α-SMA Research Articles

Inhibitory effect of rapamycin on proliferation, migration and fibrosis of human pterygium fibroblasts in vitro

Objective To investigate the inhibitory effect of rapamycin, an mammalian target of rapamycin (mTOR) pathway inhibitor, on the proliferation, migration and fibrosis of human pterygium fibroblasts (PFBs). Methods Pterygium tissues were collected from patients with primary pterygium who underwent surgical excision in Shenyang Fourth People's Hospital from May to July 2015.The tissues were cultured in vitro and the PFBs were identified by anti-human vimentin immunofluorescence assay.The 3 to 5 generation cells were used for the experiments.The viability of cells treated with different concentrations of rapamycin was detected by methyl thiazolyl tetrazolium (MTT). The cells were divided into normal control group and rapamycin group, and the scratch wound healing test was used to evaluate migration of the PFBs.The expressions of MKI67, α-smooth muscle actin (α-SMA), fibronectin, caspase3, mammalian target of rapamycin (mTOR) and LC3B mRNA were detected by real-time quantitative PCR. Results The cultured cells showed morphology of long spindle and were vimentin immunopositive.The cell viability in rapamycin treated PFBs demonstrated a dose-dependent decrease.At 24 hours after culture, The cell viability in 30 μmol/L rapamycin group was (76.67±8.84)% of that in 0 μmol/L rapamycin group (P<0.001). The relative residual scratch width in 30 μmol/L rapamycin group was (35.40±11.62)% 48 hours after scratch, which was significantly greater than (2.45±0.76)% in the normal control group (P<0.05). Real-time quantitative PCR showed that the mRNA expressions of MKI67, α-SMA, fibronectin and mTOR in rapamycin group were significantly decreased when compared with those in normal control group (all at P<0.05). The expression of LC3B mRNA in rapamycin group was significantly higher than that in normal control group (P<0.05). The mRNA expression of caspase3 was not significantly different between the two groups (P=0.861). Conclusions Rapamycin can effectively inhibit the proliferation, migration and fibrosis of PFBs without affecting the cell survival.Detailed mechanism remains to be further studied.Rapamycin may serve as an anti-fibrosis agent to prevent the progression and recurrence of pterygium in the future. Key words: Pterygium; Fibroblasts; Rapamycin; Anti-fibrosis

Inhibition of hepatocyte growth factor on proliferation and transdifferentiation of human Tenon capsule fibroblasts triggered by transforming growth factor-β1in vitro

Objective To investigate the effects of hepatocyte growth factor (HGF) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor -β1 (TGF-β1) in vitro. Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group, TGF-β1 treated group and different concentrations HGF+ TGF-β1 groups.The TGF-β1 at 10 μg/L was added into culture medium of the TGF-β1 treated group, and different concentrations of HGF (25, 50, 100, 200 μg/L) and 10 μg/L TGF-β1 were added into culture medium of the HGF25 μg/L+ TGF-β1, HGF50 μg/L+ TGF-β1, HGF100 μg/L+ TGF-β1, HGF200 μg/L+ TGF-β1 group respectively, Methyl thiazolyl tetrazolium (MTT) was employed to measure the cell proliferation (absorbance at 560 nm). Immunofluorescence staining was used to evaluate and locate the expression of α-smooth muscle action (α-SMA) in the cells.The expression of α-SMA protein in the cells was detected by Western blot assay. Results Cultured cells showed fusiform in shape with the positive response for vimentin.The proliferation value of the cells was 0.203±0.025, 0.497±0.101, 0.426±0.062, 0.354±0.040, 0.272±0.084, 0.241±0.011 in the blank control group, TGF-β1 treated group, HGF25 μg/L+ TGF-β1 group, HGF50 μg/L+ TGF-β1 group, HGF100 μg/L+ TGF-β1 group and HGF200 μg/L+ TGF-β1 group, respectively, showing a significant difference among the groups (F=9.210, P=0.003). Compared with the TGF-β1 treated group, the proliferation values of the cells were significantly reduced in the blank control group and HGF+ TGF-β1 group (all at P<0.05). Immunofluorescence staining showed that α-SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF-β1 treated group and weak red fluorescence in HGF+ TGF-β1 group, and the expression of α-SMA was absent in the blank control group.The percentage of α-SMA-positive cells was (60.0±4.7)% in the TGF-β1 treated group and (14.3±3.1)% in the HGF+ TGF-β1 group, with significant difference between the two groups (t=19.856, P<0.001). The relative expression levels of the α-SMA protein in the cells were 0.642±0.032, 1.330±0.069 and 0.884±0.040 in the blank control group, TGF-β1 group and HGF100 μg/L+ TGF-β1 group, respectively, showing a significant difference among the groups (F=13.370, P<0.001), and relative expression levels of the α-SMA protein in the cells were significantly lower in the blank control group and HGF100 μg/L+ TGF-β1 group than that in the TGF-β1 treated group (all at P<0.05). Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts, down-regulate the expression of α-SMA protein induced by TGF-β1 and arrest the phenotype transformation of fibroblasts in vitro. Key words: Tenon capsule/cytology; Fibroblasts/metabolism; Hepatocyte growth factor/administration & dosage; Transforming growth factor beta type 1; Fibrosis/prevention & control; α-Smooth muscular actin; Humans; Cells, cultured

