependent manner (rPDGFR-α=0.952, rPDGFR-ß=0.953, P<0.05). Conclusions: LOX inhibition can inhibit the expression and enzyme activity of MMP-2 and MMP-9 in gastric cancer tissues and cells, and the probable mechanism is through its effects on the PDGF-PDGFR signaling pathway.

Lysyl oxidase inhibition via ß-aminoproprionitrile hampers human umbilical vein endothelial cell angiogenesis and migration in vitro.

Lysyl oxidase (LOX) is an enzyme that oxidizes lysine residues in collagens and elastin. It stabilizes or remodels the extracellular matrix and basement membrane of blood vessels. Current oncology studies have revealed that LOX is upregulated in invasive cancer cells and bolstered cell movement, and LOX was observed to promote the angiogenesis and migration of endothelial cells. In the present study, angiogenesis and migration were examined in human umbilical vein endothelial cells (HUVECs). Following cell treatment with 0.1-0.4 mM ß-aminoproprionitrile (BAPN), a specific inhibitor of LOX, angiogenesis was analyzed with a fibrin gel in vitro angiogenesis assay kit and migration was examined via a Boyden Chamber assay. Angiogenesis-associated gene expression was investigated with a microarray assay and confirmed with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results showed that HUVEC angiogenesis substantially increased in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and phorbol 12-myristate 13-acetate (PMA). In addition, LOX inhibition blocked the angiogenesis stimulated by VEGF bFGF and PMA, and the inhibition of LOX reduced the migration of HUVECs. Furthermore, the microarray and RT-qPCR revealed that BAPN downregulated myeloid progenitor inhibitory factor 1, and western blot analysis demonstrated that BAPN decreased the phosphorylation of MAPK and Akt, suggesting that the specific inhibitor of LOX, BAPN, may serve as an alternative strategy for preventing angiogenesis.

Lysyl oxidase is involved in synovial hyperplasia and angiogenesis in rats with collagen­induced arthritis.

Lysyl oxidase (LOX) serves an important role in remodeling the extracellular matrix and angiogenesis in various types of cancer; however, whether LOX is involved in the pathogenesis of rheumatoid arthritis remains unknown. In order to investigate this in the present study, ß­aminopropionitrile, an inhibitor of LOX, was injected intraperitoneally into rats with type II collagen­induced arthritis (CIA). Subsequently, synovial hyperplasia was examined by hematoxyl in and eosin staining, and the microvascular density (MVD) and expression levels of LOX, matrix metalloproteinase (MMP)­2 and MMP­9 in the synovial membrane and fluid were determined by immunohistochemistry and ELISA, respectively. The enzyme activity of LOX was evaluated by the Amplex Red Hydrogen Peroxide method. The results demonstrated an increased amount of rough synovial membranes, higher MVD in these membranes and more synovial cell layers in CIA rats compared with in the control rats. In addition, higher enzymatic activity of LOX and higher expression levels of MMP­2 and MMP­9 were revealed in CIA rats compared with in the control rats. Notably, ß­aminopropionitrile inhibited paw swelling and the decreased the arthritis index, the MVD in the synovial membranes and the expression levels of MMP­2 and MMP­9. Furthermore, the expression level of LOX in the synovial membranes was positively associated with the MVD and the expression levels of MMP­2 and MMP­9, suggesting that LOX promotes synovial hyperplasia and angiogenesis and that LOX may be a potential therapeutic target for rheumatoid arthritis.

Citrullinated fibronectin inhibits apoptosis and promotes the secretion of pro-inflammatory cytokines in fibroblast-like synoviocytes in rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is characterized by synovial lining hyperplasia, in which there may be an imbalance between the growth and death of fibroblast-like synoviocytes (FLSs). Antibodies against citrullinated proteins are proposed to induce RA. This study aimed to investigate the pathogenic role of citrullinated fibronectin (cFn) in RA. METHODS: The distribution of fibronectin (Fn) and cFn in synovial tissues from RA and osteoarthritis (OA) patients was examined by immunohistochemical and double immunofluorescence analysis. FLSs were isolated from RA and OA patients and treated with Fn or cFn. Apoptosis was detected by flow cytometry and TUNEL assay. The expression of survivin, caspase-3, cyclin-B1, Bcl-2 and Bax was detected by real-time PCR. The secretion of proinflammatory cytokines was measured by ELISA. RESULTS: Fn formed extracellular aggregates that were specifically citrullinated in synovial tissues of RA patients, but no Fn deposits were observed in those of OA patients. Fn induced the apoptosis of RA and OA FLSs while cFn inhibited the apoptosis of RA and OA FLSs. Fn significantly increased the expression of caspase-3 and decreased the expression of survivin and cyclin-B1 in FLSs from RA and OA patients. cFn significantly increased the expression of survivin in RA FLSs. Furthermore, cFn increased the secretion of TNF-α and IL-1 by FLSs. CONCLUSIONS: cFn plays a potential pathophysiologic role in RA by inhibiting apoptosis and increasing proinflammatory cytokine secretion of FLSs.

