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PubMed1999

Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion.

The expression pattern of laminin (Ln) alpha1 chain has been a controversial topic due to discrepancies between mRNA and protein studies. Recently it was reported that the monoclonal antibody 4C7, previously thought to recognize Ln alpha1 chain, actually detects Ln alpha5 chain. This finding makes it necessary to reestimate the role of Ln alpha1 chain and to compare the expression and functions of Ln alpha1 and alpha5 chains. We studied the expression of Ln alpha1 and alpha5 chains and production of Ln-1 and Ln-10 in cultured human carcinoma cells. Ln alpha1 chain mRNA was detected in JAR choriocarcinoma cells and in all four renal cell carcinoma cell lines studied. In contrast, pancreatic, colon, and lung alveolar carcinoma cell lines did not express or produce Ln alpha1 chain, suggesting that Ln-1 (alpha1 beta1 gamma1) is produced only by certain carcinoma cells. Ln alpha5 chain mRNA was expressed in all carcinoma cells, but was not incorporated into extracellular matrix in vitro, as shown with JAR cells. Immunoprecipitation of metabolically labeled cells showed that cells expressing Ln alpha1 mRNA also produced 400-kDa Ln alpha1 chain, whereas all cells produced 380-kDa Ln alpha5 chain. Adhesion to Ln-1 was inhibited by a functionally blocking antibody against alpha6-integrin subunit, whereas adhesion to Ln-10 was inhibited by an antibody against alpha6-integrin in JAR cells and by an antibody against alpha3-integrin in PANC-1 cells. The results suggest that Ln-10 is a ubiquitously expressed Ln isoform in carcinoma cells, and the mechanism of adhesion to Ln-10 is cell-type specific.

Blotting, Northern↗
PubMed2002

The chain register in heterotrimeric collagen peptides affects triple helix stability and folding kinetics.

Collagen type IV is a highly specialized form of collagen found only in basement membranes, where it provides mechanical stability and structural integrity to tissues and organs, and binding sites for cell adhesion. In its ubiquitous form, collagen type IV consists of two alpha1 chains and one alpha2 chain, whose internal alignment within the triple helix seems to exert a strong influence on the binding affinity to alpha1beta1 integrin receptor. This has been assessed recently using two synthetic collagen peptides that contain the cell adhesion epitope of collagen type IV and are assembled into the most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. In the present study, the effects of the chain register on the stability of the triple helix and the folding kinetics of these collagen peptides were investigated by CD spectroscopy and microcalorimetry. The results revealed a multi-domain structural organization for both trimers, with an unexpected strong effect of the chain alignment on the conformational stability. Molecular dynamics simulations served to rationalize more properly the experimental results.

Binding Sites↗
PubMed2004

Induction of anoikis by doxazosin in prostate cancer cells is associated with activation of caspase-3 and a reduction of focal adhesion kinase.

The quinazoline family of alpha1-blockers (prazosin, doxazosin, and terazosin) induce apoptosis of prostate cells through an alpha1-adrenoceptor-independent mechanism. The objective of this study was to gain insight into the non-adrenergic, apoptotic mechanism of action of doxazosin in the prostate and the induction of anoikis by doxazosin. Primary cultures of benign prostate stromal and epithelial cells and the LNCaP (androgen sensitive) and PC-3 (androgen insensitive) prostate carcinoma cell lines were treated with doxazosin (0-50 microM). The effects of doxazosin on cell morphology, caspase-3 activity, and the expression levels of focal adhesion kinase (FAK) and integrin-linked kinase (ILK) were examined. Doxazosin induced changes in morphology consistent with anoikis in both benign and cancerous prostatic cells and increased caspase-3 activity. The effects were similar comparing benign cells (which express alpha1-adrenoceptors) and cancer cells (which do not express alpha1-adrenoceptors), but were more robust in benign cells. Norepinephrine had no effect on doxazosin-induced cell morphology or caspase-3 activity. Treatment of PC-3 cells with doxazosin significantly reduced the protein levels of FAK but did not significantly affect the levels of ILK. These findings suggest that doxazosin induces apoptosis and anoikis of prostate cancer cells by a mechanism of action that is alpha1-adrenoceptor independent. The apoptosis of cancer cells induced by doxazosin counteracts cell proliferation and may have the potential of retarding or reversing prostate cancer cell growth.