Mesenchymal stem cells local transplantation in abdominal aorta in rabbit and TIS effect on stenosis of the aorta andα-smooth muscle actin expression

Objective The aim of this study was to evaluate the effect of human umbilical cord mesenchymal stem cell transplantation on the degree of intima, middle membrane and lumen stenosis after abdominal aorta injury in rabbits. Methods Twenty male 20 New Zealand white rabbits were divided into control group and experimental group with each 10 rabbits. The abdominal aorta injury models were made by balloon dilation in two groups. In the experimental group, the 1×107/kg umbilical cord mesenchymal stem cells were injected into the vascular of the abdominal aorta of the rabbit from the side hole of the balloon catheter. The control group was injected with the same amount of normal saline by the same method. 4 weeks after, the rabbit were killed, rabbit abdominal aorta intimal thickness (IT), intimal area (IA), vascular media thickness (MT) and area (MA) and the degree of stenosis were measured and compared between the two groups; The α-smooth muscle actin (α-SMA) expression in rabbit vascular smooth muscle were compared between two groups. Results The IT [(1.63±0.29) mm vs. (0.68±0.11) mm, t=9.680, P=0.0001], IA [(4.11±1.23) mm2 vs. (1.42±0.56) mm2,t=6.290, P=0.0001], MT [(2.31±0.47) mm vs. (1.88±0.31) mm, t=2.420, P=0.026] and MA [(6.32±1.66) mm2 vs. (4.02±1.06) mm2,t=3.690, P=0.0017] in experiment group were significant lower than that of control group. The degree of stenosis in the control group and the test group were (72.2±8.4)% and (35.0±8.6)% respectively with statistical difference (t=9.790, P=0.000); α-SMA was brownish brown granules under optical microscope with the positive expression rate of 0.12±0.04 and 0.48±0.05 in the control and experiment groupers respectively with statistical difference (t=17.780, P=0.0001). Conclusion Human umbilical cord mesenchymal stem cell transplantation can significantly reduce the degree of vascular stenosis after abdominal aorta injury in rabbit and promote the expression of alpha -SM-actin in the membrane. Key words: Umbilical cord mesenchymal stem cells; Abdominal aorta injury; Arterial stenosis

Histological study on the safety of the controllable ileostomy with pipe

To investigate the safety of the controllable ileostomy with pipe in view of histology. Twenty-eight Beagle dogs undergoing controllable ileostomy with pipe were studied. The special fistula tube with balloon was placed into the hole locating at the cecal root opposing the mesenteric side, and fixed by double knot compression method. The fistula tube was removed 14 days after surgery, then the safety of the procedure was preliminarily evaluated by gastrointestinal radiography and anatomical observation. The small intestine tissue at the compression suture was used as the experimental segment, and the small intestine tissue at the proximal non-compression suture was used as the control segment. The histological staining and the immunohistochemical staining of S-100 protein, c-kit protein and α-smooth muscle actin(α-SMA) protein between two segment were compared, while quantitative comparison of myenteric plexus, intestinal Cajal cell(ICC) and smooth muscle cells in intestinal wall was carried out. After removal of fistula tube at 14 days postoperative, the dogs were normal in feeding and defecation. The digestive tract radiography showed that the intestine was patent without obvious stenosis and obstruction. The dogs were dissected 21 days after operation. The abdominal sinus ostium was well healed and the internal sinus was well formed. Under gross inspection, blood supply, morphology and motor function of experimental intestine segment were similar from the proximal and distal segments of control intestine. S-100 immunohistochemical staining showed that the morphology and distribution of S-100 protein positive cells and "blank area" cells in the experimental and control segments were consistent. Myenteric plexus counting showed that the experimental segment was 3.62±1.82/field and the control segment was 3.27±1.62/field, whose difference was not statistically significant(t=1.30, P=0.20). Immunohistochemical staining of c-kit showed that the distribution of c-kit positive cells in both segments was consistent. Counting of the number of ICCs in myenteric plexus revealed that experimental segment was 2.96±2.57/plexus, and control segment was 2.49±1.80/plexus without significant difference(t=1.81, P=0.07). Immunohistochemical staining of α-SMA showed that the morphology and distribution of smooth muscle cells in whole intestinal wall(muscle layer, longitudinal muscle, ring muscle) in experimental and control segments were consistent. The average absorbance(A) value of α-SMA staining in ring muscle layer was detected and quantified. The experimental segment was 0.15±0.03 and control segment was 0.14±0.04 without significant difference(t=1.16, P=0.25). The technique of controllable ileostomy with pipe is safe in view of histology, which may replace the traditional protective ileostomy.

