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Osteoarthritis (OA) is the most common chronic disabling joint disease, and currently there is no effective treatment for the cause. Necroptosis plays a key role in many diseases, and receptor-interacting protein kinase 3 (RIP3) is a key regulator during necroptosis process. Studies have shown that the expression level of RIP3 was significantly upregulated in human and mouse OA degenerative cartilage tissues, suggesting the occurrence of necroptosis. However, the specific pathophysiological role of RIP3 in cartilage is still unclear. This study intends to sequence and analyze the transcriptome of chondrocytes before and after RIP3 overexpression, and explore the specific functional mechanism of RIP3 in OA pathogenesis. RNA sequencing results showed that overexpression of RIP3 induced upregulation of 244 genes and downregulation of 277 genes in chondrocytes. Sixteen candidate target genes were screened out by constructing gene co-expression network for further verification at mRNA level, and the results suggested that RIP3 had the most significant inductive effect on the expression of phosphoinositide-3kinase, regulatory subunit 5 (Pik3r5), integrin subunit beta 3 (Itgb3) and MYB proto-oncogene like 2 (Mybl2). Results from CCK-8 and lactate dehydrogenase activity analysis showed that silencing the expression of Itgb3 by siRNA significantly rescued chondrocyte viability decline and necroptosis induced by RIP3, and it also inhibited the upregulating effect of RIP3 on the expression of catabolism-related genes Mmp1, Mmp13 and Il6, as well as the downregulating effect of RIP3 on the expression of anabolism-related genes Acan, Col2a1 and Sox9. This study has demonstrated that RIP3 promotes chondrocyte necrosis and cartilage matrix metabolism disorders by upregulating the expression of Itgb3 in chondrocytes, and ultimately leads to cartilage degeneration. These findings provided potential novel targets for the clinical treatment of OA, and further clarified the pathophysiological significance of necroptosis.
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Objective To explore the molecular pathogenesis of 3 Glanzmann's thrombasthenia pedigree by using bioinformatics software and provide evidence for in vitro experiments. Methods The genetic analysis of 3 pedigree diagnosed as Glanzmann's thrombasthenia was carried out. Clustalx-2.1 win software was used to analyze the conservatism of mutant sites in homologous sequences. Bioinformatics software such as PolyPhen-2, PROVEAN, SIFT and Mutationtaster was used to analyze the biological effect of mutation. SPDBV software constructed the molecular structure model of mutant protein and evaluated the influence of mutation on protein structure. Results The "new mutations" found in 3 Glanzmann's thrombasthenia pedigree were ITGA2B:c. 814G>C (p. Val272Leu), ITGA2B:c. 432G>A (p. Trp144Ter) and ACTN1:c. 2458A>G (p. Ile820Val). All three mutations were highly conserved among homologous species. Mutationtaster software showed that 3 new mutations were likely pathogenic. PolyPhen-2 and PROVEAN software showed ITGA2B p.Val272Leu and ACTN1 p.Ile820Val were benign and SIFT software showed that ITGA2B p. Val272Leu were likely pathogenic, while ACTN1 p. Ile820Val is benign. The result of SPDBV software showed that the Val272 of ITGA2B was transformed to Leu, neutralizing all the original hydrogen bond. The Trp144 of ITGA2B is transformed to Ter, resulting in the truncated proteins with only 113 amino acid residues. All these mutations affected the molecular structure of GPⅡb, resulting in a decrease ofGPⅡb/Ⅲa expression. When the Ile820 of ACTN1 is transformed to Val, onlyretained the hydrogen bond of Ile820 and Asp822, neutralized the rest hydrogen bond, whichaffected the molecular structure and protein function of ACTN1. Conclusion The mutations of ITGA2B:c.814G>C (p.VAL272LEU), ITGA2B:c.432G>A (p.Trp144Ter) and ACTN1:c.2458A>G (p.Ile820Val) are pathogenic.
