The ω-3 epoxide of eicosapentaenoic acid inhibits endothelial cell proliferation by p38 MAP kinase activation and cyclin D1/CDK4 down-regulation

BACKGROUND AND PURPOSE

Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation.

EXPERIMENTAL APPROACH

Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids.

KEY RESULTS

The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive.

CONCLUSIONS AND IMPLICATIONS

The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation.     

Flavonone treatment reverses airway inflammation and remodelling in an asthma murine model

Background and Purpose

Asthma is an inflammatory disease that involves airway hyperresponsiveness and remodelling. Flavonoids have been associated to anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment of asthma. Our aim was to evaluate the effects of the sakuranetin treatment in several aspects of experimental asthma model in mice.

Experimental Approach

Male BALB/c mice received ovalbumin (i.p.) on days 0 and 14, and were challenged with aerolized ovalbumin 1% on days 24, 26 and 28. Ovalbumin-sensitized animals received vehicle (saline and dimethyl sulfoxide, DMSO), sakuranetin (20 mg kg^–1 per mice) or dexamethasone (5 mg kg^–1 per mice) daily beginning from 24th to 29th day. Control group received saline inhalation and nasal drop vehicle. On day 29, we determined the airway hyperresponsiveness, inflammation and remodelling as well as specific IgE antibody. RANTES, IL-5, IL-4, Eotaxin, IL-10, TNF-α, IFN-γ and GMC-SF content in lung homogenate was performed by Bioplex assay, and 8-isoprostane and NF-kB activations were visualized in inflammatory cells by immunohistochemistry.

Key Results

We have demonstrated that sakuranetin treatment attenuated airway hyperresponsiveness, inflammation and remodelling; and these effects could be attributed to Th2 pro-inflammatory cytokines and oxidative stress reduction as well as control of NF-kB activation.

Conclusions and Implications

These results highlighted the importance of counteracting oxidative stress by flavonoids in this asthma model and suggest sakuranetin as a potential candidate for studies of treatment of asthma.     

Eicosapentaenoic acid suppression of systemic inflammatory responses and inverse up-regulation of 15-deoxyΔ12,14 Prostaglandin J2 production

Background and Purpose

Eicosapentaenoic acid (EPA) has been shown to suppress immune cell responses, such as cytokine production and downstream PG production in vitro. Studies in vivo, however, have used EPA as a minor constituent of fish oil with variable results. We investigated the effects of EPA on systemic inflammatory responses as pure EPA has not been evaluated on immune/inflammatory responses in vivo.

Experimental Approach

Rabbits were administered polyinosinic: polycytidylic acid (poly I:C) i.v. before and after oral treatment with EPA for 42 days (given daily). The responses to IL-1β and TNF-α were also studied. Immediately following administration of poly I:C, body temperature was continuously monitored and blood samples were taken. Plasma levels of IL-1β, PGE₂ (PGE₂), and 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) were measured by enzyme immunoassay.

Key Results

Following EPA treatment, the fever response to poly I:C was markedly suppressed compared with pretreatment responses. This was accompanied by a parallel reduction in the poly I:C-stimulated elevation in plasma levels of IL-1β and PGE₂. Paradoxically, the levels of 15d-PGJ₂ were higher following EPA treatment. EPA treatment did not significantly alter the fever response or plasma levels of PGE₂ in response to either IL-1β or TNF-α.

Conclusion and Implications

Oral treatment with EPA can suppress immune/inflammatory responses in vivo via a suppression of upstream cytokine production resulting in a decreased fever response and indirectly reducing circulating levels of PGE₂. EPA also enhances the production of the cytoprotective prostanoid 15d-PGJ₂ indicating the therapeutic benefit of EPA.     

α-Galactosylceramide suppresses murine eosinophil production through interferon-γ-dependent induction of NO synthase and CD95

Background and Purpose

α-Galactosylceramide (α-GalCer), a pleiotropic immunomodulator with therapeutic potential in neoplastic, autoimmune and allergic diseases, activates invariant natural killer T-cells throughCD1-restricted receptors for α-GalCer on antigen-presenting cells, inducing cytokine secretion. However the haemopoietic effects of α-GalCer remain little explored.