Anoikis↗
PubMed2003

Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and -9.

The role of proteases in the tumor cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of extracellular matrix (ECM) components. Differentiating between the up-regulation of MMP production and the presence of activated MMPs can be difficult but may well dictate which MMPs are critical to invasion. Because the hydrolysis of collagens is one of the committed steps in ECM turnover, we have investigated selective MMP action on collagenous substrates as a means to evaluate active MMPs. Two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen, alpha1(V)436-450 THP and alpha1(V)436-447 fTHP, were hydrolyzed by MMP-2 and MMP-9 at the Gly-Val bond, analogous to the bond cleaved by MMP-9 in the corresponding native collagen. Kinetic analyses showed k(cat)/K(m) values of 14,002 and 5,449 s(-1)m(-1) for MMP-2 and -9 hydrolysis of alpha1(V)436-447 fTHP, respectively. These values, along with individual k(cat) and K(m) values, are comparable with collagen hydrolysis by MMP-2 and -9. Neither THP was hydrolyzed by MMP-1, -3, -13, or -14. alpha1(V)436-447 fTHP and a general fluorogenic THP were used to screen for triple-helical peptidase activity in alpha(2)beta(1) integrin-stimulated melanoma cells. Binding of the alpha(2)beta(1) integrin resulted in the production of substantial triple-helical peptidase activity, the majority (>95%) of which was non-MMP-2/-9. THPs were found to provide highly selective substrates for members of the MMP family and can be used to evaluate active MMP production in cellular systems.

Circular Dichroism↗
PubMed1998

Role of laminin-1, collagen IV, and an autocrine factor(s) in regulated secretion by lacrimal acinar cells.

Adhesion to novel basement membrane component BM180 in the presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 (Cell Physiol. 39): C1743-C1750, 1996]. The identity of the active laminin-1 site and the possibility that other promoters of coupling are present in the acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the beta1 and gamma1 chains, but not alpha1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the beta1-integrin subunit, and several growth factors. Increasing cell number in serum-free medium revealed an unknown, serum-maskable, secretion-enhancing activity with a remarkable specificity for regulated secretion. Stimulus-secretion coupling, therefore, appears to be modulated by several extracellular factors whose relative contributions remain to be determined.

Animals↗
PubMed2006

How the headpiece hinge angle is opened: New insights into the dynamics of integrin activation.

How the integrin head transitions to the high-affinity conformation is debated. Although experiments link activation with the opening of the hinge angle between the betaA and hybrid domains in the ligand-binding headpiece, this hinge is closed in the liganded alpha(v)beta3 integrin crystal structure. We replaced the RGD peptide ligand of this structure with the 10th type III fibronectin module (FnIII10) and discovered through molecular dynamics (MD) equilibrations that when the conformational constraints of the leg domains are lifted, the betaA/hybrid hinge opens spontaneously. Together with additional equilibrations on the same nanosecond timescale in which small structural variations impeded hinge-angle opening, these simulations allowed us to identify the allosteric pathway along which ligand-induced strain propagates via elastic distortions of the alpha1 helix to the betaA/hybrid domain hinge. Finally, we show with steered MD how force accelerates hinge-angle opening along the same allosteric pathway. Together with available experimental data, these predictions provide a novel framework for understanding integrin activation.

Allosteric Regulation↗
PubMed2001

The role of the extracellular matrix in articular chondrocyte regulation.

