Β1 Expression Research Articles

Store-operated Ca2+ entry-sensitive glycolysis regulates neutrophil adhesion and phagocytosis in dairy cows with subclinical hypocalcemia

Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca2+ entry (SOCE)-related molecules. It is well-established in non-ruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca2+ < 2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (HYP, average 2.83 ± 0.42 lactations, n = 6). In the first experiment, neutrophils were isolated from blood of CON and HYP cows and used to analyze aspects of adhesion and phagocytosis function through QRT-PCR along with confocal laser scanning microscopy, mRNA expression of the glycolysis-related gene hexokinase 2 (HKII) and components of the SOCE moiety ORAI calcium release-activated calcium modulator 1 [ORAI1, ORAI2, ORAI3, stromal interaction molecule 1 (STIM1) and STIM2]. Results showed that adhesion and phagocytosis function were reduced in HYP cows. The mRNA expression of adhesion-related syndecan-4 (SDC4), integrin β9 (ITGB9) and integrin β3 (ITGB3) and phagocytosis-related molecules complement component 1 R subcomponent (C1R), CD36, tubulinß1 (TUBB1) were significantly decreased in the HYP group. In the second experiment, to address how glycolysis affects neutrophil adhesion and phagocytosis, neutrophils isolated from CON and HYP cows were treated with 2 μM of the HKII inhibitor Benserazide-d3 or 1 μM of the fructose-bisphosphatase 1 (FBP1) inhibitor MB05032 for 1 h. Results revealed that the HKII inhibitor Benserazide-d3 reduced phagocytosis and the mRNA abundance of ITGB9, and CD36 in the HYP group. The FBP1 inhibitor MB05032 increased adhesion and phagocytosis and increased mRNA abundance of HKII, ITGB9, and CD36 in the HYP group. Lastly, to investigate the mechanism whereby SOCE-sensitive glycolysis affects neutrophil adhesion and phagocytosis, isolated neutrophils were treated with 1 μM of the SOCE activator thapsigagin or 50 μM of the inhibitor 2-APB for 1 h. Results showed that thapsigargin increased mRNA abundance of HKII, ITGB9, and CD36, and increased adhesion and phagocytosis in the HYP group. In contrast, 2-APB decreased mRNA abundance of HKII and both adhesion and phagocytosis of neutrophils in the CON group. Overall, the data indicated that SOCE-sensitive intracellular Ca2+ levels affect glycolysis and help regulate adhesion and phagocytosis of neutrophils during hypocalcemia in dairy cows.

Expression levels of β1, β3 integrins and MMP2 and features of morphological heterogeneity of pancreatic ductal adenocarcinoma associated with distant metastasis

Background. Pancreatic cancer is characterized by pronounced invasive growth and a high tendency to lymphogenous and hematogenous metastasis. Molecular mechanisms underlying the epithelial-mesenchymal transition, in particular, changes in the expression of β1, β3 integrins and MMP2, contribute to the invasion and metastasis of pancreatic cancer. A phenotypic manifestation of invasive potential of pancreatic cancer may be the heterogeneity of the morphological structure of the parenchymal component of ductal adenocarcinoma.The aim of the study was to analyze the expression levels of β1 integrin, β3 integrin, MMP2 and the morphological heterogeneity in the tissue of pancreatic ductal adenocarcinoma, as well as to compare the data obtained with distant metastasis.Material and Methods. The study group included 84 patients with morphologically verifed pancreatic ductal adenocarcinoma (T1–4N0–2M0–1). The median age of the patients was 58.6 ± 9.1 years. All patients underwent surgery, during which an incisional biopsy of the primary tumor tissue was performed. Expression of integrin β1, integrin β3, and MMP2 markers was assessed by immunohistochemistry.Results. The positive expression of β3 integrin in glandular, trabecular, and solid structures was observed more frequently in patients with hematogenous metastases than in patients without distant metastases (87 vs 12 %, р=0.001; 100 vs 22 %, р=0.004; 100 vs 8 %, р=0.001, respectively). The positive expression of MMP2 in glandular and solid structures was also observed more often in patients with hematogenous metastases than in patients without distant metastases (87 vs 32 %, р=0.001 and 100 vs 27 %, р=0.001, respectively). The positive expression of integrin β1 in glandular, trabecular, solid structures and tumor cells was observed less frequently in patients with hematogenous metastases than in patients without distant metastases (27 vs 86 %, р=0.001; 25 vs 79 %, р=0.007; 0 vs 93 %, р=0.001 and 18 vs 60 %, respectively). The logistic regression model for predicting the risk of hematogenous metastasis of pancreatic ductal adenocarcinoma was developed: Y=(-6.71 + 30.9×X1 – 27.2×X2), where X1 – the presence or absence of β3 integrin expression in the cells of the glandular structures, X2 – the presence or absence of MMP2 in the cells of the glandular structures. The risk of developing hematogenous metastasis was determined by the formula: Р=еY/(1+еY). Model confdence interval: χ2 =34.0; p&lt;0.001, sensitivity: 89 %, specifcity: 87 %. Conclusion. Expression of integrin β1, integrin β3, and MMP2 markers in various structures of the parenchymal component of pancreatic ductal adenocarcinoma is associated with distant metastasis. The localization of the expression of the studied markers in the glandular structures of tumor tissue is of particular importance. The presence of positive expression of integrin β3 and MMP2 in almost all types of tumor structures is associated with the highest frequency of distant dissemination. Key words: ductal pancreatic adenocarcinoma, β1 integrin, β3 integrin, MMP2, tumor morphological heterogeneity, distant metastases&gt; ˂ 0.001, sensitivity: 89 %, specifcity: 87 %.Conclusion. Expression of integrin β1, integrin β3, and MMP2 markers in various structures of the parenchymal component of pancreatic ductal adenocarcinoma is associated with distant metastasis. The localization of the expression of the studied markers in the glandular structures of tumor tissue is of particular importance. The presence of positive expression of integrin β3 and MMP2 in almost all types of tumor structures is associated with the highest frequency of distant dissemination.