Experimental Approach

α-GalCer-induced modulation of eosinophil production in IL-5-stimulated bone marrow cultures was examined in wild-type (BALB/c, C57BL/6) mice and their mutants lacking CD1, inducible NOS (iNOS), CD95 and IFN-γ, along with the effects of lymphocytes; IFN-γ; caspase and iNOS inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs) and LTD₄; and dexamethasone.

Key Results

α-GalCer (10⁻⁶–10⁻⁸M) suppressed IL-5-stimulated eosinopoiesis by inducing apoptosis. α-GalCer pretreatment in vivo (100 μg·kg⁻¹, i.v.) suppressed colony formation by GM-CSF-stimulated bone marrow progenitors in semi-solid cultures. α-GalCer and dexamethasone synergistically promoted eosinophil maturation. Suppression of eosinophil production by α-GalCer was prevented by aminoguanidine and was undetectable in bone marrow lacking iNOS, CD95, CD28; or CD1d. Separation on Percoll gradients and depletion of CD3+ cells made bone marrow precursors unresponsive to α-GalCer. Responsiveness was restored with splenic lymphocytes. Experiments with (i) IFN-γ-deficient bone marrow, alone or co-cultured with spleen T-cells from wild-type, but not from CD1d-deficient, donors; (ii) IFN-γ neutralization; and (iii) recombinant IFN-γ, showed that these effects of α-GalCer were mediated by IFN-γ. Effects of α-GalCer on eosinophil production were blocked by LTD₄ and NSAIDs.

Conclusions and Implications

α-GalCer activation of IFN-γ-secreting, CD1d-restricted lymphocytes induced iNOS-CD95-dependent apoptosis in developing eosinophils. This pathway is initiated by endogenous regulatory lymphocytes, antagonised by LTD₄, NSAIDs and aminoguanidine, and modified by dexamethasone.     

Human umbilical cord blood mesenchymal stem cell transplantation suppresses inflammatory responses and neuronal apoptosis during early stage of focal cerebral ischemia in rabbits

Aim： Human umbilical cord blood mesenchymal stem cells （hUCB-MSCs） have been shown to ameliorate cerebral ischemia in animal models. In this study we investigated the effects of hUCB-MSCs on inflammatory responses and neuronal apoptosis during the early stage of focal cerebral ischemia in rabbits. Methods： Focal cerebral ischemia was induced in male New Zealand rabbits by occlusion of MCA for 2 h. The blood samples were collected at different time points prior and during MCAO-reperfusion. The animals were euthanized 3 d after MCAO, and the protein levels of IL-113, IL-6, IL-IO, and TNF-a in the serum and peri-ischemic brain tissues were detected using Western blot and ELISA, respectively. Inflammatory cell infiltration, neuronal apoptosis and neuronal density were measured morphologically, hUCB-MSCs （5x106） were iv injected a few minutes after MCAO. Results： The serum levels of IL-113, IL-6, and TNF-~ were rapidly increased, and peaked at 2 h after the start of MCAO. hUCB-MSC transplantation markedly and progressively suppressed the ischemia-induced increases of serum IL-113, IL-6, and TNF-a levels within 6 h MCAO-reperfusion. Focal cerebral ischemia decreased the serum level of IL-IO, which was prevented by hUCB-MSC transplantation The expression of IL-113, IL-6, IL-IO, and TNF-a in the peri-ischemic brain tissues showed similar changes as in the serum, hUCB-MSC transplantation markedly suppressed the infiltration of inflammatory cells, and increased the neuronal density around the ischemic region. Furthermore, hUCB-MSC transplantation significantly decreased the percentage of apoptosis around the ischemic region. Conclusion： hUCB-MSCs transplantation suppresses inflammatory responses and neuronal apoptosis during the early stage focal cerebral ischemia in rabbits.     

Antiviral and anti-inflammatory activity of arbidol hydrochloride in influenza A (H1N1) virus infection

Aim:

To investigate the effects of arbidol hydrochloride (ARB), a widely used antiviral agent, on the inflammation induced by influenza virus.