The in vivo role of the extracellular matrix and the manner in which it interfaces with soluble regulators remains largely unknown. The current study reports the extracellular Type II collagen modulation of transforming growth factor-beta 1-stimulated proliferation, proteoglycan synthesis, messenger ribonucleic acid expression for transforming growth factor-beta 1, and integrin messenger ribonucleic acid expression in articular chondrocytes from adults. This study shows that this cytokine modulation occurs through a mechanism initiated by the attachment of Type II collagen to the beta1-integrin. Transforming growth factor-beta 1 stimulated deoxyribonucleic acid and proteoglycan synthesis in a bimodal fashion. Extracellular Type II collagen increased transforming growth factor-beta 1-stimulated deoxyribonucleic acid and proteoglycan synthesis, aggrecan gene expression as much as 400%, and alpha1(II) procollagen gene expression as much as 180% in a dose-dependent fashion. Heat inactivation of the Type II collagen abrogated the observed effects on deoxyribonucleic acid and proteoglycan synthesis. In contrast to Type II collagen, heat-denatured collagen and bovine serum albumin showed none of the observed effects. The presence of Type II collagen in the alginate bead cultures was found to diminish the messenger ribonucleic acid expression for alpha2 integrin and alter the cellular distribution pattern of the beta1 integrin receptors. Blocking of the beta1-integrin with cyclic-peptides containing the Arg-Gly-Asp sequences and antibodies reduced chondrocyte attachment to Type II collagen by 93%. The physiologic effects shown by the chondrocyte as a result of blocking this attachment to Type II collagen were a significant reduction in transforming growth factor-beta 1-stimulated deoxyribonucleic acid and proteoglycan synthesis. The conclusions elucidate the role played by the extracellular matrix in cytokine-specific regulation of the articular chondrocyte. The authors have shown that extracellular Type II collagen acts through a beta1-integrin mediated mechanism to modulate the chondrocyte response to transforming growth factor-beta 1.

Aggrecans↗
PubMed1999

Functional expression of beta1 and beta2 integrins on tumor infiltrating lymphocytes (TILs) in colorectal cancer.

Integrins play an important role in various lymphocyte functions. In this study, tumor-infiltrating lymphocytes (TIL) were isolated from colorectal cancer tissues and the expression of beta1 and beta2 integrins on the TIL was quantitatively examined with two-color flow cytometry. In comparison with peripheral blood lymphocytes (PBL), TIL expressed a lower level of common beta1 chain (CD29) in both CD4 and CD8 subpopulations. Among the associated alpha chains, the expressions of alpha1 (CD49a) and alpha2 (CD49b) were slightly higher in TIL than in PBL, whereas alpha4 (CD49d) and alpha6 (CD49f) were markedly downregulated in TIL. Both alphaL (CD11a) and beta2 (CD18) were reduced in CD8(+) TIL but not in CD4(+) TIL. TIL with the CD8(+) cytotoxic phenotype showed significantly decreased binding to purified intracellular adhesion molecules (ICAM)-1, and vascular adhesion cell molecule (VCAM)-1, and HT29 colon cancer cells, compared with the in counterparts in PBL. The peculiar expression pattern and functional down regulation of these integrins may explain why TIL in colorectal cancer cannot eradicate the malignant cells.

Aged↗
PubMed2004

Regulation of gene expression by alpha-tocopherol.

Several genes are regulated by tocopherols which can be categorized, based on their function, into five groups: genes that are involved in the uptake and degradation of tocopherols (Group 1) include alpha-tocopherol transfer protein (alpha-TTP) and cytochrome P450 (CYP3A); genes that are associated with lipid uptake and atherosclerosis (Group 2) include CD36, SR-BI and SR-AI/II. Genes that modulate the expression of extracellular proteins (Group 3) include tropomyosin, collagen(alpha1), MMP-1, MMP-19 and connective tissue growth factor (CTGF). Genes that are related to inflammation, cell adhesion and platelet aggregation (Group 4) include E-selectin, ICAM-1, integrins, glycoprotein IIb, II-2, IL-4 and IL-beta. Group 5 comprises genes coding for proteins involved in cell signaling and cell cycle regulation and consists of PPAR-gamma, cyclin D1, cyclin E, Bcl2-L1, p27 and CD95 (Apo-1/Fas ligand). The expression of P27, Bcl2, alpha-TTP, CYP3A, tropomyosin, II-2, PPAR-gamma, and CTGF appears to be up-regulated by one or more tocopherols whereas all other listed genes are down-regulated. Several mechanisms may underlie tocopherol-dependent gene regulation. In some cases protein kinase C has been implicated due to its deactivation by alpha-tocopherol and its participation in the regulation of a number of transcription factors (NF-kappaB, AP-1). In other cases a direct involvement of PXR/RXR has been documented. The antioxidant responsive element (ARE) appears in some cases to be involved as well as the transforming growth factor beta responsive element (TGF-beta-RE). This heterogeneity of mediators of tocopherol action suggests the need of a common element that could be a receptor or a co-receptor, able to interact with tocopherol and with transcription factors directed toward specific regions of promoter sequences of sensitive genes. Here we review recent results of the search for molecular mechanisms underpinning the central signaling mechanism.