Protective effects of astragalosides IV on renal proximal tubular epithelial cell injury induced by high glucose

Objective To investigate the protective effects of astragalosides Ⅳ (ASI) on high glucose-induced renal proximal tubular epithelial cells (NRK-52E). Methods NRK-52E were cultured and divided randomly into three groups: control group, high glucose group (HG in short), ASI groups (with various doses). Cells were treated with increasing concentrations of ASI (20, 40, 80 and 100 μg/ml) for 24 h in high glucose and we also stimulated cells in ASI 100 μg/ml with high glucose for various lengths of time. The apoptosis rate was detected by flow cytometric analysis. The mRNA and protein expression of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), Smad2, Smad3 and their phosphorylated forms were detected by real-time polymerase chain reaction (PCR) and Western blot, respectively. Results Compared with the control group, apoptosis was increased in the high glucose group (P<0.01). However, ASI inhibited high glucose-induced cell apoptosis in a dose-dependent manner, with a maximal inhibitory effect achieved at 100 μg/ml (P<0.05). The significant inhibition caused by ASI was observed at 8h after the start of pretreatment (P<0.05) and increased in a time-dependent manner. ASI can inhibit the expression of TGF-β1, α-SMA, Smad2, Smad3 both at mRNA and protein level. Conclusions ASI inhibited NRK-52E cells apoptosis induced by high glucose and reduced expression of TGF-β1, α-SMA, Smad2, Smad3 both at the mRNA and protein level in NRK-52E cells, thus delayed epithelial-to-mesenchymal transition progress. Key words: Astragalus membranaceus; Saponins/PD; Kidney tubules; Epithelial cells/DE; Diabetic nephropathies; Rats

Role of chemokine CXC-ligand 16 in renal fibrosis following renal ischemia-reperfusion injury in mice

Objective To evaluate the role of chemokine CXC-ligand 16(CXCL16) in renal fibrosis following renal ischemia-reperfusion(I/R) injury in mice. Methods Twenty-four healthy male C57BL/6 mice, aged 8-10 weeks, weighing 20-30 g, were divided into 4 groups(n=6 each) using a random number table method: sham operation group(group S), sham operation plus anti-CXCL16 antibody group (group S+ A), group I/R, and I/R plus anti-CXCL16 antibody group(group I/R+ A). Renal ischemia was induced by occlusion of the left renal artery for 30 min followed by 72-h reperfusion, and the right kidney was removed on 5th day in anesthetized mice. CXCL16 antibody 5 mg/kg and the equal volume of normal saline were intraperitoneally injected at 72 h after reperfusion twice a week for 2 consecutive weeks in I/R+ A and I/R groups, respectively. Sham operation was performed, and the equal volume of CXCL16 antibody 5 mg/kg and normal saline were intraperitoneally injected at 72 h after reperfusion twice a week for 2 consecutive weeks in S+ A and S groups, respectively. Orbital venous blood samples were collected at day 14 after reperfusion for determination of serum blood urea nitrogen(BUN) and creatinine(Cr) concentrations. Animals were then sacrificed and the left kidney was removed for measurement of the renal fibrosis size(using Sirius red staining), expression of α-smooth muscle actin(α-SMA), fibronectin(FN) and type Ⅰ collagen(Col-Ⅰ) in renal tissues by Western blot. Results Compared with group S, the serum BUN and Cr concentrations and renal fibrosis size were significantly increased, and the expression of α-SMA, FN and Col-Ⅰ was up-regulated in group I/R(P<0.05). Compared with group I/R, the serum BUN and Cr concentrations and renal fibrosis size were significantly decreased, and the expression of α-SMA, FN and Col-Ⅰ was down-regulated in group I/R+ A(P<0.05). Conclusion CXCL16 is involved in the process of renal fibrosis following renal I/R injury in mice. Key words: Chemokines, CXC; Kidney; Reperfusion injury; Fibrosis

Locating human splenic capillary sheaths in virtual reality

Stromal capillary sheath cells in human spleens strongly express CD271, the low affinity nerve growth factor receptor p75. Serial sections of a representative adult human spleen were double-stained for CD271 versus smooth muscle alpha actin (SMA) plus CD34 to visualise capillary sheaths, the arterial tree and endothelial cells by transmitted light. Preliminary three-dimensional (3D) reconstructions of single regions were inspected in virtual reality (VR). This method showed that a large number of CD271+ sheaths occur in a post-arteriolar position often surrounding capillaries located close to divisions of arterioles. The length and diameter of capillary sheaths are rather heterogeneous. Long sheaths were observed to accompany one or two generations of capillary branches. We hypothesise that human splenic capillary sheaths may attract recirculating B-lymphocytes from the open circulation of the red pulp to start their migration into white pulp follicles along branches of the arterial tree. In addition, they may provide sites of interaction among sheath macrophages and B-lymphocytes. Our innovative approach allows stringent quality control by inserting the original immunostained serial sections into the 3D model for viewing and annotation in VR. Longer series of sections will allow to unequivocally localise most of the capillary sheaths in a given volume.