Zinc-Doped Titanium Dioxide Microporous Coating Promotes Osteoblast Adhesion Through the Integrin β1/FAK/RhoA Signaling Pathway

Titanium and titanium alloys are widely used bone repair materials in clinical practice. However, titanium and titanium alloys are biologically inert and cannot induce bone formation, resulting in poor integration between titanium-based implants and the surrounding natural bone tissue. We used early-stage plasma oxidation to prepare a zinc doped TiO2 microporous structure (ZnTMS) coating on a titanium surface with good surface morphology. In vitro studies have shown that this coating can promote osteoblast proliferation and differentiation and has good biological activity. However, the specific molecular mechanism by which the ZnTMS coating affects cell regulation is still unclear. In this study, we aimed to further investigate the effect of the ZnTMS coating on the adhesion and extension of MC3T3-E1 cells through cellular and molecular biology experiments and to explore the molecular mechanism underlying the enhanced cell adhesion.We found that the ZnTMS coating can not only promote the adhesion and extension of MC3T3-E1 cells but can also promote the expression of actin and Vinculin. Further research showed that the ZnTMS coating can upregulate the protein expression of integrin β1, FAK, pFAK, and RhoA in MC3T3-E1 cells. Therefore, the integrin β1/FAK/RhoA signaling pathway may play important roles in the promotion of MC3T3-E1 cell adhesion by the ZnTMS coating.

Impact of β3-adrenergic receptor modulation on vascular function during experimental septic shock

Dysautonomia is an adverse event in septic shock (SC) associated with loss of cardiovascular variability. Modulation by β1-blockers is associated with cardiovascular benefits. Unlike β1-adrenergic (AR) receptors, β3-ARs are widely expressed on vascular level and, when stimulated, cause vasorelaxation. We hypothesize that during SC, vascular function could also be mediated by β3-ARs. The aim of the study is to assess the impact of β3-ARs modulation at the level of endothelial cells on vascular function during SC. We developed an inflammatory model using commercial human aortic endothelial cells (HAEC) stimulated with lipopolysaccharides (LPS, 10 μg/mL) to mimic SC conditions. These HAECs were treated 6 h for the transcriptional evaluations (PCR) and 24 h for the protein evaluations (WB). Then, the β3-AR was modulated with an agonist (CL316243, 1 μM) and antagonist (SR59230A, 1 μM) and its impact on different markers was assessed as inflammatory markers (VCAM-1, ICAM-1), expression of other β-ARs, NO synthases (eNOS, iNOS) and cell permeability. Finally, the expression of β3-AR was evaluated using murine vascular tissue samples (thoracic aorta and mesenteric arteries) lysed for PCR and WB detection. The expression of endothelial surface inflammatory markers (VCAM-1, ICAM-1) and the activation of NFκB were increased in LPS condition, validating the inflammatory model. Additionally, LPS induced a decreased transcriptional expression of β1 and 2-AR. In LPS condition, eNOS was decreased, while iNOS as well as cell permeability were increased. However, β3-AR expression was not demonstrated on HEACs, and the addition of agonist or antagonist had not induced any change on the above mentioned markers. Although, we demonstrated the expression of β3-AR in mouse aortic and mesenteric vessels at the genetic and protein level, allowing the study of the modulation of this receptor in vivo. The concept of decatecholamines emerged in order to minimize the use of catecholamines in SC management. Blockade of β-AR is a therapeutic approach aimed at downregulating adrenergic stimulation during SC. While β1-ARs have been widely studied, there are no data on β3-ARs, so characterization of the β3-AR is required and could play a significant role in septic vascular dysfunction.