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Objective@#To investigate the expression of integrin β3 in the endometrial implantation window of patients with cesarean scar and infertility, and its correlation with endometrial receptivity.@*Methods@#A total of 40 patients with previous cesarean scar defect (PCSD) secondary infertility treated in Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine from April 2018 to December 2019 were enrolled as the observation group.The natural cycle of 40 normal women who were examined in our hospital at the same time were selected as the control group.The two groups were enrolled in the endometrium of the glandular epithelial cells for MMP-9, TIMP-1 and LIF immunohistochemical staining, and statistical analysis was performed.The serum E2 and P levels of the implantation group were compared.The E2/P ratio was measured and compared, and the expression level of integrin β3 endometrial planting window was compared.@*Results@#The MMP-9, TIMP-1 and LIF in the observation group [(175.31±56.36), (201.46±51.34), (209.23±45.23)] were significantly lower than those in the control group [(252.35±78.43), (257.23±74.13), (298.34±72.35)] (t=5.334, 5.766, 6.023, all P<0.05), but the E2, P and E2/P in the observation group [(515.31±56.36)pmol/L, (53.71±8.34)pmol/L, (13.23±5.23)] were higher than those in the control group[(352.35±78.43)pmol/L, (33.13±4.13)pmol/L, (8.17±2.91)] (t=7.334, 4.251, 3.241, all P<0.05). The level of beta 3 in the observation group (0.163±0.013) was significantly lower than that in the control group (0.253±0.031) (t=4.342, P<0.05). The thickness of endometrium in the observation group [(9.12±2.03)mm] was significantly thinner than that in the control group [(12.24±2.45)mm] (t=3.226, P<0.05).@*Conclusion@#The expression of integrin β3 in the endometrial implantation window of patients with cesarean section scar infertility has a certain influence on endometrial receptivity.
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Objective To explore whether Angiopoietin-like protein 3 (ANGPTL3) is involved in podocyte actin rearrangement,and to analyze whether integrin β3 signal pathway is a key in ANGPTL3 inducing actin rearrangement.Methods The cultured podocytes were divided into six groups:wild type,ADR treated,ADR+ Dex,MOCK,ANGPTL3-cDNA,miRNA,and AD +miRNA group.(1) We observed actin cytoskeleton using Invitrogen reagents with confocal microscopy;(2) Actin cytoskeleton after blocking β3 on podocytes was;(3) The expression of total FAK and p-FAK was through Western blotting.Results (1) The wild type podocyte's cytoskeleton is arranged orderly.After ADR treatment,podocyte's actin are rearranged and weaken (P ＜ 0.05).There was no significant difference in actin arrangement between knock-down and MOCK group.In ANGPTL3-cDNA group the podocyte actin was also significantly rearranged;on the contrary,in miRNA +ADR group,the actin rearrangement never obviously happened (P ＜ 0.05).(2) Over-expression of ANGPTL3 podocytes blocked integrin β3 did not happen actin rearrangement.(3) The expression of p-FAK significantly increased in over-expression ANGPTL3 podocytes.Conclusion ANGPTL3 is a key in inducing actin rearrangement.Intergrin β3 maybe a central pathway in ANGPTL3's role with podocytes.
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Objective@#To explore whether Angiopoietin-like protein 3 (ANGPTL3) is involved in podocyte actin rearrangement, and to analyze whether integrin β3 signal pathway is a key in ANGPTL3 inducing actin rearrangement.@*Methods@#The cultured podocytes were divided into six groups: wild type, ADR treated, ADR+Dex, MOCK, ANGPTL3-cDNA, miRNA, and AD+miRNA group. (1) We observed actin cytoskeleton using Invitrogen reagents with confocal microscopy; (2) Actin cytoskeleton after blocking β3 on podocytes was; (3) The expression of total FAK and p-FAK was through Western blotting.@*Results@#(1) The wild type podocyte's cytoskeleton is arranged orderly. After ADR treatment, podocyte's actin are rearranged and weaken (P<0.05). There was no significant difference in actin arrangement between knock-down and MOCK group. In ANGPTL3-cDNA group the podocyte actin was also significantly rearranged; on the contrary, in miRNA+ADR group, the actin rearrangement never obviously happened (P<0.05). (2) Over-expression of ANGPTL3 podocytes blocked integrin β3 did not happen actin rearrangement. (3) The expression of p-FAK significantly increased in over-expression ANGPTL3 podocytes.@*Conclusion@#ANGPTL3 is a key in inducing actin rearrangement. Intergrin β3 maybe a central pathway in ANGPTL3's role with podocytes.