Pathological changes of breast tissue after long-term polyacrylamide hydrogel injection for augmentation mammoplasty

Objective To study the pathological mechanism of aseptic necrosis of breast tissue after long-term breast augmentation injected with polyacrylamide hydrogel (PAHG). Methods We included patients who had no obvious clinical symptoms and underwent surgical removal of the PAHG in our hospital, excluding patients with a history of breast prosthesis implantation. Extracts were collected, and then underwent a careful gross observation and serial sections followed by HE, Mallory, and toluidine blue staining, to observe the histological structure of collagen fibers and inflammatory cells. Immunohistochemistry for CD31 and α-smooth muscle actin (α-SMA) antibody was used to further identify vascular endothelial cells and vascular smooth muscle cells, respectively. Analysis of the correlation between injection time and the incidence of different pathological changes were done. Results We included 22 females (mean age 39.14 years, range 22-55 years) who requested surgical removal of the PAHG. The mean duration of PAHG injection was 9.27 years (range 4-14 years). They had no clinical symptoms or only had induration, deformation and displacement. The basic pathological changes of local tissue included foreign body reaction and chronic inflammation, fibrosis and tissue necrosis; and half of the tissue had also histological changes, such as small vascular smooth muscle layer thickening, lumen stenosis, etc. No significant correlation was found between the pathological changes and the duration of the disease and the clinical symptoms. Conclusions Patients with PAHG injection for augmentation mammoplasty can histopathological changes of local tissues even without experiencing discomfort. The tissue necrosis in some patients may be caused by foreign body reaction and inflammatory reaction. It may also be due to local ischemia caused by small vascular smooth muscle hyperplasia, luminal stenosis etc. Key words: Polyacrylamide hydrogel; Breast augmentation, Injection; Long-term; Histopathology; Small vessel

Smooth Muscle Alpha Actin Immunoexpression (α-Sma) and CD-117 Antibody (C-Kit) in Capsules Formed by Polyurethane Foam-Coated Silicone Implants and with Textured Surface: A Study on Rats.

One of the undesirable complications that might occur after breast augmentation with silicone implants is capsular contracture. In its etiology, the relations between mast cells and myofibroblasts play an important role in collagen synthesis. Mast cells are able to activate fibroblasts into myofibroblasts, through paracrine secretions, inducing collagen production. The objectives of this study were to analyze the myofibroblast concentration through the α-SMA immunomarker and evaluate the intensity of mast cell expression against the C-Kit immunomarker. Sixty-four Wistar rats were used, divided into two groups (polyurethane foam and textured surface) with 32 animals in each. The animals received silicone implants on the back, below the panniculus carnosus, and after the determined period, they were killed and the capsules formed around the implants were studied. The capsules were analyzed employing the immunohistochemical technique, with the α-SMA and C-Kit immunomarkers in subgroups of 30, 50, 70 and 90days. The myofibroblast concentration was higher in the polyurethane group when compared to the textured group (30days p = 0.105; 50days p = 0.247; 70days p = 0.014 and 90days p = 0.536). The intensity of mast cell expression was more pronounced in the polyurethane group when compared to the textured group (30days p = 0.798; 50days p = 0.537; 70days p = 0.094 and 90days p = 0.536). Polyurethane-coated implants induced higher concentrations of myofibroblasts and higher expression of mast cells, when compared to the textured surface implants. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Pseudomonas aeruginosa recombinant protein PA3611 induces epithelial-mesenchymal transformation of bronchial epithelial cells

Objective To investigate whether Pseudomonas aeruginosa recombinant protein PA3611 can induce the in vitro epithelial-mesenchymal transformation (EMT) of rat bronchial epithelial cells. Methods A series of processes including DNA synthesis, gene amplification, vector construction, induction of expression and protein purification was used to synthesize the recombinant protein PA3611. CCK-8 kit was used to detect the proliferation of bronchial epithelial cells after stimulation with the protein PA3611. Morphological changes in bronchial epithelial cells were observed. Western blot and qPCR were performed to measure the expression of E-cadherin (E-CAD) and α-smooth muscle actin (α-SMA). T test was used for statistical analysis. Results DNA sequencing verified the successful preparation of the recombinant protein PA3611. PA3611 inhibited the proliferation (P<0.05) and induced the EMT of bronchial epithelial cells. Moreover, it also inhibited the expression of E-CAD protein, but promoted the expression of α-SMA (P<0.05). Conclusion The recombinant protein PA3611 can induce the EMT of bronchial epithelial cells and the underlying mechanisms are worthy of further investigation. Key words: Recombinant protein PA3611; Pseudomonas aeruginosa; Epithelial-mesenchymal transformation

Assembly of Tissue-Engineered Blood Vessels with Spatially Controlled Heterogeneities.

