Loss of β<Subscript>1</Subscript>D-integrin function in human ischemic cardiomyopathy

Integrins play a pivotal role in cardiomyocyte survival and function, with integrin loss leading to myocyte apoptosis and heart failure. The aim of this study was to characterize whether regulation of integrins may contribute to cardiac remodeling in human ischemic cardiomyopathy (ICM). Myocardial tissues of the left ventricle were obtained from patients with ICM (n = 8) undergoing cardiac transplantation and from unused donor hearts (NF, n = 8). In addition, tissue samples from patients with dilated cardiomyopathy (DCM, n = 5) were analyzed. Expression of integrins β₁D and β₃, the effector proteins focal adhesion kinase (FAK) and melusin, and FAK phosphorylation were examined by Western blotting, real-time-PCR and immunofluorescence analysis, respectively. β₁D-integrin protein was decreased in ICM vs. NF by 36%. β₁D-integrin mRNA levels and β₁D-integrin shedding were unchanged. Corresponding to β₁D-integrin regulation, FAK and phosphorylated FAK were decreased in ICM vs. NF by 54% and 49%, respectively. β₃-integrin and melusin were not altered in ICM. As a mediator of integrin effects, AKT kinase activity was examined. In parallel to β₁D-integrin and FAK, AKT activity was decreased in ICM by 44%. In contrast, none of the proteins were significantly altered in DCM compared to NF. Integrins and integrin signaling are regulated differentially in ICM and DCM with a decrease of β₁D-integrin and FAK in ICM. The loss of the β₁Dintegrin-FAK-complex in ICM was paralleled by a reduced AKT activity supporting in vitro data which demonstrate the pivotal role of intact integrin function in anti-apoptotic signaling and cell survival.     

整合素家族与肿瘤骨转移相关性研究进展

整合素家族作为黏附分子,主要介导细胞与细胞外基质间的黏附,调节许多类型肿瘤细胞的迁移、存活、增殖和血管生成等。近期许多研究证实,整合素家族还和恶性肿瘤的骨转移有着密切的关系。本文就整合素家族结构与功能、整合素家族信号通路与肿瘤转移关系、整合素与肿瘤骨转移的形成等方面进行综述。     

Pathway inhibition: emerging molecular targets for treating glioblastoma

Insights into the molecular pathogenesis of glioblastoma have not yet resulted in relevant clinical improvement. With standard therapy, which consists of surgical resection with concomitant temozolomide in addition to radiotherapy followed by adjuvant temozolomide, the median duration of survival is 12-14 months. Therefore, the identification of novel molecular targets and inhibitory agents has become a focus of research for glioblastoma treatment. Recent results of bevacizumab may represent a proof of principle that treatment with targeted agents can result in clinical benefits for patients with glioblastoma. This review discusses limitations in the existing therapy for glioblastoma and provides an overview of current efforts to identify molecular targets using large-scale screening of glioblastoma cell lines and tumor samples. We discuss preclinical and clinical data for several novel molecular targets, including growth factor receptors, phosphatidylinositol-3 kinase, SRC-family kinases, integrins, and CD95 ligand and agents that inhibit these targets, including erlotinib, enzastaurin, dasatinib, sorafenib, cilengitide, AMG102, and APG101. By combining advances in tumor screening with novel targeted therapies, it is hoped that new treatment options will emerge for this challenging tumor type.     

Structure–activity relationship of human bone sialoprotein peptides

In the current study, the relationship between the structure of the RGD‐containing human bone sialoprotein (hBSP) peptide 278–293 and its attachment activity toward osteoblast‐like (MC3T3) cells was investigated. This goal was accomplished by examining the comparative cell‐attachment activities of several truncated forms of peptide 278–293. Computer modeling of the various peptides was also performed to assess the role of secondary structure in peptide bioactivity. Elimination of tyrosine‐278 at the N‐terminus resulted in a more dramatic loss of cell‐attachment activity compared with the removal of either tyrosine‐293 or the arg‐ala‐tyr (291–293) tripeptide. Although replacement of the RGD (arg‐gly‐asp) peptide moiety with peptide KAE (lys‐ala‐glu) resulted in a dramatic loss of cell‐attachment activity, a peptide containing RGE (arg‐gly‐glu) in place of RGD retained 70–85% of the parental peptide's attachment activity. These results suggest that the N‐terminal RGD‐flanking region of hBSP peptide 278–293, in particular the tyrosine‐278 residue, represents a second cell‐attachment site that stabilizes the RGD–integrin receptor complex. Computer modeling also suggested that a β‐turn encompassing RGD or RGE in some of the hBSP peptides may facilitate its binding to integrins by increasing the exposure of the tripeptide. This knowledge may be useful in the future design of biomimetic peptides which are more effective in promoting the attachment of osteogenic cells to implant surfaces in vivo.     