Autoantibodies to the high-affinity IgE receptor in patients with asthma.

Autoimmune diseases have been implicated as a cause of intrinsic asthma; however, there is little data on the role of autoimmunity in the pathogenesis of asthma. The purpose of this study was to investigate circulating autoantibodies against the high-affinity IgE receptor Fc(epsilon)RI in patients with asthma. Seventy-eight patients with asthma and 32 healthy individuals as control subjects were included. All subjects were tested with basophil histamine releasing assay and immunoblotting to assess for the potential presence of receptor Fc(epsilon)RI autoantibodies. Of the 78 asthma patients total subjects, 25 (32.1%) had a positive by basophil histamine releasing assay and 23 (29.5%) by immunoblotting. Both of them were significant higher than the positive rate, 9.4% (p < 0.05) and 9.4% (p < 0.05), respectively. Our data demonstrated that aberrant autoantibodies against the high-affinity IgE receptor Fc(epsilon)RI were found in some patients with asthma implies that the autoimmunity may be one factor in intrinsic asthma pathogenesis.

Hair follicle reformation induced by dermal papilla cells from human scalp skin.

To investigate the possibility of hair follicle reformation induced by dermal papilla cells in vivo and in vitro. Dermal papilla cells, dermal sheath cells obtained from human scalp skin by enzyme digestion were mixed with collagen to form mesenchymal cell-populated collagen gels. Superior and inferior epithelial cells and bulb matrical cells were then cultured on these gels by organotypic culture to recombine bilayer artificial skins. Dermal papilla cells and outer root sheath keratinocytes were mingled together and transplanted under subcutaneous tissue of the dorsal skin of nude mice. The results of histologic examination was observed with HE stain. These recombinants by organotypic culture all reformed bilayer structure like nature skin. Hair follicle-like structure reformation was found in dermal sheath cell-populated collagen gel when combined with superior or inferior epithelial cells. Dermal papilla cells also induced superior and inferior epithelial cells to form hair follicle on nude mice. Low passage dermal papilla cells mixed with hair follicle epithelial cells reformed many typical hair follicle structures and produced hair fibres after transplantation on nude mice. The dermal part of hair follicle, such as dermal papilla cells and dermal sheath cells, has the ability to induce hair follicle formation by interaction with the epithelial cells of hair follicle.

Enzyme digestion to isolate and culture human scalp dermal papilla cells: a more efficient method.

In this study, we show a more efficient method for isolation and cultivation of dermal papilla cells from hair follicles of human scalp skin. The dermal partments of low hair follicles were pulled out from cutaneous fat and the bulb epithelium was teased out from the fibrous sheath with attached dermal papilla by applying gentle pressure with the tip of an occal forceps. When these fibrous sheaths were entirely digested into isolated cells by collagenase D but the dermal papillae were justly to be digested, collagenase D was discarded and the dermal papillae were isolated completely out from the resuspension solution by repeated low-speed centrifugation and transferred to another dish for free-floating culture. This procedure markedly simplifies the steps of isolated dermal papilla operation and relieves the laborious tension. Furthermore, dermal papillae could be isolated on a large-scale and remained intact. After collagenase digestion, the dermal papillae showed very high adherent rate and quicker growth than that of microdissection, which suggests that the definition factor of dermal papilla cell migration was relaxed and some structure had been activated or exposed. The cells exhibited a multi-layer forming property and spread-out growth style. They showed positive with alcian blue, with toluidine blue O for different gradient pH and PAS, which was similar to the staining results of in situ dermal papilla. It suggests that the culture papilla cells still synthesize and excrete neutral and acid mucopolysaccharides. Our results demonstrate that the papilla cells in culture condition still remain the ability to synthesize the specific extracellular matrix components of in situ dermal papilla, which supports the concept that the dermal papilla cell, a highly specialized fibroblast, especially is involved in hair growth regulation.