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Objective To investigate the mRNA and protein expressions of nine integrin subunits in human keloid-derived mesenchymal stem cells (KD-MSCs).Methods Cultured KD-MSCs and normal skin-derived MSCs (NS-MSCs) served as the experiment group and control group respectively.Real-time quantitative PCR and Western blot were performed to measure the mRNA and protein expressions of nine integrin subunits in the two groups respectively.Statistical analysis was carried out by t test.Results Real-time quantitative PCR showed no significant difference in the mRNA expression level of any of the integrin units α2,α3,α5,αV,α10,α11 or β1 between KD-MSCs and NS-MSCs (all P ＞ 0.05).The mRNA expression level of integrin α8 was decreased,while that of integrin β3 was significantly increased in KD-MSCs compared with NS-MSCs (both P ＜ 0.01).Western blot revealed that the changes in protein expression levels of integrin units α8 and β3 were consistent with those in their mRNA expression levels in both KDMSCs and NS-MSCs (both P ＜ 0.01).Conclusions Integrin units α8 and β3 may be involved in the occurrence and development of keloid,and the receptors made up of them may play important roles in the pathogenesis of keloid.
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Objective To investigate the expression of integrin β3 and osteopontin(OPN) in eutopic and ectopic endometrium of adenomyosis. Methods From January 2007 to July 2008, the endometrium specimens were collected from 43 patients with adenomyosis undergoing hysterectomy in Peking University First Hospital. Eutopic endometrium were 11 in proliferative phase and 32 in secretory phase (18 cases in mid-secretory phase) were collected. Ectopic endometriums were also collected. In the mean time, it was chosen 41 cases with pure subserous uterine myoma or cervical intraepithelial neoplasia (CIN) Ⅱ-Ⅲ treated by hysterectomy as controls including 12 endometrium in proliferative phase and 29 endometrium in secretory phase (19 cases in mid-secretory phase). The expression of Integrin β3 subunit and OPN in the endometrium were assessed by immunohistochemical staining and quantitative real-time polymerase chain reaction. Results (1)Immunohistochemical staining showed that positive staining of integrin β3 and OPN were present predominantly in eutopic and ectopic endometrial glandular epithelium. There was significant different protein expression of integrin β3 and OPN, which were 1.6±0.8 and 1.7±0.7 in eutopic endometrium,1.7±0.7 and 1.8±0.9 in ectopic endometrium,2.1±0.9 and 2.0±0.9 in control endometrium (P<0.05). The protein expression of integrin β3 and OPN in eutopic endometrium of adenomyosis in the proliferative phase(0.8±0.4 and 0.7±0.3) were remarkably lower than those of the secretory phase(1.8±0.8 and 1.9±0.8,P<0.01). The protein expression of integrin β3 and OPN in the endometrium of controls in the proliferative phase(1.0±0.4 and 1.0±0.4) were significantly lower than those of the secretory phase(2.5±0.7 and 2.5±0.7)(P=0.000). In the mid-secretory phase, the protein expression of integrin β3(2.0±0.9) and OPN (2.1±0.8)in eutopic endometrium of adenomyosis were significantly lower than that of control endometrium(2.7±0.5 and 2.7±0.7)(P<0.01). (2)The mRNA expression level of integrin β and OPN in eutopic and ectopic endometrium were assessed by quantitative real-time PCR(result was shown by median index). It was observed that integrin β3 mRNA and OPN mRNA were significantly lower in the eutopic endometrium of adenomyosis (4.69 and 4.23), when compared with ectopic endometrium(7.96 and 14.84)and controls (13.47 and 17.40) (P<0.05). Eutopic endometrium had higher mRNA expression of integrin β and OPN mRNA in the secretory phase (5.54 and 11.40) than that in the proliferative phase(2.69 and 3.30) (P<0.01).The mRNA expression level of integrin β and OPN of control endometrium in the proliferative phase (3.12 and 4.75)were significantly lower than that in the secretory phase(19.94 and 21.00, P=0.000). The mRNA expression of integrin β and OPN were 10.10 and 14.34 in the mid-secretory phase, which were significantly lower than 21.50 and 24.18 in control endometrium(P<0.05). Conclusions High expression of integrin β3 and OPN in ectopic endometrium of adenomyosis may cause endometriotic lesions; abnormal expression of integrin β3 and OPN in the endometrium of adenomyosis during the implantation window may contribute to infertility in some patients.