Tissue-engineered human blood vessels may enable in vitro disease modeling and drug screening to accelerate advances in vascular medicine. Existing methods for tissue-engineered blood vessel (TEBV) fabrication create homogenous tubes not conducive to modeling the focal pathologies characteristic of certain vascular diseases. We developed a system for generating self-assembled human smooth muscle cell (SMC) ring units, which were fused together into TEBVs. The goal of this study was to assess the feasibility of modular assembly and fusion of ring building units to fabricate spatially controlled, heterogeneous tissue tubes. We first aimed to enhance fusion and reduce total culture time, and determined that reducing ring preculture duration improved tube fusion. Next, we incorporated electrospun polymer ring units onto tube ends as reinforced extensions, which allowed us to cannulate tubes after only 7 days of fusion, and culture tubes with luminal flow in a custom bioreactor. To create focal heterogeneities, we incorporated gelatin microspheres into select ring units during self-assembly, and fused these rings between ring units without microspheres. Cells within rings maintained their spatial position along tissue tubes after fusion. Because tubes fabricated from primary SMCs did not express contractile proteins, we also fabricated tubes from human mesenchymal stem cells, which expressed smooth muscle alpha actin and SM22-α. This work describes a platform approach for creating modular TEBVs with spatially defined structural heterogeneities, which may ultimately be applied to mimic focal diseases such as intimal hyperplasia or aneurysm.

The effect of age factor on telomerase reverse transcriptase activity in pulmonary fibrosis model in mice

Objective To explore the effect of age factor on the degree of pulmonary fibrosis and on the change in telomerase reverse transcriptase(TERT)activity in mice. Methods A total of 80 healthy male C57BL/6 mice aged 20-weeks were randomized into 4 groups: a young pulmonary fibrosis model group(n=20), a young control group(n=20), a senile pulmonary fibrosis model group(n=20), and an elderly control group(n=20). Two model groups were induced by an intratracheally injected bleomycin in 5 mg/kg, and two control groups by an intratracheally injected normal saline.The elderly were defined as 26 weeks old mice.Five mouse pulmonary fibrosis models and five mouse controls in four groups were randomly selected and killed at 7, 14, 21, 28 days.Lung tissue specimens were stained with hematoxylin eosin(HE)and Masson trichrome(Masson)method, and then pathological changes were observed.In addition, immunohisto-chemical staining was used to observe the expression levels of epithelial cell marker protein E-cadherin(E-cad), stromal cell marker protein Vimentin, and alpha smooth muscle actin(α-SMA). Finally, the expression level of telomerase reverse transcriptase(TERT)protein was detected by Western blotting. Results A bleomycin-induced pulmonary fibrosis mice model was successfully prepared.The degree of pulmonary fibrosis was more severe in old mice than in young mice.Compared with the young pulmonary fibrosis model group, the expression level of E-Cad was decreased in the senile pulmonary fibrosis model group(P<0.05). Compared with the young control group and the elderly control group, the expression levels of E-Cad in the young pulmonary fibrosis model group and the senile pulmonary fibrosis model group were decreased(P<0.05), and decreased along with the prolongation of the modeling time.Compared with the young pulmonary fibrosis model group, the expression levels of alpha-SMA and vimentin were increased in the senile pulmonary fibrosis model group(P<0.05). The expression levels of alpha-SMA and vimentin were increased in the young pulmonary fibrosis model group and the senile pulmonary fibrosis model group, as compared to the young control group and the elderly control group(P<0.05), and increased along with the prolongation of the modeling time.The activity of TERT in lung tissue of mice was increased at first and then decreased.Compared with the young pulmonary fibrosis model group, the activity of TERT in the senile pulmonary fibrosis model group significantly fluctuated(P<0.05). Conclusions Age factor can affect the severity of pulmonary fibrosis by affecting the activity of telomerase reverse transcriptase in mouse models of pulmonary fibrosis. Key words: Age factors; Idiopathic pulmonary fibrosis; Telomerase reverse transcriptase

A new method for separation of human pancreatic stellate cells

Objective To explore a new method for the separation of human pancreatic stellate cells. Methods Single-cell suspension of normal pancreatic tissue and pancreatic cancer tissue was prepared by gentle MACS™ tissue processor-constant temperature shaking digestion. Human pancreatic stellate cells of quiescent and activated state were isolated by density gradient centrifugation. Results A new type of isolation method could obtain about (2.6±0.7) ×106 quiescent pancreatic stellate cells in 1 g of human normal pancreatic tissue, with a viability of about 90.0%. The morphology of the cells were conformed to the representative for the quiescent state characteristics and transient blue-green autofluorescence was observed at the 328 nm excitation wavelength; 1 g of human pancreatic cancer was able to obtain approximately (4.1±1.1)×106 activated PSCs with a viability of 92.0%, and all of the activated cells expressed α-SMA vimentin, FSP-1 and other characteristic markers. Conclusions The new separation method of this experiment is suitable for both human resting and activated human pancreatic stellate cells. At the same time, the purity is high and the separation time is greatly shortened, which is worth promoting. Key words: Pancreatic stellate cells, human; Separation method; Technological improvement; Density gradient centrifugation

Effects of long noncoding RNA-MIAT on lens epithelial to mesenchymal transition induced by transforming growth factor-β2