Animals↗
PubMed1997

Integrin up-regulation as marker of neuroblastoma cell differentiation: correlation with neurite extension.

We have characterized the adhesion properties, integrin expression, and morphological changes due to extracellular matrix (ECM)-integrin interactions in a neuronal model. We showed that a modulation of some integrin heterodimers occurs during interferon-gamma (IFN-gamma) induced neuroblastoma (NB) cell differentiation. To better elucidate the possible implication and function of integrin receptors during neuronal maturation, we analyzed the changes in integrin expression in two human NB cell lines, LAN-5 and GI-LI-N, which represent different stages of neuronal differentiation. These models show opposite morphological maturation after interferon-gamma and tumor necrosis factor-alpha (IFN-gamma+TNF) treatment. While LAN-5 cells acquired the ability to extend long and branched neurites, GI-LI-N cells did not. Both cell lines showed enhanced expression of phenotypical and biochemical markers of neural maturation. Moreover, retinoic acid (RA) had different effects on the two NB cell lines: on LAN-5 cells it acts as a differentiation-promoting agent, while on GI-LI-N cells it has an antiproliferative effect, driving them to apoptosis. RT-PCR experiments and immunoprecipitation assays showed a late but marked increase in the expression of alpha1, alpha2, alpha3, and beta1 chains after IFN-gamma+TNF treatment of LAN-5 cells, and only alpha1 and beta1 chains upon RA induction. Treatment with IFN-gamma+TNF induced GI-LI-N cells to show only a late and remarkable increase of alpha1/beta1 heterodimer; on the contrary, RA treatment caused a decrease in all integrin chains. These changes are accompanied in differentiated cells by substantial increases in cell attachment to all purified ECM components tested and an increase of neurite-bearing cells and of average neurite length. In conclusion, these findings indicate a close correlation between up-regulation of integrins and neuronal morphogenesis.

Journal Article↗
PubMed2007

Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans.

Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proalpha1(I) and proalpha2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype-phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in alpha1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691-823 and 910-964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril-matrix interactions. Recurrences at the same site in alpha2(I) are generally concordant for outcome, unlike alpha1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In alpha2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype-phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events.

Binding Sites↗
PubMed2003

Analysis of retinal pigment epithelium integrin expression and adhesion to aged submacular human Bruch's membrane.

PURPOSE: Uncultured aged retinal pigment epithelium (RPE) does not resurface aged Bruch's membrane after 24 hours in organ culture. These experiments assess whether culturing alters RPE integrin expression and resurfacing of Bruch's membrane. METHODS: RNA was isolated from uncultured and cultured RPE of aged adult donor and fetal eyes. Integrin subunit messenger RNA (mRNA) expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and semiquantitative analysis of the amplified products. Cell surface integrin expression was assessed using flow cytometry. Passaged cultured fetal RPE and primary cultured aged RPE were seeded onto Bruch's membrane, and resurfacing was assessed with scanning electron microscopy. RESULTS: Uncultured fetal RPE had low levels of alpha3 and beta5 mRNA compared to passaged cultured fetal RPE. Uncultured aged RPE had decreased alpha1-5 mRNA compared to primary cultured aged RPE. Cultured aged RPE had decreased beta4 and beta5 mRNA compared to passaged cultured fetal RPE. Flow cytometry confirmed the expression of alpha1-5, alphav, and beta1 protein on cultured fetal RPE and alpha1-3 and beta1 protein on cultured aged RPE. Twenty-four hours after seeding, cultured fetal and aged RPE resurfaced 99% +/- 1.3% and 76% +/- 22%, respectively, of aged submacular Bruch's membrane specimens from which native RPE had been debrided, exposing the native RPE basement membrane. Cultured fetal and aged RPE resurfaced 97% +/- 3.1% and 39% +/- 35%, respectively, of specimens in which the inner collagenous layer was exposed. CONCLUSIONS: Uncultured aged RPE has low amounts of integrin subunits that form receptors for laminin, fibronectin, and collagens. Culturing up-regulates integrins and promotes more efficient aged RPE attachment to and survival on aged Bruch's membrane.