Impact of IH-induced sympathetic hyperactivity and metabolic dysfunctions on ischemic cardiomyopathy progression

Patients with obstructive sleep apnea (OSA) exhibit poor prognosis after myocardial infarction (MI). Intermittent hypoxia (IH), the hallmark feature of OSA, promotes sympathetic hyperactivity and systemic insulin resistance, and has been identified as a major contributor to post-MI cardiac remodeling and contractile dysfunction. We hypothesize that sympathetic hyperactivity and metabolic alterations induced by IH promote desensitization of cardiac adrenergic and insulin pathways via activation of G protein-coupled receptors (GRK2), and subsequently participate in the aggravation of ischemic cardiomyopathy. MI is induced in C57bl6 mice by permanent ligation of the left coronary artery (MI). Mice are then randomized to IH (21–5% FiO2, 60 s cycle, 8 h/day) or normoxia for up to 6 weeks. After two weeks, mice are treated with a GRK2 inhibitor, Paroxetine (5 mg/kg/d), or 25% DMSO (Alzet® pumps). Longitudinal follow-up of mice includes evaluation of sympathetic activity (spectral analysis of heart rate variability (HRV), systemic insulin sensitivity (dynamic insulin tolerance test) and determination of cardiac function/remodeling (echocardiography). At the end of the protocol, cardiac interstitial fibrosis and hypertrophy are evaluated by RT-qPCR and histology (Sirius Red and WGA staining, respectively). Assessment of insulin signaling pathway is performed by Western blot. IH induces sympathetic hyperactivity in MI mice, demonstrated by a modification of the sympathovagal balance (LF increase). Paroxetine treatment abolishes the increase in LF in MI-IH condition and restores mRNA expression of β1 and β2 adrenergic receptors. MI-IH induces systemic insulin resistance compared to MI-N condition, which is prevented by Paroxetine treatment. In addition, Paroxetine treatment improves insulin signaling in the liver of MI-IH mice (pIRβ and pAKT). IH exacerbates cardiac interstitial fibrosis and worsens contractile dysfunction in MI mice, resulting in a significant decrease in ejection fraction (19.4 ± 2.1% vs. 29.9 ± 3.6% in IH vs. N respectively, P < 0.05). The effect of paroxetine on cardiac remodeling and function is currently being analyzed. These findings demonstrate that IH induces cardiac sympathetic hyperactivity and insulin resistance, which could contribute to the worsening of ischemic heart disease via GRK2.

Poly-D,L-Lactic Acid Stimulates Angiogenesis and Collagen Synthesis in Aged Animal Skin.

Angiogenesis promotes rejuvenation in multiple organs, including the skin. Heat shock protein 90 (HSP90), hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF) are proangiogenic factors that stimulate the activities of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase 1/2 (ERK1/2). Poly-D,L-lactic acid (PDLLA), polynucleotide (PN), and calcium hydroxyapatite (CaHA) are dermal fillers that stimulate the synthesis of dermal collagen. However, it is not yet known whether these compounds promote angiogenesis, which leads to skin rejuvenation. Here, we evaluated whether PDLLA, PN, and CaHA stimulate angiogenesis and skin rejuvenation using H2O2-treated senescent macrophages and endothelial cells as an in vitro model for skin aging, and we used young and aged C57BL/6 mice as an in vivo model. Angiogenesis was evaluated via endothelial cell migration length, proliferation, and tube formation after conditioned media (CM) from senescent macrophages was treated with PDLLA, PN, or CaHA. Western blot showed decreased expression levels of HSP90, HIF-1α, and VEGF in senescent macrophages, but higher expression levels of these factors were found after treatment with PDLLA, PN, or CaHA. In addition, after exposure to CM from senescent macrophages treated with PDLLA, PN, or CaHA, senescent endothelial cells expressed higher levels of VEGF receptor 2 (VEGFR2), PI3K, phosphorylated AKT (pAKT), and phosphorylated ERK1/2 (pERK1/2) and demonstrated greater capacities for cell migration, cell proliferation, and tube formation. Based on the levels of 4-hydroxy-2-nonenal, the oxidative stress level was lower in the skin of aged mice injected with PDLLA, PN, or CaHA, while the tumor growth factor (TGF)-β1, TGF-β2, and TGF-β3 expression levels; the density of collagen fibers; and the skin elasticity were higher in the skin of aged mice injected with PDLLA, PN, or CaHA. These effects were greater in PDLLA than in PN or CaHA. In conclusion, our results are consistent with the hypothesis that PDLLA stimulates angiogenesis, leading to the rejuvenation of aged skin. Our study is the first to show that PDLLA, PN, or CaHA can result in angiogenesis in the aged skin, possibly by increasing the levels of HSP90, HIF-1α, and VEGF and increasing collagen synthesis.