Objective To investigate the roles of long noncoding RNA-myocardial infarction-associated transcript (MIAT) on lens epithelial cells (LECs) fibrosis induced by transforming growth factor-β2 (TGF-β2). Methods LECs line (SRA01/04) was cultured in conventional DMEM (normal control group) and DMEM containing 10 ng/ml TGF-β2(TGF-β2 induced group) for 48 hours.The morphology of the cells was observed under the optical microscope, and the relative expression levels of MIAT, E-cadherin(E-cad), α-smooth muscle action (α-SMA), collagenⅠ(CollⅠ) in protein level and mRNA level were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The cells cultured in DMEM or DMEM containing 10 ng/ml TGF-β2 were transfected by siNRA empty carrier (siNRA group, siNRA+ TGF-β2 group) and siRNA-MIAT (siRNA-MIAT group, siNRA-MIAT+ TGF-β2 group) for 48 hours, and the morphology of the cells was observed under the optical microscope, and the relative expression levels of MIAT, E-cadherin(E-cad), α-smooth muscle action (α-SMA), collagenⅠ(CollⅠ) in protein level and mRNA level were detected by real-time fluorescence quantitative PCR and Western blot. Results The cells in the normal control group showed the round and polygon in shape, and those in the TGF-β2 induced group showed the spindle-like.Compared with the normal control group, the relative expression levels of MIAT mRNA, α-SMA mRNA and CollⅠmRNA were significantly elevated (2.497±0.644 vs.0.827±0.062; 2.951±0.146 vs.1.085±0.517; 2.115±0.090 vs.1.002±0.088), and the expression of E-Cad mRNA was significantly reduced (0.102±0.027 vs.1.020±0.262) in the TGF-β2 induced group (P=0.045, 0.004, 0.000, 0.025). The expressions of MIAT, α-SMA, CollⅠ and E-Cad showed a similar trend between two groups.The relative expressions of MIAT protein and mRNA were evidently reduced in the SiRNA-MIAT group compared with the siRNA empty vector group (all at P<0.05). Compared with the siRNA+ TGF-β2 group, the relative expressions of α-SMA and CollⅠin protein and mRNA levels were significantly reduced, and the expressions of E-cad protwin and mRNA were elevated in the siRNA-MIAT+ TGF-β2 group (all at P<0.01). Conclusions MIAT might participate in TGF-β2-induced LECs-EMT.The down-regulation of MIAT in the LECs inhibits the fibrosis of LECs. Key words: Myocardial infarction-associated transcript; Long non-coding RNA; Lens epithelial cells; Epithelial to mesenchymal transition; Posterior capsule opacification

The value of diffusion tensor imaging in early diagnosis of chronic allograft nephropathy and monitoring graft fibrosis in rats

Objective To estimate the value of diffusion tensor imaging (DTI) in early diagnosis of chronic allograft nephropathy (CAN) and monitoring of graft fibrosis in rat models. Methods Thirty CAN rat models were established as experimental group by transplanting Fisher donor kidneys into Lewis recipients. Thirty Lewis rats that received Lewis kidneys served as control group. Serum creatinine (SCr) was monitored regularly every two weeks from 14 days after transplantation. Eight rats were randomly selected by random number table method and underwent DTI examination at 4, 12, 20 weeks after modeling. DTI scans were performed on the renal cortex and medulla to measure apparent diffusion coefficient (ADC) and fractional anisotropy (FA). From the remaining 22 rats in each group, 6 rats were randomly selected and underwent pathological analysis at 4, 12, 20 weeks after modeling. Histological changes in the kidney were evaluated by chronic allograft damage index (CADI) scores. The expression of alpha-smooth muscle actin (α-SMA) and Vimentin were quantitatively measured. The differences in creatinine, DTI parameters, CADI score, α-SMA, Vimentin expression level were analyzed by two independent samples t test in two groups, the differences among CADI score, α-SMA, Vimentin expression level of the experimental group were compared using ANOVA. The correlations among DTI parameters and CADI score, α-SMA and Vimentin expression level were analyzed using Pearson analysis. Results The creatinine in the experimental group increased continuously, and the creatinine in the control group showed no significant increase. The difference in creatinine between the two groups was statistically significant from 8th week after operation (P<0.01). There was no obvious difference in the size and signal intensity of transplanted kidneys in control group at different time points. Compared with the control group, the graft kidney in the experimental group at the 4 weeks demonstrated increased signal intensity with mild increased volume of kidney, and the boundaries between cortex and medulla were not clear. The cortex and medulla showed gradually increased signal intensity, heterogeneous signal distribution and marginal haziness over time. The ADC and FA value of renal cortex and medulla in experimental group were significantly lower than those in control group at 4, 12, 20 weeks (P<0.05). The ADC and FA values of the cortex and medulla gradually decreased in the experimental group over time, while the values of the parameters in the control group did not show a significant decrease. The ADC and FA values of the cortex and medulla were negatively correlated with the scores of CADI, and the expression level of α-SMA, Vimentin in the experimental group(r=-0.50 to-0.85, P<0.01). Conclusion DTI can be an effective technique for early diagnosis of CAN and monitoring of graft fibrosis process. Key words: Kidney transplantation; Chronic allograft nephropathy; Diffusion tensor imaging; Comparative study

What is the pathogenesis of proximal hypospadias?