Methods:

MDCK cells were infected with seasonal influenza A/FM/1/47 (H1N1) or pandemic influenza A/Hubei/71/2009 (H1N1). In vitro cytotoxicity and antiviral activity of ARB was determined using MTT assay. BALB/c mice were infected with A/FM/1/47 (H1N1). Four hours later the mice were administered ARB (45, 90, and 180 mg·kg⁻¹·d⁻¹) or the neuraminidase inhibitor oseltamivir (22.5 mg·kg⁻¹·d⁻¹) via oral gavage once a day for 5 d. Body-weight, median survival time, viral titer, and lung index of the mice were measured. The levels of inflammatory cytokines were examined using real-time RT-PCR and ELISA.

Results:

Both H1N1 stains were equally sensitive to ARB as tested in vitro. In the infected mice, ARB (90 and 180 mg·kg⁻¹·d⁻¹) significantly decreased the mortality, alleviated virus-induced lung lesions and viral titers. Furthermore, ARB suppressed the levels of IL-1β, IL-6, IL-12, and TNF-α, and elevated the level of IL-10 in the bronchoalveolar lavage fluids and lung tissues. However, ARB did not significantly affect the levels of IFN-α and IFN-γ, but reduced the level of IFN-β1 in lung tissues at 5 dpi. In peritoneal macrophages challenged with A/FM/1/47 (H1N1) or poly I:C, ARB (20 μmol/L) suppressed the levels of IL-1β, IL-6, IL-12, and TNF-α, and elevated the level of IL-10. Oseltamivir produced comparable alleviation of virus-induced lung lesions with more reduction in the viral titers, but less effective modulation of the inflammatory cytokines.

Conclusion:

ARB efficiently inhibits both H1N1 stains and diminishes both viral replication and acute inflammation through modulating the expression of inflammatory cytokines.     

Effects of Risperidone on Cytokine Profile in Drug-Na?ve First-Episode Psychosis

Background:

There is robust evidence that schizophrenia is characterized by immune-inflammatory abnormalities, including variations on cytokine levels. The results of previous studies, however, are heterogeneous due to several confounding factors, such as the effects of antipsychotic drugs. Therefore, research on drug-naïve first-episode psychosis (FEP) patients is essential to elucidate the role of immune processes in that disorder.

Methods:

The aim of this study is to compare cytokine levels (IL-2, IL-10, IL-4, IL-6, IFN-γ, TNF-α, and IL-17) in drug-naïve FEP patients both before and after treatment with risperidone for 10 weeks, and to investigate possible associations between cytokine levels and clinical responses to treatment and presence of depressive symptoms. It this study, we included 55 drug-naïve FEP patients who had repeated measurements of cytokine levels and 57 healthy controls.

Results:

We found that FEP patients had significantly higher IL-6, IL-10 and TNF-α levels than healthy controls. After risperidone treatment, these three cytokines and additionally IL-4 decreased significantly. No significant difference was found between the post-treatment cytokine levels in FEP patients and in healthy controls, suggesting that these alterations in cytokine profiles are a state marker of FEP. No significant association was found between risperidone-induced changes in cytokines and the clinical response to treatment or the presence of depression. There was a significant inverse association between the risperidone-induced changes in IL-10 and the negative symptoms.

Conclusions:

In conclusion, our results show a specific cytokine profile in FEP patients (monocytic and regulatory T-cell activation) and suggest immunoregulatory effects of risperidone treatment, characterized by suppressant effects on monocytic, Th2, and T-regulatory functions.     

Betulinic acid regulates generation of neuroinflammatory mediators responsible for tissue destruction in multiple sclerosis in vitro

Aim:

To investigate the influences of betulinic acid (BA), a triterpenoid isolated from birch bark, on neuroinflammatory mediators involved in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis in vitro.

Methods:

Encephalitogenic T cells were prepared from draining lymph nodes and spinal cords of Dark Agouti rats 8 to 10 d after immunization with myelin basic protein (MBP) and complete Freund''s adjuvant. Macrophages were isolated from the peritoneal cavity of adult untreated rats. Astrocytes were isolated from neonatal rat brains. The cells were cultured and then treated with different agents. IFN-γ, IL-17, iNOS and CXCL12 mRNA levels in the cells were analyzed with RT-PCR. iNOS and CXCL12 protein levels were detected using immunoblot. NO and ROS generation was measured using Griess reaction and flow cytometry, respectively.