Deletion of Cd44 Inhibits Metastasis Formation of Liver Cancer in Nf2-Mutant Mice.

Primary liver cancer is the third leading cause of cancer-related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-like protein) encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be crucial for its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (cluster of differentiation 44). Mutations within the CD44-binding domain of Merlin have been reported in many human cancers. This study evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with a liver-specific deletion of the Nf2 gene were crossed with Cd44-knockout mice and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and analyzed by in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs), as well as mixed hepatocellular cholangiocarcinomas. Whilst deletion of Cd44 had no influence on liver size or primary liver tumor development, it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin β2 and promotes transendothelial migration of liver cancer cells, which may facilitate metastatic spreading. Overall, our results suggest that CD44 may be a promising target for intervening with metastatic spreading of liver cancer.

The Effect of Titanium Surface Topography on Adherent Macrophage Integrin and Cytokine Expression

Immunomodulatory biomaterials have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling, as opposed to persistent inflammation and scar tissue formation. This study examined the effects of titanium surface modification on integrin expression and concurrent cytokine secretion by adherent macrophages in vitro in an attempt to delineate the molecular events involved in biomaterial-mediated immunomodulation. Non-polarised (M0) and inflammatory polarised (M1) macrophages were cultured on a relatively smooth (machined) titanium surface and two proprietary modified rough titanium surfaces (blasted and fluoride-modified) for 24 h. The physiochemical characteristics of the titanium surfaces were assessed by microscopy and profilometry, while macrophage integrin expression and cytokine secretion were determined using PCR and ELISA, respectively. After 24 h adhesion onto titanium, integrin α1 expression was downregulated in both M0 and M1 cells on all titanium surfaces. Expression of integrins α2, αM, β1 and β2 increased in M0 cells cultured on the machined surface only, whereas in M1 cells, expression of integrins α2, αM and β1 all increased with culture on both the machined and rough titanium surfaces. These results correlated with a cytokine secretory response whereby levels of IL-1β, IL-31 and TNF-α increased significantly in M1 cells cultured on the titanium surfaces. These results show that adherent inflammatory macrophages interact with titanium in a surface-dependent manner such that increased levels of inflammatory cytokines IL-1β, TNF-α and IL-31 secreted by M1 cells were associated with higher expression of integrins α2, αM and β1.

Detrimental effects of vitrification on integrin genes (α9 and β1) and in vitro fertilization in mouse oocytes.

Integrins are known as key molecules that importantly involve in fertilization. This study aimed to evaluate effects of vitrification on fertilization rate and expression of integrin genes, α9 and β1, on mice oocytes in GV and MІІ stages. From the ovarian tissue and fallopian tube of NMRI mice, germinal vesicle (GV, n = 200) and metaphase II (MII, n = 200) oocytes were obtained. Then, oocytes were distributed into 4 groups including non-vitrified GV, non-vitrified MII, vitrified GV, and vitrified MII. Cryotop method was used for vitrification and oocytes (for 4 weeks) were kept in liquid nitrogen. After that, by using an inverted microscope, the rate of survived oocytes was assessed. Also, in vitro fertilization (IVF) for oocytes, obtained from in vitro maturated MII and mice ovaries (ovulated MII), was done to assess embryos at differenced stages (2-cells, morula, and hatched). Finally, RT-qPCR was performed to investigate the mRNA expression of integrin genes (α9 and β1). After vitrification, the rate of survived oocytes, 68.65%for GV and 65.07% % for MII, did not show a remarkable difference related to non-vitrified groups, while the fertilization rate in vitrified groups remarkably decrease compared to non-vitrified groups (p < 0.05). Also, the expression of α9 and β1 genes was significantly altered in vitrified groups when compared to non-vitrified groups (p < 0.05). There was no significant difference in embryo developmental rates for non-vitrified and vitrified groups. Cryotop method for vitrification caused an alternation in oocyte quality by reducing fertilization rate and integrin gene expression.

