Fibronectin-Mediated Inflammatory Signaling Through Integrin α5 in Vascular Remodeling.

Background Adhesion of vascular endothelial cells to the underlying basement membrane potently modulates endothelial cells to cells' inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin α2ß1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes endothelial cells to inflammatory stimuli through integrins α5ß1and and αvß3. A chimeric integrin in which the cytoplasmic domain of α5 is replaced with that of α2 pairs with ß1 and binds fibronectin but signals like α2ß1. Methods and Results Here, we examined mice in which integrin α5 is replaced with the α5/2 chimera, using the transverse aortic constriction and partial carotid ligation models of vessel remodeling. Following transverse aortic constriction and partial carotid ligation surgery, wild-type mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. α5/2 mice also showed reduced artery wall hypertrophy in the transverse aortic constriction model and diminished inward remodeling in the partial carotid ligation model. Acute atherosclerosis after partial carotid ligation in hyperlipidemic ApoE-/- mice on a high fat diet was dramatically decreased in α5/2 mice. Conclusions Fibronectin and integrin α5 signaling is a key element of pathological vascular remodeling in acute models of both hypertension and disturbed flow. These results underscore the key role for integrin α5 signaling in pathological vascular remodeling associated with hypertension and atherosclerosis and support its potential as a therapeutic target.

IL-10 from dendritic cells but not from T regulatory cells protects against cisplatin-induced nephrotoxicity.

Interleukin-10 (IL-10), a cytokine with anti-inflammatory effects, is produced by renal parenchymal cells and bone marrow derived cells. Both endogenous and exogenous IL-10 are protective in cisplatin-induced acute kidney injury. However, the source of endogenous IL-10 in cisplatin-induced nephrotoxicity is not clear. Bone marrow chimera experiments in IL10-KO mice indicated that bone marrow derived cells were the primary source of IL-10 in cisplatin nephrotoxicity. Cell specific deletion of IL-10 in T regulatory cells and dendritic cells was accomplished using Foxp3 and CD11c driven cre recombination in IL10flox/flox mice, respectively. Upon treatment with cisplatin, both the IL10flox/flox and the Foxp3YFP-Cre x IL10flox/flox mice developed similar degrees of kidney injury. However, mice with the dendritic cell deletion of IL-10 showed more severe structural and functional changes in the kidney compared to the IL10flox/flox mice. These results indicate that IL-10 from dendritic cells but not from T regulatory cells offers significant endogenous protection against cisplatin induced nephrotoxicity.

Inhibiting Integrin α5 Cytoplasmic Domain Signaling Reduces Atherosclerosis and Promotes Arteriogenesis.

BACKGROUND: Fibronectin in endothelial basement membranes promotes endothelial inflammatory activation and atherosclerosis but also promotes plaque stability and vascular remodeling. The fibronectin receptor α5 subunit is proinflammatory through binding to and activating phosphodiesterase 4D5, which inhibits anti-inflammatory cyclic adenosine monophosphate and protein kinase A. Replacing the α5 cytoplasmic domain with that of α2 resulted in smaller atherosclerotic plaques. Here, we further assessed plaque phenotype and compensatory vascular remodeling in this model. METHODS AND RESULTS: α5/2 mice in the hyperlipidemic apolipoprotein E null background had smaller plaques in the aortic root, with reduced endothelial NF-κB activation and inflammatory gene expression, reduced leukocyte content, and much lower metalloproteinase expression. However, smooth muscle cell content, fibrous cap thickness, and fibrillar collagen were unchanged, indicating no shift toward vulnerability. In vivo knockdown of phosphodiesterase 4D5 also decreased endothelial inflammatory activation and atherosclerotic plaque size. α5/2 mice showed improved recovery from hindlimb ischemia after femoral artery ligation. CONCLUSIONS: Blocking the fibronectin-Integrin α5 pathway reduces atherosclerotic plaque size, maintains plaque stability, and improves compensatory remodeling. This pathway is therefore a potential therapeutic target for treatment of atherosclerosis.

VE-Cadherin Phosphorylation Regulates Endothelial Fluid Shear Stress Responses through the Polarity Protein LGN.

