Exocytosis of mesoporous silica nanoparticles from mammalian cells: from asymmetric cell-to-cell transfer to protein harvesting

The exocytosis of mesoporous silica nanoparticles (MSNs) from mammalian cells is demonstrated for the first time. The differences in the degree of exocytosis of MSNs between healthy and cancer cells are shown to be responsible for the asymmetric transfer of the particles between both cell types. The exo­cytosis of highly adsorbent magnetic MSNs proves to be useful as a means for harvesting biomolecules from living cells

PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

Insulin increases GLUT4 at the muscle cell surface, and this process requires actin remodeling. We show that a dynamic cycle of actin polymerization and severing is induced by insulin, governed by Arp2/3 and dephosphorylation of cofilin, respectively. The cycle is self-perpetuating and is essential for GLUT4 translocation

The myofibroblast matrix: implications for tissue repair and fibrosis

Myofibroblasts, and the extracellular matrix ( ECM ) in which they reside, are critical components of wound healing and fibrosis. The ECM , traditionally viewed as the structural elements within which cells reside, is actually a functional tissue whose components possess not only scaffolding characteristics, but also growth factor, mitogenic, and other bioactive properties. Although it has been suggested that tissue fibrosis simply reflects an ‘exuberant’ wound‐healing response, examination of the ECM and the roles of myofibroblasts during fibrogenesis instead suggest that the organism may be attempting to recapitulate developmental programmes designed to regenerate functional tissue. Evidence of this is provided by the temporospatial re‐emergence of embryonic ECM proteins by fibroblasts and myofibroblasts that induce cellular programmatic responses intended to produce a functional tissue. In the setting of wound healing (or physiological fibrosis), this occurs in a highly regulated and exquisitely choreographed fashion which results in cessation of haemorrhage, restoration of barrier integrity, and re‐establishment of tissue function. However, pathological tissue fibrosis, which oftentimes causes organ dysfunction and significant morbidity or mortality, likely results from dysregulation of normal wound‐healing processes or abnormalities of the process itself. This review will focus on the myofibroblast ECM and its role in both physiological and pathological fibrosis, and will discuss the potential for therapeutically targeting ECM proteins for treatment of fibrotic disorders.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94657/1/path4104.pd

Integrin alpha 11 in the regulation of the myofibroblast phenotype: implications for fibrotic diseases

Tissue fibrosis, characterized by excessive accumulation of aberrant extracellular matrix (ECM) produced by myofibroblasts, is a growing cause of mortality worldwide. Understanding the factors that induce myofibroblastic differentiation is paramount to prevent or reverse the fibrogenic process. Integrin-mediated interaction between the ECM and cytoskeleton promotes myofibroblast differentiation. In the present study, we explored the significance of integrin alpha 11 (ITGA11), the integrin alpha subunit that selectively binds to type I collagen during tissue fibrosis in the liver, lungs and kidneys. We showed that ITGA11 was co-localized with α-smooth muscle actin-positive myofibroblasts and was correlatively induced with increasing fibrogenesis in mouse models and human fibrotic organs. Furthermore, transcriptome and protein expression analysis revealed that ITGA11 knockdown in hepatic stellate cells (liver-specific myofibroblasts) markedly reduced transforming growth factor β-induced differentiation and fibrotic parameters. Moreover, ITGA11 knockdown dramatically altered the myofibroblast phenotype, as indicated by the loss of protrusions, attenuated adhesion and migration, and impaired contractility of collagen I matrices. Furthermore, we demonstrated that ITGA11 was regulated by the hedgehog signaling pathway, and inhibition of the hedgehog pathway reduced ITGA11 expression and fibrotic parameters in human hepatic stellate cells in vitro, in liver fibrosis mouse model in vivo and in human liver slices ex vivo. Therefore, we speculated that ITGA11 might be involved in fibrogenic signaling and might act downstream of the hedgehog signaling pathway. These findings highlight the significance of the ITGA11 receptor as a highly promising therapeutic target in organ fibrosis