The potential curative and hepatoprotective effects of platelet rich plasma on liver fibrosis in Schistosoma mansoni experimentally infected mice.

Trapped Schistosoma mansoni eggs trigger fibrotic liver disease that can continue to liver cirrhosis and failure. This work evaluates the outcome of platelet rich plasma (PRP) on S. mansoni-induced liver fibrosis by intraperitoneal (IP) and intrahepatic (IH) routes with/without Praziquantel (PZQ) treatment. Swiss albino mice (n = 162) were divided into non-infected (n = 66) and infected (n = 96) groups, then subdivided into non-treated and treated subgroups with PRP(IP), PRP(IH) 6th and 10th weeks post-infection, PZQ, PZQ + PRP(IP) and PZQ + PRP(IH) 6th and 10th weeks post-infection. Effects of treatments were evaluated by parasitological, histopathological and Immunohistochemical assessments. In the early assessment (12th week post-infection) of infected-treated groups, the mean granuloma number showed significant reduction in groups treated with PZQ + PRP (IH) 10th week, PRP (IP), PZQ + PRP (IP) and PZQ + PRP (IH) 6th week (33.33%, 33%, 27.77% and 27.22%, respectively). Furthermore, the mean granuloma diameter showed significant reduction in groups treated with PRP (IH) 10th week and PZQ + PRP (IP) (24.17% and 15.5%, respectively). Also, the fibrotic index showed significant reduction in groups treated with PZQ + PRP (IP), PRP (IP) and PZQ + PRP (IH) 6th week (48.18%, 46.81% and 41.36%, respectively). Transforming growth factor β1(TGF-β1) expression was in correlation with parasitological and histopathological results. Diminished TGF-β1 expression was mostly in infected groups treated with PZQ + PRP (IP), PZQ + PRP (IH) 6th week and PRP (IP) (88.63%, 88.63% and 77.27%, respectively). In the late assessment (14th week post-infection) of infected treated groups, TGF-β1expression was reduced in groups treated with PZQ, PRP (IH) 10th weeks, PRP (IP) (83.33%, 66.66%, 33.33% respectively). PRP showed promising anti-fibrotic effects on S. mansoni-induced liver fibrosis.

Integrative Analysis of the AMPK subunits in Colorectal Adeno Carcinoma.

The 5-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an emerging cancer treatment and therapeutic target. Due to the enzyme's complexity and dual nature, it is a confounding target in the treatment strategy. The study aimed to conduct an integrative analysis of the seven subunits and twelve isoforms of AMPK, which is not reported so far in colorectal adenocarcinoma patients. The web-based tools UALCAN, Timer 2.0, KM Plotter, cBioPortal, COSMIC, and STRING were used to investigate the differential expression of AMPK subunits, protein-level Expression, promoter methylation status, survival analyses, Enrichment analysis, and protein-protein interaction. The mRNA expression of AMPK subunits are upregulated in Colorectal Adeno Carcinoma (COAD), while the protein expression is comparatively reduced in colon tumors. The protein-level expression of α2 and β2 is decreased significantly in COAD patients. The γ3 subunit in colon tumor is hypermethylated. The study also reports that Liver Kinase B1 mutation in 7% of CRC patients, which might be the reason for downregulation of the gene and the protein expression of AMPK subunits in COAD. The Overall analysis of the subunits affirms that AMPK expression is beneficial in cancer.

Inhibition of Integrin αvβ3-FAK-MAPK signaling constrains the invasion of T-ALL cells

ABSTRACT The role of adhesion receptor integrin αvβ3 in T-ALL was unclear. Firstly, we performed quantitative real-time PCR to assess medullary expression of integrin β3(ITGB3) in T-ALL patients and high ITGB3 expression was relevant with the central nervous system leukemia(CNSL) incidence. Decreasing of cell invasion was observed in Jurkat and Molt4 treated with integrin αvβ3 specific antibody and inhibitor as well as cells with ITGB3 interference. Further, phosphorylation of FAK, cRAF, MEK and ERK decreased in cells with integrin αvβ3 inhibition or interference. Invasion decreased in T-ALL cells treated with FAK and ERK inhibitors. In conclusion, inhibition of integrin αvβ3 signals significantly limits the cell invasion of T-ALL cells.

Effect of acupuncture on the opening time of implantation window and endometrial receptivity in controlled ovarian hyperstimulation rats during peri-implantation period.