Fluid shear stress due to blood flow on the vascular endothelium regulates blood vessel development, remodeling, physiology, and pathology [1, 2]. A complex consisting of PECAM-1, VE-cadherin, and vascular endothelial growth factor receptors (VEGFRs) that resides at endothelial cell-cell junctions transduces signals important for flow-dependent vasodilation, blood vessel remodeling, and atherosclerosis. PECAM-1 transduces forces to activate src family kinases (SFKs), which phosphorylate and transactivate VEGFRs [3-5]. By contrast, VE-cadherin functions as an adaptor that interacts with VEGFRs through their respective cytoplasmic domains and promotes VEGFR activation in flow [6]. Indeed, shear stress triggers rapid increases in force across PECAM-1 but decreases the force across VE-cadherin, in close association with downstream signaling [5]. Interestingly, VE-cadherin cytoplasmic tyrosine Y658 can be phosphorylated by SFKs [7], which is maximally induced by low shear stress in vitro and in vivo [8]. These considerations prompted us to address the involvement of VE-cadherin cytoplasmic tyrosines in flow sensing. We found that phosphorylation of a small pool of VE-cadherin on Y658 is essential for flow sensing through the junctional complex. Y658 phosphorylation induces dissociation of p120ctn, which allows binding of the polarity protein LGN. LGN is then required for multiple flow responses in vitro and in vivo, including activation of inflammatory signaling at regions of disturbed flow, and flow-dependent vascular remodeling. Thus, endothelial flow mechanotransduction through the junctional complex is mediated by a specific pool of VE-cadherin that is phosphorylated on Y658 and bound to LGN.

Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling.

Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin-collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets.

Endothelial-to-mesenchymal transition drives atherosclerosis progression.

The molecular mechanisms responsible for the development and progression of atherosclerotic lesions have not been fully established. Here, we investigated the role played by endothelial-to-mesenchymal transition (EndMT) and its key regulator FGF receptor 1 (FGFR1) in atherosclerosis. In cultured human endothelial cells, both inflammatory cytokines and oscillatory shear stress reduced endothelial FGFR1 expression and activated TGF-ß signaling. We further explored the link between disrupted FGF endothelial signaling and progression of atherosclerosis by introducing endothelial-specific deletion of FGF receptor substrate 2 α (Frs2a) in atherosclerotic (Apoe(-/-)) mice. When placed on a high-fat diet, these double-knockout mice developed atherosclerosis at a much earlier time point compared with that their Apoe(-/-) counterparts, eventually demonstrating an 84% increase in total plaque burden. Moreover, these animals exhibited extensive development of EndMT, deposition of fibronectin, and increased neointima formation. Additionally, we conducted a molecular and morphometric examination of left main coronary arteries from 43 patients with various levels of coronary disease to assess the clinical relevance of these findings. The extent of coronary atherosclerosis in this patient set strongly correlated with loss of endothelial FGFR1 expression, activation of endothelial TGF-ß signaling, and the extent of EndMT. These data demonstrate a link between loss of protective endothelial FGFR signaling, development of EndMT, and progression of atherosclerosis.

Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Endothelial responses to fluid shear stress are essential for vascular development and physiology, and determine the formation of atherosclerotic plaques at regions of disturbed flow. Previous work identified VE-cadherin as an essential component, along with PECAM-1 and VEGFR2, of a complex that mediates flow signaling. However, VE-cadherin's precise role is poorly understood. We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex. VEGFR2 and VEGFR3 signal redundantly downstream of VE-cadherin. Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo. In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

Is hexamerin receptor a GPI-anchored protein in Achaea janata (Lepidoptera: Noctuidae)?