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Objective: To use three dimensional model in vitro to mimic solid tumor growth in vivo and explore the relationship between the multicellular resistance of colon carcinoma cell and the over-expression of cell adhesion molecules (integrin α V and integrin β3). Methods: The colon carcinoma HT-29 multicellular spheroids (MCS) model were constructed with three dimensional cell culture methods. The mRNA and protein expressions of integrin α V and integrin β3 was detected by RT-PCR and immunofluorescence analysis and compared between monolayer cells (MC) and MCS. The viability and apoptotic rate of MC and MCS were detected by flow cytometry (FCM) after treatment with 5-fluorouracil (5-FU). Results: The expression levels of integrin α V and integrin β3 in three dimensional cell culture model of HT29 MCS were much higher than those in MC (P < 0.01). Compared with MC, the sensitivity of MCS to the anticancer drug 5-FU significantly decreased ( P < 0.05), and the rate of 5-FU-induced apoptosis significantly decreased (0.346 ± 0.035 vs 0.235 ± 0.024, P < 0.05). Conclusion: Over-expression of cell adhesion molecules integrin α V and integrin β3 may increase the multi-cellular resistance of colon carcinoma cells.
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Objective To identify the gene mutations of platelet membrane glycoprotein Ⅱ b,Ⅲa(GPⅡb/Ⅲa)in three Chinese pedigrees with Glanzmann thrombastIlenia.Methods All exons and exonintron boundaries of GP Ⅱ b/Ⅲ a gene were amplified by PCR analysis followed by DNA sequencing.DNA sequencing was used to exclude gene polymorphisms.Results The probands in the three pedigrees had a normal platelet count,coagulation profiles,scattered platelets on the blood film,a prolonged cutaneous bleeding time,and impaired or minimal ex vivo platelet aggregation in response to ADP,thrombin,collagen,adrenaline and arachidonic acid,but normal platelet aggregation in response to ristoeetin.Both FACS and Western blotting demonstrated trace content of αⅡb in the platelets from proband 1 and proband 3,who were classified as type Ⅰ GT,and a small amount of αⅡb in the platelets from proband 2,who was classified as type Ⅱ GT.Compound heterozygous mutations,T2255G(Leu721Arg)and C2671T(Gln860Stop)were identified in proband 1.The proband 2 had homozygous A2334C(Gln747Pro)missense mutation.Nonsense mutations C1750T (Arg584Stop)and 69-79 deletion mutation were identified in proband 3. Conclusions Compound heterozygous mutations T2255G and C2671T of αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 1. Homozygous mutation A2334C of αⅡb gene leads to type Ⅱ Glanzmann thrombasthenia for proband 2. Compound heterozygous mutations C1750T and 69-79del αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 3. T2255G,C1671T and 69-79del aye novel mutations for αⅡb gene.
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Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the alpha v beta3 integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether beta3 is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of beta3 integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 +/- 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either beta3 or av prolonged survival significantly to a maximal average survival of 19.7 +/- 1.5 days (P<0.01) and 18.4 +/- 0.9 days (P<0.01), respectively. XT-199, a chemical blocker of the alpha v beta3 receptor also prolonged survival to 19.5 +/- 1.3 days (P<0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine beta1 antibody comparatively prolonged survival (19.0 +/- 1.2 days), suggesting that HTNV infection is partly mediated through integrin beta1 receptors as well as through beta3 receptors in vivo. Our data demonstrate that the beta3 receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond beta3 for cellular entry within an organism.