To report the information concerning the pathogenesis of proximal hypospadias and causes of curvature associated with it. From January 2009 to December 2015, 74 patients underwent repair of proximal hypospadias. In 70 patients we performed a systematic biopsies in the lateral areas of the urethral plate, as well as under this plate. The study of the histological structure of these areas was performed using routine staining with hematoxylin and eosin, and the Masson's trichrome which color the collagen fibers in blue and monoclonal antibody against alpha-smooth muscle actin. This prospective study shows that the fibrotic tissue abnormally present on the ventral side of the penis consists of a mixture of fibrous connective tissue, nerve nets, short vessels, and smooth muscle fibers. In contrast to the scrotal dartos, penile dartos fascia does not contain smooth muscle fibers. Therefore, these fibers may come from a blood vessel or spongy tissue which existed during neonatal period in the distal part of the penis before disappearing. In addition, in 13 cases, the presence downstream of the urethral meatus, of a bifurcation of corpus spongiosum into two branches supposes that the corpus spongiosum is form by fusion around the urethra of two mesenchymal bodies. The arterial supply of this purely masculine formation originates from a new vascularization and it is probably, developed under secretion of androgens (angiogenic substances in target tissues). These findings allow us to suggest that the proximal hypospadias is due to avascular necrosis of the distal part ie. poorly vascularized part of the corpus spongiosum.

Changes of cancer perfusion after antiangiogenesis in transplanted tumor of nude mice

Objective To analyze the feasibility of volume perfusion CT (VPCT) in the transplanted tumor of hepatocellular carcinoma after antiangiogenesis. Methods Hepatocellular carcinoma HepG-2 cells were inoculated with 40 BALB/c A-nu nude mice. The rats were divided into treatment group and control group by random number table method. The control group was injected with bevacizumab (10 mg/kg), and the control group was injected with the same amount of saline. On the 3rd, 6th and 9th day after treatment, 5 mice were randomly selected for VPCT detection. The mice were sacrificed and the α-SMA and CD31 was detected by immunohistochemistry. And changes in vascular maturity index (VMI) and microvessel density (MVD) were detected. Results The success rate of both experimental modeling was 100%, and no infection or death occurred. On the 3rd, 6th, and 9th day of treatment, the average tumor volume of the treatment group was (1.15±0.35), (1.61±0.43), and (1.76±0.59) cm3, respectively; the control group was (1.72±0.53), (2.96±0.86) and (3.98±1.02) cm3, there was a significant difference between the two groups (t=2.102, 2.251, 2.357, P=0.039, 0.025, 0.016). Before treatment, there was no significant difference in BF and BV between the two groups (P=0.358, 0.259). On the 3rd, 6th, and 9th days of treatment, the BF in the treatment group was (18.57±4.27), (26.15±10.78), and (13.05±3.72) ml/(100 ml·min), respectively. BV were (3.35±0.85), (5.56±2.38), and (3.02±0.99) ml/100 ml, respectively, and there was a significant difference from the control group (t= 2.351, 5.102, 3.561, 2.133, 2.963, 3.512, P=0.030, 0.000, 0.000, 0.038, 0.000). As time progressed, the MVD of the tumor tissues in the treatment group gradually decreased, and the MVD value of the control group gradually increased. On the 3rd, 6th, and 9th days of treatment, the MVD of the treatment group was lower than that of the control group (t=3.591, 4.632, 4.215, P=0.000, 0.000, 0.000). As time progressed, the VMI of the treatment group increased first and then decreased. The VMI value of the control group had no significant change. On the 3rd, 6th, and 9th days of treatment, the VMI of the treatment group was higher than that of the control group (t=2.351, 2.638, 2.159, P=0.032, 0.000, 0.040). Conclusion VPCT can make noninvasive evaluation of blood perfusion, tumor anatomy and vascular function, reduce the systematic error caused by heterogeneity of tumor. The blood perfusion of hepatocellular carcinoma is consistent with VMI, the potential of evaluation tools for angiogenesis therapy. Key words: Hepatocellular carcinoma; Antiangiogenesis; Tumor perfusion; CT perfusion imaging

Abstract 614: From Genotype to Phenotype: Molecular and Functional Characterization of the CVD-associated SNP rs11574 in ID3