To investigate the effect of acupuncture for improving the pregnancy rate of COH rats from the viewpoint of regulating the opening time of the implantation window and endometrial receptivity. Experimental rats were randomly divided into normal group (N), model group (M) and acupuncture group(A), and samples were collected on Day 4, 5 and 6 after mating. COH rats were treated with acupuncture at SP6, LR3, and ST36 once a day for 7 times. The pinopodes were observed under a scanning electron microscope. Serum estrogen and progesterone levels were measured via ELISA. The protein and mRNA levels of estrogen receptor (ER), progesterone receptor (PR), leukemia inhibitory factor (LIF), integrin β3, vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2) in the endometrium were evaluated via West-blot, immunohistochemistry, and PCR. Compared with group N, the pregnancy rate of group M was significantly decreased (P<0.05), and the abnormal serum hormone levels and implantation window advancement were observed. Compared with group M, the pregnancy rate of group A was significantly increased (P<0.05), the supraphysiological serum progesterone levels were restored to normalcy (P<0.05), and the advanced implantation window was restored to a certain extent. Further, the abnormal ER, PR, LIF, integrin β3, VEGF, and FGF-2 expression levels of the endometrium got recovered to varying degrees. Acupuncture may restore the estrogen and progesterone balance in COH rats and the forward shift of the implantation window to a certain extent, improving the endometrial receptivity and finally improving the pregnancy rate of COH rats.

Anti-proliferation and anti-migration effects of Yishen Tongbi decoction in experimental rheumatoid arthritis by suppressing SLC3A2/integrin β3 signaling pathways

BackgroundYishen Tongbi (YSTB) decoction is a patented herbal formula that is used in China to treat rheumatoid arthritis (RA); however, the exact mechanism of its anti-synovial hyperplasia efficacy has not been fully elucidated. PurposeBased on our previous proteomics study, we aimed to reveal whether YSTB inhibits the proliferation and migration of RA-FLSs through the SLC3A2/integrin β3 pathway in vivo and in vitro. Study designThe study design consists of three parts, a comparison of the expression of SLC3A2 and integrin β3 in synovial tissues of RA and OA patients; an animal experiment to verify the pharmacodynamic effect of YSTB, and in vitro experiment to elucidate the specific mechanism of YSTB. MethodsThe expression of SLC3A2 and integrin β3 in the synovial tissues of patients with RA and osteoarthritis (OA) patients were detected by immunohistochemistry (IHC). In vitro, firstly, the proliferation and migration abilities of HFLS (human fibroblast-like synoviocytes) and HFLS-RA (human fibroblast-like synoviocytes-RA) cells were compared by EdU staining and wound healing assays, respectively, and the differences in the expression and localization of SLC3A2, integrin β3, p-FAK and p-Src between HFLS and HFLS-RA cells were detected by IF and WB. In vivo, DBA/1 mice were injected with bovine collagen II to construct a CIA mouse model. Paw swelling, body weight and the arthritis index (AI) were used as basic treatment evaluation indicators for YSTB. Micro-CT and histopathological analyses of the knee and ankle joints were also performed. In addition, the expression of SLC3A2, integrin β3, p-FAK and p-Src in the synovial tissue of mice was detected by IHC. Subsequently, CCK-8 was used to screen for suitable concentrations of YSTB for use in HFLS-RA cells. EdU staining and transwell migration assays were performed to evaluate the inhibitory effect of YSTB on cell proliferation and migration, and WB was conducted to assess whether YSTB inhibited HFLS-RA migration through downregulation of the SLC3A2/integrin β3 pathways. ResultsIHC showed that the expression of SLC3A2 and integrin β3 was higher in RA synovial tissues than in OA tissues. In vivo experiments showed that YSTB inhibited synovial hyperplasia, prevented bone destruction, and reduced the expression of SLC3A2, integrin β3, p-FAK and p-Src. In vitro experiments showed that YSTB inhibited HFLS-RA migration and proliferation by inhibiting the expression of SLC3A2/integrin β3 and downstream signaling molecules. ConclusionYSTB inhibits the proliferation and migration of synovial fibroblasts in RA by downregulating the SLC3A2/integrin β3 pathways.