Aged↗
PubMed1999

Integrin subunit gene expression is regionally differentiated in adult brain.

Integrins are a diverse family of heterodimeric (alphabeta) adhesion receptors recently shown to be concentrated within synapses and involved in the consolidation of long-term potentiation. Whether neuronal types or anatomical systems in the adult rat brain are coded by integrin type was studied in the present experiments by mapping the relative densities of mRNAs for nine alpha and four beta subunits. Expression patterns were markedly different and in some regions complementary. General results and areas of notable labeling were as follows: alpha1-limited neuronal expression, neocortical layer V, hippocampal CA3; alpha3 and alpha5-diffuse neuronal and glial labeling, Purkinje cells, hippocampal stratum pyramidale, locus coeruleus (alpha3); alpha4- discrete limbic regions, olfactory cortical layer II, hippocampal CA2; alpha6-most prominently neuronal, neocortical subplate, endopiriform, subiculum; alpha7-discrete, all neocortical layers, hippocampal granule cells and CA3, cerebellar granule and Purkinje cells, all efferent cranial nerve nuclei; alpha8-discrete neuronal, deep cortex, hippocampal CA1, basolateral amygdala, striatum; alphaV-all cortical layers, striatum, Purkinje cells; beta4-dentate gyrus granule cells; beta5-broadly distributed, neocortex, medial amygdala, cerebellar granule and Purkinje cells, efferent cranial nerve nuclei; alpha2, beta2, and beta3-mRNAs not detected. These results establish that brain subfields express different balances of integrin subunits and thus different integrin receptors. Such variations will determine which matrix proteins are recognized by neurons and the types of intraneuronal signaling generated by matrix binding. They also could generate important differences in synaptic plasticity across brain systems.

Animals↗
PubMed2000

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues.

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.

Adult↗
PubMed2004

Signaling hierarchy downstream of retinoic acid that independently regulates vascular remodeling and endothelial cell proliferation.

We previously demonstrated that during vascular morphogenesis, retinoic acid (RA) is required for the control of endothelial cell proliferation and capillary plexus remodeling. Herein, we investigate the mechanisms by which RA regulates these processes in the yolk sac. We found that although the enzyme required for RA production during early embryogenesis, retinaldehyde dehydrogenase-2 (Raldh2), was expressed in the visceral endoderm, RA receptors alpha1 and alpha2 were expressed in endothelial cells in the mesoderm, indicating that they are direct targets of RA. In Raldh2(-/-) embryos, there was down-regulation of TGF-beta1, fibronectin (Fn) and integrin alpha5, which was associated with decreased visceral endoderm survival and production of VEGF-A, Indian hedgehog (IHH), and bFGF. Exogenous provision of RA or Fn to Raldh2(-/-) explants in whole mouse embryo culture restored vascular remodeling, visceral endoderm survival, as well as integrin alpha5 expression and its downstream signaling that controls endothelial growth. Exogenous provision of visceral endoderm-derived factors (VEGF-A, IHH, and bFGF) failed to rescue endothelial cell proliferative control but collectively promoted vascular remodeling, suggesting that these processes are independently regulated via a signaling hierarchy downstream of RA.

Aldehyde Oxidoreductases↗
PubMed2002

Initial interaction of rat bone marrow cells with non-coated and calcium phosphate coated titanium substrates.