Results:

In encephalitogenic T cells stimulated with MBP (10 μg/mL), addition of BA inhibited IL-17 and IFN-γ production in a dose-dependent manner. The estimated IC50 values for IL-17 and IFN γ were 11.2 and 63.8 μmol/L, respectively. When the macrophages were stimulated with LPS (10 ng/mL), addition of BA (50 μmol/L) significantly increased ROS generation, and suppressed NO generation. The astrocytes were stimulated with ConASn containing numerous inflammatory mediators, which mimicked the inflammatory milieu within CNS; addition of BA (50 μmol/L) significantly increased ROS generation, and blocked ConASn-induced increases in iNOS and CXCL12 mRNA levels, but did not affect iNOS and CXCL12 protein levels. Importantly, in both the macrophages and astrocytes, addition of BA (50 μmol/L) inhibited lipid peroxidation.

Conclusion:

Besides inhibiting encephalitogenic T cell cytokines and reducing NO generation, BA induces tissue-damaging ROS generation within CNS.     

Bioequivalence of two omega-3 fatty acid ethyl ester formulations: a case of clinical pharmacology of dietary supplements

AIM

To evaluate the bioequivalence of two omega-3 long chain polyunsaturated fatty acid (n-3 LC-PUFA) ethyl ester preparations, previously shown not to be bioequivalent in healthy subjects, with the objective of providing a guideline for future work in this area.

METHOD

A randomized double-blind crossover protocol was chosen. Volunteers with the lowest blood concentrations of n-3 LC-PUFA were selected. They received the ethyl esters in a single high dose (12 g) and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) blood concentrations were analyzed after fingerprick collection at intervals up to 24 h.

RESULTS

Differently from a prior study, the pharmacokinetic analysis indicated a satisfactory bioequivalence: for the AUC(0,24 h) 90% CI of the ratio between the two formulations were in the range for bioequivalence (for EPA 0.98, 1.04 and for DHA 0.99, 1.04) and the same was true for C_max and t_max (90% CI were 0.95, 1.14 and 1.10, 1.25 for EPA and 0.88, 1.02 and 0.84, 1.24 for DHA).

CONCLUSION

This study shows that, in order to obtain reliable bioequivalence data of products present in the daily diet, certain conditions should be met. Subjects should have low, homogeneous baseline concentrations and not be exposed to food items containing the product under evaluation, e.g. fish. Finally, as in the case of omega-3 fatty acids, selected doses should be high, eventually with appropriate conditions of intake.     

Rimonabant inhibits TNF-α-induced endothelial IL-6 secretion via CB1 receptor and cAMP-dependent protein kinase pathway

Aim:

To investigate whether rimonabant, a cannabinoid receptor antagonist, had inhibitory effects on inflammatory reactions in human umbilical vein endothelial cells (HUVEC).

Methods:

TNF-α-induced IL-6 production was measured by ELISA and effects on related signaling pathways were investigated by immunoblot analysis. Cellular cAMP level was measured using kinase-coupled luciferase reaction.

Results:

Rimonabant at 1 and 10 μmol/L significantly inhibited TNF-α-induced IL-6 production when added 15, 30 and 60 minutes before TNF-α treatment. Rimonabant also inhibited TNF-α-induced phosphorylation of IκB kinase (IKK) α/β and IκB-α degradation. ACEA, a cannabinoid receptor subtype 1 (CB1) agonist, added before rimonabant abolished the former effects of rimonabant. H-89, an inhibitor of cAMP-dependent protein kinase (PKA), abolished the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Rimonabant also increased the phosphorylation of PKA regulatory subunit II (PKA-RII), implying the essential role of PKA activation in the inhibitory effects of rimonabant. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin did not abolish the inhibitory effects of rimonabant on TNF-α induced IL-6 production.

