Type I Collagen Signaling Regulates Opposing Fibrotic Pathways through α2ß1 Integrin.

Fibrosis is characterized by fibroblast activation, leading to matrix remodeling culminating in a stiff, type I collagen-rich fibrotic matrix. Alveolar epithelial cell (AEC) apoptosis is also a major feature of fibrogenesis, and AEC apoptosis is sufficient to initiate a robust lung fibrotic response. TGF-ß (transforming growth factor-ß) is a major driver of fibrosis and can induce both AEC apoptosis and fibroblast activation. We and others have previously shown that changes in extracellular matrix stiffness and composition can regulate the cellular response to TGF-ß. In the present study, we find that type I collagen signaling promotes TGF-ß-mediated fibroblast activation and inhibits TGF-ß-induced AEC death. Fibroblasts cultured on type I collagen or fibrotic decellularized lung matrix had augmented activation in response to TGF-ß, whereas AECs on cultured on type I collagen or fibrotic lung matrix were more resistant to TGF-ß-induced apoptosis. Both of these responses were mediated by integrin α2ß1, a major collagen receptor. AECs treated with an α2 integrin inhibitor or with deletion of α2 integrin had loss of collagen-mediated protection from apoptosis. We found that mice with fibroblast-specific deletion of α2 integrin were protected from fibrosis whereas mice with AEC-specific deletion of α2 integrin had more lung injury and a greater fibrotic response to bleomycin. Intrapulmonary delivery of an α2 integrin-activating collagen peptide inhibited AEC apoptosis in vitro and in vivo and attenuated the fibrotic response. These studies underscore the need for a thorough understanding of the divergent response to matrix signaling.

Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions.

The platelet receptors glycoprotein (Gp)VI, integrin α2ß1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca2+ ([Ca2+]i) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca2+]i measurements using confocal imaging. All three collagen receptors coupled to [Ca2+]i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca2+]i signals leading to real-time increases in integrins α2ß1- and αIIbß3-mediated platelet adhesion. αIIbß3-dependent platelet aggregation was dependent on P2Y12 signalling. Co-engagement of α2ß1 and GpIb/V/IX generated transient [Ca2+]i spikes and low amplitude [Ca2+]i responses that potentiated GpVI-dependent [Ca2+]i signalling. Therefore α2ß1, GpIb/V/IX and GpVI synergise to generate [Ca2+]i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for αIIbß3 in stable platelet adhesion and aggregation.

alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

The integrin alpha2beta1 agonist, aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production.

BACKGROUND AND PURPOSE: During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin alpha2beta1 agonist. EXPERIMENTAL APPROACH: Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques. KEY RESULTS: Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin alpha2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-beta. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by alpha2beta1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-beta mAb, or a phosphoinositide-3 kinase inhibitor, LY294002. Aggretin stimulated the phosphorylation of PDGFR-beta, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, U73122 and PD98059. CONCLUSIONS AND IMPLICATIONS: Aggretin induces VSMC proliferation and migration mainly through binding to integrin alpha2beta1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.

TPA primes alpha2beta1 integrins for cell adhesion.

Integrin avidity is regulated by changes in the conformation of the heterodimer and cluster formation. We measured cell adhesion by integrin alpha2beta1 (CHO-alpha2) to collagen at short contact times (0.5-60s) by single cell force spectroscopy (SCFS). The adhesion increased rapidly with contact time and was further strengthened by the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) and integrin activator. TPA also improved the strength of adhesive units. Furthermore, changes in membrane nanotube properties indicated better coupling of integrins to the cell cytoskeleton. We conclude that in addition to increasing integrin avidity TPA strengthens integrin-cytoskeletal linkage.

Characterization of a novel and potent collagen antagonist, caffeic acid phenethyl ester, in human platelets: in vitro and in vivo studies.

OBJECTIVE: Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been demonstrated to possess multiple pharmacological activities. In the present study, CAPE (6-25 microM) specifically inhibited collagen-induced platelet aggregation and the ATP release reaction in platelet suspensions. METHODS: Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance (ESR) were used to assess the anti-platelet activity of CAPE. Fluorescein sodium-induced platelet thrombi in mesenteric microvessels of mice were used for an in vivo study. RESULTS: CAPE (15-100 microM) produced a concentration-related rightward displacement of the collagen concentration-response curve, and the Schild plot gave pA(2) and pA(10) values of 4.28+/-0.07 and 3.14+/-0.73, respectively, with a slope of -0.83+/-0.16, indicating specific antagonism. CAPE (25 microM) also inhibited platelet aggregation stimulated by the glycoprotein VI agonist, convulxin, and the alpha(2)beta(1) integrin agonist, aggretin. CAPE (25 microM) also markedly interfered with FITC-collagen binding to platelet membranes. CAPE (15 and 25 microM) concentration-dependently inhibited collagen-induced platelet activation accompanied by [Ca(+2)](i) mobilization, phosphoinositide breakdown, activation of protein kinase C and mitogen-activated protein kinases (i.e., ERK2, JNK, and p38 MAPK), Akt phosphorylation, and thromboxane A(2) formation. In the ESR study, CAPE (15 and 25 microM) markedly reduced hydroxyl radical (OH) formation in collagen-activated platelets. In an in vivo study, CAPE (5 mg/kg) significantly prolonged the latency in inducing platelet plug formation in mesenteric venules of mice. CONCLUSIONS: The most important findings of this study suggest that CAPE specifically inhibits collagen-induced platelet activation. Thus, CAPE treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.

Aggretin, a snake venom-derived endothelial integrin alpha 2 beta 1 agonist, induces angiogenesis via expression of vascular endothelial growth factor.

Aggretin, a collagen-like alpha 2 beta 1 agonist purified from Calloselasma rhodostoma venom, was shown to increase human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC migration toward immobilized aggretin was also increased. These effects were blocked by A2-IIE10, an antibody raised against integrin alpha 2. Aggretin bound to HUVECs in a dose-dependent and saturable manner, which was specifically inhibited by A2-IIE10, as examined by flow cytometry. Aggretin elicited significant angiogenic effects in both in vivo and in vitro angiogenesis assays, and incubation of HUVECs with aggretin activated phosphatidylinositol 3-kinase (PI3K), Akt, and extracellular-regulated kinase 1/2 (ERK1/2); these effects were blocked by A2-IIE10 or vascular endothelial growth factor (VEGF) monoclonal antibody (mAb). The angiogenic effect induced by aggretin may be via the production of VEGF because the VEGF level was elevated and VEGF mAb pretreatment inhibited Akt/ERK1/2 activation as well as the in vivo angiogenesis induced by aggretin. The VEGF production induced by aggretin can be blocked by A2-IIE10 mAb pretreatment. In conclusion, aggretin induces endothelial cell proliferation, migration, and angiogenesis by interacting with integrin alpha 2 beta 1 leading to activation of PI3K, Akt, and ERK1/2 pathways, and the increased expression of VEGF may be responsible for its angiogenic activity.