Stepping up regulatory mechanisms of GLUT4 traffic in L6 skeletal muscle cellss

Insulin increases glucose uptake into muscle and fat by enhancing GLUT4 glucose transporter externalization; a process requiring input from Akt and actin. Downstream of phosphatidylinositol-3-kinase, insulin signaling bifurcates into Akt and actin activating arms. Akt-mediated phosphorylation of the Rab-GAP AS160 is required for gain in surface GLUT4 by insulin. However, little is known of the mechanism(s) by which AS160 and/or actin dynamics modulate GLUT4 traffic in muscle. We recently showed that GLUT4 arrival and/or fusion can be regulated by insulin signaling molecules and phospholipids. Using ‘rounded up’ L6 myoblasts stably expressing GLUT4myc, we find that transient expression of a non-phosphorylatable mutant of AS160 (AS160-4P) abrogates the surface fusion of GLUT4myc and partially reduces its sub-membranous accumulation. In contrast, tetanus toxin-mediated cleavage of VAMP2 inhibits GLUT4myc fusion but not arrival to the plasma membrane. Conversely, disrupting actin dynamics with Latrunculin B or silencing expression of a cytoskeletal protein a-actinin4 precludes the insulin-induced cortical build-up of GLUT4myc. These data suggest that AS160 and actin dynamics impinge on distinct stages of insulin-regulated GLUT4 traffic: AS160 may contribute to peripheral retention and is essential for GLUT4myc vesicle docking/fusion. It will be interesting to note which Rabs facilitate these AS160-dependent events. Actin dynamics instead may allow GLUT4 vesicle movement to the cell surface and/or its retention, presumably via cortical anchoring mechanisms involving a-actinin4, whilst VAMP2 has a major role in GLUT4 vesicle fusion. Indeed, defects in AS160 phosphorylation and actin dynamics are associated with insulin resistant states. Thus, discerning which steps of GLUT4 traffic are modulated by these inputs may help elucidate strategies to bypass insulin resistance

Overexpression of integrin α

AIM: To understand the role of the collagen-binding integrin α11 in vivo, we have used a classical approach of creating a mouse strain overexpressing integrin α11. A transgenic mouse strain overexpressing α11 in muscle tissues was analysed in the current study with special reference to the heart tissue. METHODS: We generated and phenotyped integrin α11 transgenic (TG) mice by echocardiography, magnetic resonance imaging and histology. Wild-type (WT) mice were subjected to aortic banding (AB) and the expression of integrin α11 was measured in flow cytometry-sorted cardiomyocytes and non-myocytes. RESULTS: TG mice developed left ventricular concentric hypertrophy by 6 months, with increased collagen deposition and reactivation of mRNA encoding foetal genes associated with cardiovascular pathological remodelling compared to WT mice. Masson's trichrome staining revealed interstitial fibrosis, confirmed additionally by magnetic resonance imaging and was found to be most prominent in the cardiac septum of TG but not WT mice. TG hearts expressed increased levels of transforming growth factor-β2 and transforming growth factor-β3 and upregulated smooth muscle actin. Macrophage infiltration coincided with increased NF-κB signalling in TG but not WT hearts. Integrin α11 expression was increased in both cardiomyocytes and non-myocyte cells from WT AB hearts compared to sham-operated animals. CONCLUSION: We report for the first time that overexpression of integrin α11 induces cardiac fibrosis and left ventricular hypertrophy. This is a result of changes in intracellular hypertrophic signalling and secretion of soluble factors that increase collagen production in the heart

Glycated collagen induces a11 integrin expression through TGF-ß2 and Smad3

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the a11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates a11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased a11 integrin and a-smooth muscle actin expression. Compared with native collagen, binding of purified a11b1 integrin to glycated collagen was reduced by fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-b2 but not TGF-b1 or TGF-b3. The increased expression of TGF-b2 was inhibited by triple helical collagen peptides that mimic the a11b1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with a11 integrin luciferase promoter constructs, glycated collagen activated the a11 integrin promoter. Analysis of a11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between 809 and 1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-b2 signaling pathway that stimulates a11 integrin expression through Smad2/3 binding elements in the a11 integrin promoter, which is important for myofibroblast formation and fibrosis.This work was sponsored by the Canadian Institutes of Health Research