Alterations of neutrophil L-selection and CD18 expression by tobacco smoke: implications for periodontal diseases

Alterations in neutrophil functions by both chronic low levels of tobacco and by acute short-term higher levels of tobacco smoke, as encountered during the act of smoking, may play a role in the pathogenesis of periodontal diseases in smokers. Among the early migration events of neutrophil function is the alteration in surface expression of L-selectin and the CD11/18 integrins. In the present study we examined the effect of in vitro smoke exposure and nicotine alone on the expression of these 2 adhesion molecules in neutrophils from smokers and non-smokers. We also determined the physiological relevance of this in vitro system by assessing the levels of nicotine exposure in this in vitro system and comparing these levels to acute and chronic levels of nicotine in saliva and gingival crevicular fluid. Peripheral neutrophils were isolated from the blood of smokers (> 1 pack/d) and non-smokers and incubated in vitro with either cigarette smoke (0–5 min), 10^?7 M F-met-leu-phe, or nicotine alone at 1.62mg/ml to 162ng/ml (10^?2 M-10^?6 M). The neutrophils were then incubated with fluoresceine conjugated anti-Leu8 (L-selectin), anti-CD18 (CD18 integrin), or γ-4 (non-specific control), fixed and analyzed by flow cytometry. With cigarette smoke exposure, there was an approximate 75% shedding of L-selectin in both smokers and non-smokers with no marked difference between groups at 1–5 min of smoke exposure. Cigarette smoke exposure resulted in a 15–20% increase in CD 18 expression in both smokers and non-smokers. At all time points, there was slightly greater but statistically insignificant expression of CD18 integrin in non-smokers when compared to smokers. These patterns of CD18 increases and L-selectin shedding were similar in magnitude to incubations with 10^?7 M F-met-leu-phe. Acute smoke exposure resulted in elevation of nicotine in the smoke box to 529 ng/ml at 5 min, in saliva from 109.2 ng/ml before smoking to 1821.4 ng/ml after smoking, and in gingival crevicular fluid to 5961 ng/ml after smoking. No significant alterations in L-selectin or CD 18 expression were noted with in vitro nicotine from 1.62 mg/ml to 162 ng/ml.     

Adhesion Molecules Involved in Macrophage Responses to Wallerian Degeneration in the Murine Peripheral Nervous System

When a peripheral nerve is damaged the severed axon undergoes Wallerian degeneration. The distal nerve is infiltrated by large numbers of monocyte-derived macrophages which participate in the phagocytosis of degenerating myelin. In other tissues, adhesion molecules play a crucial role in leukocyte recruitment during inflammation. Blood-borne cells enter damaged tissue by interacting with adhesion molecules expressed on activated endothelium. Having crossed the endothelium, leukocytes must adhere and migrate within the tissue. We investigated the adhesion molecules involved in both stages of the macrophage response to transection of one sciatic nerve of BALB/c mice. By injecting monoclonal antibodies in vivo, before and after peripheral nerve injury, we showed that intercellular adhesion molecule-1 (ICAM-1) and integrins α₄β₁ (VLA-4) and α_Mβ₂ (type 3 complement receptor) are unlikely to be involved in the transendothelial migration of monocytes responding to peripheral nerve degeneration. We also studied the adhesion of macrophages within the endoneurium, using an in vitro adhesion assay. Macrophages showed much greater levels of adhesion to cryostat sections of transected nerves than to control nerves. This increased adhesion was partially inhibited by antibodies to the β1 -integrin chain, and more strongly inhibited by the extracellular matrix molecules fibronectin and collagen. Adhesion was unaffected by laminin-I and by antibodies to other adhesion molecules, including α₄β₁- and α₅β₁-integrins. Thus we conclude that monocyte entry into a degenerating peripheral nerve is independent of α_Lβ₂/α_Mβ₂-ICAM-1 or α₄β₁/NCAM-1 interactions, and that adhesion within the endoneurium is mediated in part by a β1 -integrin other than α₄β₁ or α₅β₁.     

Immunolocalization of fibronectin and vitronectin receptors in human gingival fibroblasts spreading on titanium surfaces

The adhesion and spreading of human gingival fibroblasls on glass and differently processed titanium surfaces was studied by immunolocalization of vinculin and the alpha and beta subunits of the fibronectin (α₅β₁) and vitronectin (α_vβ3) receptors. Vinculin-containing focal contacts were present both at 4 and 24 h of spreading in cells grown on glass or electropolished or etched titanium surfaces but not in cells spreading on sandblasted titanium surfaces. Immunostaining for the α5 and β1 subunits of the fibronectin receptor showed only a diffuse membrane fluorescence after 4 h of cell spreading irrespective of the growth surface. The α_v and β₃ subunits of the vitronectin receptor were at this stage detected in focal contacts in cells spreading on glass or electropolished or etched titanium surfaces. In cells spreading on sandblasted titanium surfaces, however, the vitronectin receptor had only a diffuse distribution. In cells that had been allowed to spread for 24 h on glass or electropolished or etched titanium surfaces the α₅ and β₁ integrin subunits were either diffusely distributed or showed a localization typical of extracellular matrix contacts. The α_v and β₃ integrin subunits were, as earlier, localized to typical focal contacts in cells grown on glass or electropolished or etched titanium surfaces. Cells attached to sandblasted titanium surfaces still expressed all the integrin subunits only diffusely. The results show that the surface texture of the substratum can affect the expression of integrin subunits in human gingival fibroblasts. As evidenced by the recruitment of integrin subunits to focal and extracellular matrix contacts, smooth or finely grooved titanium surfaces appear to be optimal in supporting the attachment of human gingival fibroblasts.     

Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433-1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150-157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD.     

Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34⁺ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA-2), fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) during the maturation of Mks. We have shown that a significant percentage of CFU-MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM-CD34⁺ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA-4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA-4 may be involved in the adhesion of CFU-MK and immature Mks on fibronectin, then replaced by VLA-5 in the final stages of maturation. The expression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.