Abstract P6-14-01: Invasively distinct subpopulations cooperate via a laminin-332/Rac1 axis in triple-negative breast cancer

Abstract Intratumoral heterogeneity poses a significant hurdle for cancer treatment, yet is under-characterized in the context of tumor invasion. Cancer cells from solid tumors can invade through two predominant modes: collective invasion, whereby cancer cells invade in multi-cellular packs or streams marked by intact cell-cell junctions; and single-cell invasion, whereby cells invade independently without intercellular adhesion. We have observed that collective and single-cell invasion co-occur within the same tumor microenvironment in triple-negative breast cancer, suggesting that invasive heterogeneity supports cooperative behavior between tumor subpopulations. To test this, we used a novel, published technique developed by the lab (SaGA) to isolate pure subpopulations of 4T1 cells that collectively invade (collectives) or single cells that invade alone (singles). 3-D spheroids of SaGA-purified collectives and singles exhibited almost exclusively collective and single-cell invasion, respectively, and these invasive phenotypes were retained over multiple passages. Integration of RNA sequencing and methylation array data obtained from RNA and DNA isolates, respectively, of collectives and singles revealed that collectives exhibit drastic overexpression and promoter hypomethylation of two laminin genes that form the laminin-332 complex, Lama3 and Lamc2. Additionally, an unbiased proteomic analysis of secreted proteins also revealed an overabundance of these laminins in collectives media. We found that singles have increased expression of integrin α6 and β4, which together have been found to specifically bind to laminin-332 to activate the Rac1 GTPase. Interestingly, our RNA sequencing data revealed a binary overexpression of a Rac1 GTPase, Prex1 in singles, suggesting that singles have enhanced Rac1 activation when compared to collectives with the potential for hyperactivation upon laminin-332 binding. Indeed, laminin-332 resulted in higher GTP-bound Rac1 in singles and subsequently increased invasion of singles in 3-D models. Additionally, laminin-332 induced cell elongation at the leading edge of singles spheroids, which was reversable by treatment with a Rac1 inhibitor. Together, our data suggests that distinct subpopulations amidst a heterogeneous tumor cooperate via laminin-332 and Rac1 to facilitate tumor invasion in metastatic triple-negative breast cancer. Citation Format: Sung Bo (Joseph) Yoon, Janna Mouw, Luxiao Chen, Hao Wu, Adam Marcus. Invasively distinct subpopulations cooperate via a laminin-332/Rac1 axis in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-01.

T-cadherin is a novel regulator of pericyte function during angiogenesis.

Pericytes are mural cells that play an important role in regulation of angiogenesis and endothelial function. Cadherins are a superfamily of adhesion molecules mediating Ca2+-dependent homophilic cell-cell interactions that control morphogenesis and tissue remodeling. To date, classical N-cadherin is the only cadherin described on pericytes. Here, we demonstrate that pericytes also express T-cadherin (H-cadherin, CDH13), an atypical glycosyl-phosphatidylinositol (GPI)-anchored member of the superfamily that has previously been implicated in regulation of neurite guidance, endothelial angiogenic behavior, and smooth muscle cell differentiation and progression of cardiovascular disease. The aim of the study was to investigate T-cadherin function in pericytes. Expression of T-cadherin in pericytes from different tissues was performed by immunofluorescence analysis. Using lentivirus-mediated gain-of-function and loss-of-function in cultured human pericytes, we demonstrate that T-cadherin regulates pericyte proliferation, migration, invasion, and interactions with endothelial cells during angiogenesis in vitro and in vivo. T-cadherin effects are associated with the reorganization of the cytoskeleton, modulation of cyclin D1, α-smooth muscle actin (αSMA), integrin β3, metalloprotease MMP1, and collagen expression levels, and involve Akt/GSK3β and ROCK intracellular signaling pathways. We also report the development of a novel multiwell 3-D microchannel slide for easy analysis of sprouting angiogenesis from a bioengineered microvessel in vitro. In conclusion, our data identify T-cadherin as a novel regulator of pericyte function and support that it is required for pericyte proliferation and invasion during active phase of angiogenesis, while T-cadherin loss shifts pericytes toward the myofibroblast state rendering them unable to control endothelial angiogenic behavior.

Smooth muscle αv integrins regulate vascular fibrosis via CD109 downregulation of TGF-β signalling.