Atherosclerosis is the primary pathological process of CVD and is strongly controlled by heritable factors; however, the mechanisms and pathways through which many of these genetic components regulate plaque development are poorly understood. Recently, a SNP in the coding region of the gene inhibitor of differentiation 3 ( ID3 ) at rs11574 was identified in multiple studies to be associated with CVD. Mutation of rs11574 from the major allele (G) to the minor allele (A) changes the 105 th amino acid of ID3 from an alanine to a threonine and is associated with an attenuated ability of ID3 to antagonize bHLH transcription factors and to prevent them from binding to DNA and activating transcription. The current study utilized co-immunoprecipitation to demonstrate that the minor allele of rs11574 specifically impairs the ability of ID3 to bind to and sequester E12 and no other member of the bHLH family. ChIP studies further demonstrated that the impaired regulatory ability of the ID3 minor allele variant increases E12 occupancy at promoter regions and enhances transcription of E12 target genes including smooth muscle alpha actin ( Acta2 ) and p21 in murine vascular smooth muscle cells (VSMCs). To study the role of this SNP in human cells in intact chromatin under endogenous regulation, we genome edited human 293T cells using CRISPR/Cas9 to produce the allelic variants of rs11574 in the ID3 gene (GG, AG, and AA). Edited cells showed significant changes in cellular proliferation, gene expression, and promoter occupancy depending upon genotype at rs11574. Cells containing the minor allele of rs11574 displayed significantly increased p21 expression and proliferated more slowly than cells containing the major allele of rs11574; whereas, cells heterozygote at rs11574 displayed an intermediate phenotype. These data implicate rs11574 in the regulation of cellular proliferation and mature VSMC marker expression, key phenotypes regulated in the processes of lesion development and formation.

Phenotypic alteration of vascular smooth muscle cells as a sequence of mild hyper‐caloric intake in the absence of hyperglycemia: Potential modulation by antidiabetic drugs

Diabetic patients remain at a high risk of atherosclerotic cardiovascular disease in addition to hypertension and microvascular complications. A significant proportion of diabetic patients present with vascular abnormalities upon initial diagnosis implicating an early vascular insult in the course of development of diabetes. A contractile‐to‐synthetic vascular smooth muscle cell (VSMC) phenotypic switch is associated with different diseases including atherosclerosis, diabetes, hypertension, and cancer. In the present study, we examined whether early metabolic derangement in the course of diabetes development was associated with VSMC phenotypic alteration before hyperglycemia develops. For this purpose, we used a high‐calorie diet (HC) fed rat model with delayed development of hyperglycemia. Up to 12 weeks of feeding, neither increased serum glucose nor elevated mean arterial pressure was detected. Control and HC rats were sacrificed at 4, 8, and 12 weeks, and their aortae were dissected for isolation of primary VSMCs. VSMC morphology changed whereby the typical hill‐and‐valley culture pattern was gradually substituted by densely packed areas of rhomboid cells in explants for aortic tissues from HC rats. This changed appeared to be dependent on the duration of feeding. VSMC proliferation and migration increased in cultures derived from HC‐fed rats compared to controls at the same age and passage number. Moreover, VSMC contractile phenotype markers smooth muscle alpha actin and calponin gradually decreased in cells cultured from HC‐fed rats in a manner proportional to the feeding duration. In vitro, exposure to pioglitazone, but not metformin was associated with a reduction in VSMC proliferation in cells derived from HC‐fed rats. On the contrary, similar exposure to increased oleiate and palmitate concentrations in the culture medium resulted in an increased proliferation of VSMCs from control rats. Our results indicate that VSMC phenotype and function is potentially affected by the early metabolic derangement associated with the development of diabetes regardless of the blood glucose concentration. Studies to investigate the effect of chronic exposure of HC‐fed rats to metformin and pioglitazone on VSMC phenotype are underway.Support or Funding InformationSupported by AUB MPP fund #320148This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of overexpression of retinoic acid receptor α on epithelial-to-mesenchymal transition induced by hypoxia in renal tubular epithelial cells

Objective To explore the effect of overexpression of retinoic acid receptor α(RARα) on epithelial-to-mesenchymal transition (EMT) induced by hypoxia in renal tubular epithelial cells(NRK-52E). Methods The RARα lentivirus vector and negative control lentivirus vector were synthetised.The NRK-52E cells were divided into 4 groups: the normal control group, the hypoxia model group, the transfection group and the negative control group.Puromycin(2 mg/L) was added in transfection group and negative control group for screening after gene interference for 72 h. Then the 2 groups were subjected to hypoxia/reoxygenation, but the normal control group had no treatment.The change of cellular morphology was observed by using light microscope; the mRNA and protein expressions of RARα, E-cadherin, α -smooth muscle actin(α-SMA) in NRK-52E cells were detected by adopting reverse transcription-polymerase chain reaction (RT-PCR) and Western blot after hypoxia for 48 h. Results (1) Light microscope revealed that cells in both hypoxia model group and negative control group cells became atrophic and elongated, which were consistent with the morphology of myofibroblasts.But cells in transfection group cells were cubic, forming an epithelial monolayer.(2)Compared with the normal control group, the mRNA and protein expressions of RARα and E-cadherin in hypoxia model group were dramatically reduced(mRNA: 0.58±0.12 vs.1.00±0.00, 0.11±0.00 vs.1.00±0.00, t=-0.63, 767.30, all P 0.05). Conclusion Overexpression of RARα could alleviate EMT of renal tubular epithelial cells induced by hypoxia. Key words: Retinoic acid receptor α; Renal tubular epithelial cells; Epithelial-to-mesenchymal transition; Transfection; Hypoxia