Subject(s)
Animals , Mice , Animals, Newborn , Antibodies, Monoclonal/therapeutic use , Integrin beta1/metabolism , Hantaan virus/metabolism , Hemorrhagic Fever with Renal Syndrome/mortality , Imidazoles/pharmacology , Integrin alphaV/metabolism , Integrin alphaVbeta3/antagonists & inhibitors , Integrin beta3/metabolism , Receptors, Virus/metabolismABSTRACT
Objective To investigate the relationship between the expression level of platelet membrane glycoprotein?3(GPⅢa,CD61)and the severity of disease in patients with hemorrhagic fever with renal syndrome(HFRS).Methods One hundred and four patients with HFRS and 30 healthy individuals were recruited.The percentage of CD61 positive platelets and the mean fluores- cence intensities(MFI)of platelet membrane glycoprotein?3 were determined by flow cytometry (FCM).The 104 patients studied were divided into three groups based on their expression levels of platelet membrane glycoprotein?3 at oligurie phase.Clinical data and laboratory parameters in different groups were compared and analyzed.Results The expression levels of CD61 in patients with HFRS were significantly higher than those in control group,although no significant difference in the percentage of CD61 positive platelets between patients with HFRS and controls was detected.The MFI of CD61 expression in patients with HFRS at fever phase,oliguric phase and polyuric phase was 19.75?2.57,17.46?1.48 and 15.55?0.60,respectively,which was significantly higher than that in control group(3 20?0.12).The expression level of CD61 in patients with HFRS at oliguric phase was negatively correlated with platelet count and serum albumin(r=-0.637 and-0.695,respec- tively)and positively correlated with white blood cell count,blood urea nitrogen,serum creatinine and alanine aminotransferase(r=0.945,0.904,0.956 and 0.891,respectively).When the patients were compared according to the expression levels of CD61,it was indicated that the higher the expression level of CD61,the higher the incidence of uremia,hypoalbuminemia,abnormal liver func- tion and leukocytosis.Conclusions The expression levels of platelet membrane glycoprotein?3 in patients with HFRS are different in different clinical phases and are significantly correlated with the severity of the disease in the patients.It suggests that the expression levels of platelet?3 integrin are dramatically increased in patients with HFRS,which may be an indicator for the severity of disease and be helpful for monitoring the state of the patients' diseases and evaluating the severity of the disease.
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OBJECTIVE: We compared the expression pattern of progesterone receptor, integrin 3, cyclooxygenase-2 (COX-2) in in-phased endomerium of patient with the disease related implantation and control group, and tried to confirm the clinical efficacy of the immunohistochemical markers for discrimination of occult uterine receptivity defect in in-phase endometrium. STUDY DESIGN: Endometrial tissues were obtained from 60 women with normal (group 1; n = 20), uterine synechiae (group 2; n = 15), and endometriosis (group 3; n = 25). On 7 ~ 8 days after ovulation (POD 7 ~ 8), sex hormone levels were measured and immunohistochemical staining of PR, integrin 3, and COX-2 expression were performed. RESULTS: PR was decreased in the group 2 and increased in the group 3 comparing with the group 1. integrin 3 expression was significantly decreased in the group 2 and 3. COX-2 expression was significantly decreased in the group 2. But, in the group 3, COX-2 expression was slightly increased in glandular epithelial cells, and significantly increased in stromal cells. CONCLUSIONS: In-phase biopsies from patients with endometriosis and uterine synechiae showed different expression pattern of integrin 3, COX-2, and PR compared to the control. The aberrant expression of immunohistochemical markers be associated with occult uterine receptivity defect and produce the useful diagnostic method.
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Female , Humans , Biopsy , Cyclooxygenase 2 , Discrimination, Psychological , Endometriosis , Endometrium , Epithelial Cells , Gynatresia , Ovulation , Progesterone , Receptors, Progesterone , Stromal CellsABSTRACT
Vascular cells respond to multiple cytokines, they also express a variety of integrin adhesion receptor. A number of the vascular cell integrins are functionally and structurally homologous, suggesting some level of biologic redundancy. We investigated the importance of alphavbeta3 function during vascular endothelial growth factor(VEGF) induced corneal angiogenesis by examining the effects of 9D491, a monoclonal antibody against beta3 that blocks alphavbeta3-mediated cell adhesion to vitronectin and fibrinogen. A hydrogel disk containing 500ng of VEGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the first, randomized to contain either 2.6microgram of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and assigned an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 postimplantation, angiogenesis scores were not significantly lower in eyes treated with anti-alphavbeta3 mAb (averaged score=21.6) as compared to eyes treated with 6E10 (averaged score=24.0) (p<0.2, Wilcoxon rank sum test). In a rabbit corneal pocket assay, monoclonal antibody to beta3 could not inhibit corneal angiogenesis induced by VEGF.