The process of uptake of hexamerins during metamorphosis from insect haemolymph by fat body cells is reminiscent of receptor-mediated endocytosis. Previously, we had identified a hexamerin-binding protein (HBP) and reported for the first time that uptake of hexamerins is dependent on the phosphorylation of HBP partly by a tyrosine kinase, which is, in turn, activated by 20-hydroxyecdysone (20E). However, the exact nature of HBP and the mechanism of interaction are still unknown. Here we report the possibility of HBP being a GPI-anchored protein in the fat body of Achaea janata and its role in the tyrosine-kinase-mediated phosphorylation signalling. Digestion of fat body membrane preparation with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the subsequent recognition by antibodies specific for the cross-reacting determinant (CRD), revealed that HBP is glycosylphosphatidylinositol (GPI)-anchored protein and, further, that the hexamerin binding to HBP was inhibited after digestion. Hexamerin overlay assay (HOA) of co-immunoprecipitated in vitro phosphorylated HBP showed exclusive binding to ~120 kDa protein. Lectin-binding analysis of hexamerins revealed the presence of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GluNAc), whereas HBP showed the presence of GalNac alone. Mild chemical deglycosylation studies and binding interaction in the presence of sugars revealed that glycan moieties are possibly not involved in the interaction between HBP and hexamerins. Taken together, these results suggest that HBP may be a GPI-anchored protein, and interaction and activation of HBP is through lipid-linked non-receptor src tyrosine kinases. However, additional studies are needed to prove that HBP is a GPI-anchored protein.

Extracellular matrix proteases contribute to progression of pelvic organ prolapse in mice and humans.

Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, fibulin-5 inhibited the ß1 integrin-dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5RGE/RGE) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to fibulin-5 knockouts (Fbln5-/-), Fbln5RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with ß-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5-/- mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre- and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women.

Fibulin-4 deficiency results in ascending aortic aneurysms: a potential link between abnormal smooth muscle cell phenotype and aneurysm progression.

RATIONALE: Loss of fibulin-4 during embryogenesis results in perinatal lethality because of aneurysm rupture, and defective elastic fiber assembly has been proposed as an underlying cause for the aneurysm phenotype. However, aneurysms are never seen in mice deficient for elastin, or for fibulin-5, which absence also leads to compromised elastic fibers. OBJECTIVE: We sought to determine the mechanism of aneurysm development in the absence of fibulin-4 and establish the role of fibulin-4 in aortic development. METHODS AND RESULTS: We generated germline and smooth muscle cell (SMC)-specific deletion of the fibulin-4 gene in mice (Fbln4(GKO) and Fbln4(SMKO), respectively). Fbln4(GKO) and Fbln4(SMKO) aortic walls fail to fully differentiate, exhibiting reduced expression of SM-specific contractile genes and focal proliferation of SMCs accompanied by degenerative changes of the medial wall. Marked upregulation of extracellular signal-regulated kinase 1/2 signaling pathway was observed in the aneurysmal wall of Fbln4(GKO) and Fbln4(SMKO) mice and both mutants developed aneurysm predominantly in the ascending thoracic aorta. In vitro, Fbln4(GKO) SMCs exhibit an immature SMC phenotype with a marked reduction of SM-myosin heavy chain and increased proliferative capacity. CONCLUSIONS: The vascular phenotype in Fbln4 mutant mice is remarkably similar to a subset of human thoracic aortic aneurysms caused by mutations in SMC contractile genes. Our study provides a potential link between the intrinsic properties of SMCs and aneurysm progression in vivo and supports the dual role of fibulin-4 in the formation of elastic fibers as well as terminal differentiation and maturation of SMCs in the aortic wall.

Constitutive expression of Arabidopsis NPR1 confers enhanced resistance to the early instars of Spodoptera litura in transgenic tobacco.

In Arabidopsis, NPR1 (AtNPR1) regulates salicylic acid (SA)-mediated activation of PR genes at the onset of systemic acquired resistance. AtNPR1 also modulates SA-induced suppression of jasmonic acid-responsive gene expression, and npr1 mutants manifest enhanced herbivore resistance. We have raised stable transgenic tobacco lines, expressing AtNPR1 constitutively, which showed elevated expression of PR1 and PR2 genes upon SA treatment. Herbivore bioassays with a generalist polyphagous pest, Spodoptera litura, revealed that the transgenic lines exhibited enhanced resistance compared to the wild-type plants, particularly with respect to younger larval populations. Insect-mediated injury induced several protease inhibitors (PIs), more significantly a 40-kDa serine PI in all the tobacco lines, but the induction was higher in the transgenic plants. We show in this communication that heterologous expression of AtNPR1 provides enhanced resistance to early larval populations of the herbivore, Spodoptera in transgenic tobacco plants.

Characterization of Bacillus thuringiensis strain DOR4 toxic to castor semilooper Achaea janata: proteolytic processing and binding of toxins to receptors.