Conclusion:

Rimonabant had anti-inflammatory effects on endothelial cells and inhibited TNF-α-induced IKKα/β phosphorylation, IκB-α degradation and IL-6 production in HUVEC. This effect was related to CB1 antagonism and PKA activation.     

Glycyrrhizin attenuates rat ischemic spinal cord injury by suppressing inflammatory cytokines and HMGB1

Aim:

To investigate the neuroprotective effect of glycyrrhizin (Gly) against the ischemic injury of rat spinal cord and the possible role of the nuclear protein high-mobility group box 1 (HMGB1) in the process.

Methods:

Male Sprague-Dawley rats were subjected to 45 min aortic occlusion to induce transient lumbar spinal cord ischemia. The motor functions of the animals were assessed according to the modified Tarlov scale. The animals were sacrificed 72 h after reperfusion and the lumbar spinal cord segment (L2–L4) was taken out for histopathological examination and Western blotting analysis. Serum inflammatory cytokine and HMGB1 levels were analyzed using ELISA.

Results:

Gly (6 mg/kg) administered intravenously 30 min before inducing the transient lumbar spinal cord ischemia significantly improved the hind-limb motor function scores, and reduced the number of apoptotic neurons, which was accompanied by reduced levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the plasma and injured spinal cord. Moreover, the serum HMGB1 level correlated well with the serum TNF-α, IL-1β and IL-6 levels during the time period of reperfusion.

Conclusion:

The results suggest that Gly can attenuate the transient spinal cord ischemic injury in rats via reducing inflammatory cytokines and inhibiting the release of HMGB1.     

Cannabinoid CB(2) receptor attenuates morphine-induced inflammatory responses in activated microglial cells

BACKGROUND AND PURPOSE

Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB₂ and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.

EXPERIMENTAL APPROACH

We examined the effects of µ-opioid and CB₂ receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.

KEY RESULTS

Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB₂ receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.

CONCLUSIONS AND IMPLICATIONS

Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB₂ receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids.     

Stress-induced anhedonia is associated with an increase in Alzheimer's disease-related markers

BACKGROUND AND PURPOSE

Stress is believed to be associated with the development of neuropsychiatric disorders, including Alzheimer''s disease (AD). We have studied mechanisms implicated in vulnerability to stress and the relationship with changes in AD-related markers.

EXPERIMENTAL APPROACH

Anhedonia induced by a chronic mild stress (CMS) procedure, applied for 6 weeks, was used to select rats vulnerable or resistant to stress. Sucrose intake, the Porsolt forced swimming test and cognitive deficits in the novel object recognition test (NORT) were used to characterize vulnerable and resilient rats. The antidepressant venlafaxine (20 mg·kg⁻¹ p.o.) or saline was administered daily during the last 2 weeks of CMS. Biochemical markers affected by stress, PKB, ERK and synaptophysin, and those associated with AD, amyloid β-protein (Aβ), β-secretase (BACE1) and τ phosphorylation, were measured in the hippocampus.

KEY RESULTS

After CMS, 40% of rats were resistant to the development of anhedonia (CMS-resistant to stress), whereas the remaining were responsive [CMS-anhedonic (CMSA)]. Only CMSA rats displayed significant increases in immobility time in the forced swimming test and cognitive deficits in the NORT, and significant decreases in synaptophysin, phosphorylated PKB and phosphorylated ERK1/2 expression in the hippocampus. Increased levels of Aβ40, BACE1 and τ phosphorylation were also found only in CMSA rats. All these effects in CMSA rats were reverted by treatment with venlafaxine.

CONCLUSIONS AND IMPLICATIONS

Vulnerability to stress might constitute a risk factor for the development of AD, and pharmacological treatment with venlafaxine may represent a therapeutic strategy for the treatment of stress-related disorders, including AD.     

The integrin α2β1 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 α2β1 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 α2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-β. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}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 α2β1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-β mAb, or a phosphoinositide-3 kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Aggretin stimulated the phosphorylation of PDGFR-β, 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, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and PD98059.

Conclusions and implications:

Aggretin induces VSMC proliferation and migration mainly through binding to integrin α2β1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.