αv integrins are implicated in fibrosis in a number of organs through their ability to activate TGF-β. However their role in vascular fibrosis and collagen accumulation is only partially understood. Here we have used αv conditional knockout mice and cell lines to determine how αv contributes to vascular smooth muscle cell (VSMC) function in vascular fibrosis and the role of TGF-β in that process. Angiotensin II (Ang II) treatment causes upregulation of αv and β3 expression in the vessel wall, associated with increased collagen deposition. We found that deletion of αv integrin subunit from VSMCs (αv SMKO) protected mice against angiotensin II-induced collagen production and assembly. Transcriptomic analysis of the vessel wall in αv SMKO mice and controls identified a significant reduction in expression of fibrosis and related genes in αv SMKO mice. In contrast, αv SMKO mice showed prolonged expression of CD109, which is known to affect TGF-β signalling. Using cultured mouse and human VSMCs, we showed that overexpression of CD109 phenocopied knockdown of αv integrin, attenuating collagen expression, TGF-β activation, and Smad2/3 signalling in response to angiotensin II or TGF-β stimulation. CD109 and TGF-β receptor were internalized in early endosomes. We identify a role for VSMC αv integrin in vascular fibrosis and show that αv acts in concert with CD109 to regulate TGF-β signalling.

The Association of Integrins β3, β4, and αVβ5 on Exosomes, CTCs and Tumor Cells with Localization of Distant Metastasis in Breast Cancer Patients.

Integrins are cell adhesion receptors, which play a role in breast cancer invasion, angiogenesis, and metastasis. Moreover, it has been shown that exosomal integrins provide organotropic metastasis in a mouse model. In our study, we aimed to investigate the expression of integrins β3, β4, and αVβ5 on exosomes and tumor cells (circulating tumor cells and primary tumor) and their association with the localization of distant metastasis. We confirmed the association of exosomal integrin β4 with lung metastasis in breast cancer patients. However, we were unable to evaluate the role of integrin β3 in brain metastasis due to the rarity of this localization. We established no association of exosomal integrin αVβ5 with liver metastasis in our cohort of breast cancer patients. The further evaluation of β3, β4, and αVβ5 integrin expression on CTCs revealed an association of integrin β4 and αVβ5 with liver, but not the lung metastases. Integrin β4 in the primary tumor was associated with liver metastasis. Furthermore, an in-depth analysis of phenotypic characteristics of β4+ tumor cells revealed a significantly increased proportion of E-cadherin+ and CD44+CD24- cells in patients with liver metastases compared to patients with lung or no distant metastases.

Low dose TGF-β1 can improve vohwinkel syndrome by promoting the proliferation of keratinocytes

Vohwinkel syndrome (VS) is a very rare autosomal dominant disorder that can cause disability and deformity in severe cases. Mutations of the LOR (loricrin) and GJB2 (Cx26) genes have been found in VS so far. Many studies have indicated that the differentiation and growth of epidermal keratinocytes are regulated by mutant Cx26, and it may explain the pathogenesis of VS. It has been found that transforming growth factor β1 (TGF-β1) expression was lower in G130V (OE1) and D66H (OE2) mutant keratinocytes in the VS model with GJB2 mutation as compared to normal keratinocytes (NC). TGF-β is a cytokine involved in the regulation of processes like cell proliferation and differentiation in different types of cells. At present, many in vitro studies focus on TGF- β 1 inhibition of keratinocyte growth.However, the relationship between TGF-β1 and VS remains unknown. This study aimed at elucidating the role and potential pathogenic mechanism of TGF-β in VS. The results indicated that the down-regulation expression of TGF-β1 in VS was linked to cell proliferation inhibition through p-Smad3/c-myc. In contrast, low-dose TGF-β1 treatment of VS keratinocytes can improve their proliferation inhibition and up-regulate the expression Cyclin D1. This suggests that low doses of TGF-β1 can improve the proliferation of VS and provide new insights into its treatment.

Differential Gene Expression Pattern of Importin β3 and NS5 in C6/36 Cells Acutely and Persistently Infected with Dengue Virus 2.

The establishment of persistent dengue virus infection within the cells of the mosquito vector is an essential requirement for viral transmission to a new human host. The mechanisms involved in the establishment and maintenance of persistent infection are not well understood, but it has been suggested that both viral and cellular factors might play an important role. In the present work, we evaluated differential gene expression in Aedes albopictus cells acutely (C6/36-HT) and persistently infected (C6-L) with Dengue virus 2 by cDNA-AFLP. We observed that importin β3 was upregulated in noninfected cells compared with C6-L cells. Using RT-qPCR and plaque assays, we observed that Dengue virus levels in C6-L cells essentially do not vary over time, and peak viral titers in acutely infected cells are observed at 72 and 120 h postinfection. The expression level of importin β3 was higher in acutely infected cells than in persistently infected cells; this correlates with higher levels of NS5 in the nucleus of the cell. The differential pattern of importin β3 expression between acute and persistent infection with Dengue virus 2 could be a mechanism to maintain viral infection over time, reducing the antiviral response of the cell and the viral replicative rate.