We have isolated a strain of Bacillus thuringiensis (Bt) from Indian soil samples that was shown to be toxic to Achaea janata larvae. The isolate, named B. thuringiensis DOR4, serotypically identified with the standard subspecies kurstaki (H3a3b3c) and produced bipyramidal inclusions along with an amorphous type. Although the plasmid pattern of DOR4 was different from that of the reference strain, a crystal protein profile showed the presence of two major bands (130 and 65 kDa) similar to those of Bt subsp. kurstaki HD-1. To verify the cry gene content of DOR4, triplex PCR analysis was performed; it showed amplification of the cry1C gene in addition to cry1Aa, cry1Ac, cry2A, and cry2B genes, but not the cry1Ab gene. RT-PCR analysis showed the expression of cry1Aa and cry1Ac genes. In vitro proteolysis of DOR4 protoxin with midgut extract generated products of different sizes. Zymogram analysis of DOR4 protoxin as substrate pointed to a number of distinct proteases that were responsible for activation of protoxins. Furthermore, toxin overlay analysis revealed the presence of multiple toxin-binding proteins in midgut epithelium. Based on all these characterizations, we suggest that the Bt DOR4 strain can be exploited for an A. janata control program.

Identification and characterization of midgut proteases in Achaea janata and their implications.

Insect midgut proteases are excellent targets for insecticidal agents such as Bacillus thuringiensis Cry toxins and protease inhibitors. The midgut proteases of Achaea janata have been characterized and Casein zymograms indicated at least five distinct activities corresponding to approx 17, 20, 29 and 80, and 90 kDa. Using a combination of synthetic substrates and specific inhibitors in casein zymograms, photometric assays and activity blots, three trypsin-like and one elastase-like serine proteases were identified but no chymotrypsin-like activity. Various proteinase inhibitors displayed differential inhibitory effects towards the midgut proteases.

Characterization of Bacillus thuringiensis Cry toxin binding novel GPI anchored aminopeptidase from fat body of the moth Spodoptera litura.

Aminopeptidase N (APN) isoforms were identified as candidate receptors for Bacillus thuringiensis Cry toxins from the midgut of several insect species. In this study a partial cDNA encoding aminopeptidase (slfbAPN) was cloned from fat body of the moth Spodoptera litura. In the deduced amino acid sequence the characteristic metallopeptidase sequences, HEXXHX(18)E and GAMENWG were conserved but the sequence showed only 33-39% identity to other insect APNs, which were also reported to be Cry toxin receptors. The presence of a putative GPI anchor signal sequence at the C-terminus indicated that it is a membrane-anchored protein. The slfbAPN expression was restricted to the fat body as suggested by northern blot analysis of different tissues. Biochemical analyses including immunoblotting, ligand blotting and lectin blotting, demonstrated that slfbAPN is a membrane-anchored glycoprotein in the fat body and it binds to Cry toxins.

A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase.

Bacillus thuringiensis insecticidal crystal proteins bind to cell-surface receptors which represent a family of aminopeptidases [APN (aminopeptidase N)] present on the brush border membrane of insect midgut cells of susceptible insects leading to pore formation and death of the insect. We report here for the first time the presence of a novel APN in the fat body of the moth Achaea janata. Northern blotting detected at least one APN-specific transcript in the fat body, whereas two transcripts of different sizes were detected in the midgut. We have cloned two full-length APN cDNAs of 3015 bp and 2850 bp from fat body and midgut respectively, which encode proteins of 1004 and 950 amino acids. These two APNs share only 33% amino acid sequence identity, but both display the typical APN features, such as the N-terminal signal peptide, several putative glycosylation sites, C-terminal glycosylphosphatidylinositol anchor signal, the APN-specific zinc-binding/gluzincin motif HEXXHX(18)E and gluzincin motif GAMENWG. The fat body APN manifested a variation in its expression with respect to tissue and developmental stage. In spite of the abundance of the APN transcript in the fat body, fairly low APN activity was detected in this tissue. The fat-body- and midgut-specific APNs showed differential interaction with various Cry1A toxins. Besides, the level of toxicity of different Cry subtypes varied enormously with mode/site of delivery, such as intrahaemocoelic injections and feeding bioassays. These data indicate that the fat body might be a potential alternative Cry toxin target site in the moth.