[Retrospective analysis of 1905 patients with skin cancer from two general hospitals in western China from 1981 to 2000].

OBJECTIVE: To investigate the clinical features and changes in the incidence of skin cancer in two hospitals located in western China. METHODS: The patients diagnosed pathologically as skin cancer from 1981 to 2000 were retrospectively collected from the two hospitals. Clinical data of patients with skin cancer were collected and analyzed. RESULTS: (1) Of the 1 905 patients with skin cancer, squamous cell carcinoma accounted for 29.4%(560 patients), basal cell carcinoma 28.0% (534), and cutaneous malignant melanoma(CMM) 16.0% (305). (2) There were 591 patients with skin cancer diagnosed between 1980 and 1990, and 1 314 between 1991 and 2000, and accounted for 0.34% and 0.58% of all biopsy cases, respectively. The number of total biopsy patients increased 1.6% every year during the 20 years. The number of biopsy patients with skin cancer and with CMM increased 3.5% and 3.9% every year,respectively. (3) Of the 305 CMM patients, 63.3% located on the acra. These patients were elder, and have a higher rate of trauma and a higher incidence in the male than that in patients with CMM located on the other sites. (4) Of the 305 CMM patients, 64 (21%) had history of trauma at the primary onset sites, and 47 (15.4%) had history of small congenital nevi at the primary sites. CONCLUSION: There are some differences in the clinical features such as location and age between the skin cancer patients in our study and those in white population. The incidence of skin cancer in the two hospitals had been increasing in the 20 years (between 1981 and 2000). Both trauma and small congenital nevi are important risk factors of CMM.

[Expressions of bFGF, ET-1 and SCF in dermal papilla cells and the relation to their biological properties].

OBJECTIVE: To investigate the expression of bFGF, ET-1 and SCF in different passages of cultured dermal papilla cells (DPC), and their possible effect on biological behaviour of DPC. METHODS: The expression of bFGF, ET-1 and SCF in different passages of cultured DPC was detected by immunocytochemistry and in situ hybridization. RESULT: The expression of ET-1 and SCF in early passages of cultured DPC was stronger, but became negative in late passages (>6 passages). The stronger the expression of ET-1 and SCF in DPC, the higher ability of DPC to induce hair follicle regeneration. CONCLUSION: The expression strength of ET-1 and SCF is related to the ability of DPC inducing hair follicle regeneration.

[Factors related to collagen gel contraction in hair follicle organotypic culture].

OBJECTIVE: To investigate the effects of rat tail collagen, hair follicle dermal papilla cells and hair follicle epithelium cells on collagen gel contraction in organotypic culture. METHODS: The hair follicle organotypic culture was prepared with different concentrations of rat tail collagen, different number of dermal papilla cells and hair follicle epithelium cells in DMEM medium, after cultured for 10 days the diameter of collagen gel was measured. RESULT: The concentration of rat tail collagen, hair follicle dermal papilla cells and hair follicle epithelium cells significantly influenced on collagen gel contraction in organotypic culture (P<0.01). The contraction of collagen gel was negatively related to the concentration of rat tail collagen, while the concentration of dermal papilla cells and hair follicle epithelium cells was positively related to the contraction of collagen gel. CONCLUSION: The key factor influencing collagen gel contraction in organotypic culture is the concentration of rat tail collagen, hair follicle dermal papilla cells and hair follicle epithelium cells.

[Study on point mutations in mitochondrial DNA control region for replication DLP(6) of cultured dermal fibroblast with 8-MOP/UVA treatment].

OBJECTIVE: To investigate the relationships between skin photoaging and point mutations in mitochondrial DNA (mtDNA) control region for replication of dermal fibroblast. METHODS: Cultured dermal fibroblasts were treated by 8-methoxypsora len /ultraviolet A (8-MOP/UVA). mtDNA was extracted by one-step-method and th e PCR products of D-loop and adjacent transcription promoter (DLP(6)) fragment of mtDNA control region for replication were detected by polymerase chain reaction-single strain conformation polymorphism and direct sequencing. RESULTS: After treated by 8-MOP/UVA, point mutations of 414 T-->G of DLP(6) fragment of mtDNA control region for replication largely accumulated. CONCLUSION: Accumulation of point mutations of DLP(6) fragment of mtDNA control region for replication may be closely associated with skin photoaging.