Initial interactions of rat bone marrow (RBM) cells with smooth titanium, rough titanium or calcium phosphate coated substrates were tested. Cells were seeded onto the substrates, and attachment, integrin expression and spreading and morphology were studied. We found no difference in attachment of RBM cells to the different materials. We did find differences in the percentage of attached cells within a certain time between replicate runs of the experiments. RBM cells on all materials express alpha1, alpha3, alpha5, alpha6 and beta1 subunits. Again there was a large difference in expression patterns on RBM cells in different runs. No difference was found in expression on the various materials. For alpha1, alpha5, alpha6 and beta1, no difference was found in expression between attached and unattached cells. Expression of alpha3 was similar on attached and unattached cells during early culture. At the end of culture, alpha3 expression was downregulated for attached cells and not for unattached cells. This resulted in a higher expression of alpha3 for unattached cells compared to attached cells. Cells did spread on all materials, and reached a larger cell size on smooth titanium than on the rough materials. Morphology of the cells on the materials differed. On smooth titanium, cells usually showed a compact cell body with short cellular extensions. On the rough materials, cells often showed elongated shapes, with many thin cellular extensions. From this we conclude that the substrate surface characteristics of the materials we used do not influence attachment or integrin expression during the initial cell-material interactions. On the other hand, spreading behavior and cell morphology do depend on substrate surface characteristics.

Animals↗
PubMed2005

Gene profiling changes in cytoskeletal proteins during clinical recovery after left ventricular-assist device support.

BACKGROUND: After left ventricular-assist device (LVAD) support, a proportion of patients recover sufficient ventricular function to enable explantation of the device. The exact molecular mechanisms involved in myocardial recovery remain unknown. Cytoskeletal proteins are essential for the structure and function of the cardiac myocyte and might play a major role. METHODS AND RESULTS: A total of 15 patients with nonischemic cardiomyopathy who required LVAD implantation were studied; 6 recovered sufficiently to allow explantation of the device compared with 9 who did not recover and required transplantation. LV myocardial samples were collected at implantation and explantation/transplantation. Affymetrix microarray analysis was performed on the paired samples and analyzed with reference to sarcomeric and nonsarcomeric cytoskeletal proteins. In the recovery group, of the nonsarcomeric proteins, lamin A/C increased 1.5-fold (P<0.05) and spectrin 1.6-fold (P<0.05) between the times of implantation and explantation. Integrins beta1, beta6, and alpha7 decreased 1.7-fold (P<0.05), 2.4-fold (P<0.05), and 1.5-fold (P<0.05), respectively, but integrins alpha5 and beta5 increased 2.3-fold (P<0.01) and 1.2-fold (P<0.01) at explantation. The following sarcomeric proteins changed in the recovered group only: beta-actin increased 1.4-fold (P<0.05); alpha-tropomyosin, 1.3-fold (P<0.05); alpha1-actinin, 1.8-fold (P<0.01); and alpha-filamin A, 1.6-fold (P<0.05). Both troponin T3 and alpha2-actinin decreased by 1.6-fold at the time of explantation (P<0.05). Vinculin decreased 1.7-fold (P=0.001) in the recovered group but increased by 1.7-fold (P<0.05) in the nonrecovered group. Vinculin protein levels decreased 4.1-fold in the recovered group. CONCLUSIONS: Myocardial recovery was associated with a specific pattern of changes in sarcomeric, nonsarcomeric, and membrane-associated proteins, which could have important implications in understanding the mechanisms involved.

Adrenergic beta-2 Receptor Agonists↗
PubMed1999

A peptide of the alpha3 chain of type IV collagen protects basement membrane against damage by PMN.

We have shown that basement membrane (BM) collagen (type IV), and specifically the peptide CNYYSNSYSFWLASLNPER (a.a. 185-203), from the non-collagenous domain of the alpha3 chain inhibits PMN. We examined the role of this peptide on PMN damage to BM in a vessel wall model. The presence of the endothelial monolayer as well as treatment of PMN with the alpha3(IV) 185-203 peptide reduced damage to BM by non-activated but not by activated PMN. The damage inhibition is unique to the alpha3(IV) peptide and not exhibited by comparable alpha1(IV) and alpha2(IV) chain peptides. A shorter peptide alpha3(IV) 185-191, containing the -SNS- triplet, reduced damage, whereas the one lacking the triplet, residues 194-203, was not effective. The CD47-alphavbeta3 integrin complex is the receptor for the alpha3(IV) peptide. Incubation of PMN with CD47 reactive mAb followed by the alpha3(IV) peptide abolished its protective effect on BM damage.

Amino Acid Sequence↗