αv integrins: key regulators of tissue fibrosis

Chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction and eventual organ failure. Therefore, the development of effective anti-fibrotic therapies is urgently required. During fibrogenesis, complex interplay occurs between cellular and extracellular matrix components of the wound healing response. Integrins, a family of transmembrane cell adhesion molecules, play a key role in mediating intercellular and cell-matrix interactions. Thus, integrins provide a major node of communication between the extracellular matrix, inflammatory cells, fibroblasts and parenchymal cells and, as such, are intimately involved in the initiation, maintenance and resolution of tissue fibrosis. Modulation of members of the αv integrin family has exhibited profound effects on fibrosis in multiple organs and disease states. In this review, we discuss the current knowledge of the mechanisms of αv-integrin-mediated regulation of fibrogenesis and show that the therapeutic targeting of specific αv integrins represents a promising avenue to treat patients with a broad range of fibrotic diseases

Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice

© The Author(s) 2017.Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-? B and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-? B and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes

The immunobiology of primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease histologically characterized by the presence of intrahepatic and/or extrahepatic biliary duct concentric, obliterative fibrosis, eventually leading to cirrhosis. Approximately 75% of patients with PSC have inflammatory bowel disease. The male predominance of PSC, the lack of a defined, pathogenic autoantigen, and the potential role of the innate immune system suggest that it may be due to dysregulation of immunity rather than a classic autoimmune disease. However, PSC is associated with several classic autoimmune diseases, and the strongest genetic link to PSC identified to date is with the human leukocyte antigen DRB01*03 haplotype. The precise immunopathogenesis of PSC is largely unknown but likely involves activation of the innate immune system by bacterial components delivered to the liver via the portal vein. Induction of adhesion molecules and chemokines leads to the recruitment of intestinal lymphocytes. Bile duct injury results from the sustained inflammation and production of inflammatory cytokines. Biliary strictures may cause further damage as a result of bile stasis and recurrent secondary bacterial cholangitis. Currently, there is no effective therapy for PSC and developing a rational therapeutic strategy demands a better understanding of the disease

Targeting of alpha(v) integrin identifies a core molecular pathway that regulates fibrosis in several organs

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. We report that Pdgfrb-Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the αv integrin subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Depletion of the αv integrin subunit in HSCs protected mice from CCl(4)-induced hepatic fibrosis, whereas global loss of αvβ3, αvβ5 or αvβ6 or conditional loss of αvβ8 on HSCs did not. Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of αv integrins using this system was also protective in models of pulmonary and renal fibrosis. Critically, pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. These data identify a core pathway that regulates fibrosis, and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases

J- vs. H-type assembly: pentamethine cyanine (Cy5) as a near-IR chiroptical reporter

The DNA-enabled dimerization of pentamethine cyanine (Cy5) dyes was studied by optical methods. The value of cyanine as a chiroptical reporter using a monomer-to-dimer switch is demonstrated. The specific shape of the CD signal and its high intensity are a result of J-type assembly

ВПЛИВ КОНФОРМАЦІЙНИХ ЗМІН У МОЛЕКУЛІ АЛЬБУМІНУ (BSA) НА СПЕКТРАЛЬНІ ВЛАСТИВОСТІ СКВАРАЇНОВОГО ТА ДИЦІАНОМЕТИЛЕН-СКВАРАЇНОВОГО БАРВНИКІВ

The work was to develop a highly sensitive, fluorescence based method to monitor conformational changes in proteins. With this aim, the influence of unfolding of bovine serum albumin (BSA) molecules on the spectral properties of squaraine dye Sq1 and dicyanomethylene squaraine dye Sq2 was investigated. The conformational changes were initiated by urea in the concentration of up to 8 M. The following three intensity-based methods of using these dyes were evaluated: (i) dye Sq1 was covalently bound to BSA; (ii) dye Sq1 was noncovalently bound to BSA; and (iii) dye Sq2 was noncovalently bound to BSA. The change in the fluorescence intensity of these dyes upon the BSA unfolding was measured. An additional, fourth method was based on the measurements of the change in the Förster Resonance Energy Transfer (FRET) between Sq1 (donor) covalently bound to BSA and Sq2 (acceptor) able to noncovalent binding with BSA. The change in the FRET was due to the BSA unfolding initiated by urea. The absorption and emission maxima measured in water were 635 / 645 nm for dye Sq1 and 654 / 670 nm for Sq2. Addition of 7–8 M urea to aqueous solutions of dyes Sq1 and Sq2 increased their fluorescence intensities by factor of 1.5 and 2.3 while the quantum yield increased by factor of 1.8 and 1.9, respectively. The absorption intensity of Sq1 in the presence of urea had a tendency to decrease while for Sq2 it increased by factor of 1.3. The formation of complexes of dyes Sq1 and Sq2 with BSA as well as the covalent conjugation of Sq1 with BSA caused the red-shift in the absorption and emission maxima and an increase in the quantum yields. Unfolding of BSA molecule resulted in a blue-shift of the absorption and emission bands and a 3-fold decrease in the fluorescence intensity of Sq2–BSA complex. Unfolding of protein in Sq1–BSA conjugate had almost no effect on the position and shape of the absorption and emission bands but decreased the fluorescence intensity by about 20%. At the same time, according to the fourth method the efficiency of FRET from Sq1 conjugated with BSA, to non-conjugated Sq2 exhibited much more pronounced decrease (~10 times) upon BSA unfolding. Thus, the FRET based approach was found to be more sensitive compared to the intensity based methods and therefore can changes in BSA.Досліджено вплив конформаційних змін в молекулі сироваткового альбуміну бика (BSA) на спектральні властивості сквараїнового барвника Sq1 при ковалентному та нековалентному зв’язуванні з BSA; диціанометиленового сквараїнового барвника Sq2 при нековалентному зв’язуванні з BSA; а також можливість використання ферстерівського резонансного переносу енергії (FRET) між барвниками Sq1 (донор) і Sq2 (акцептор) для флуоресцентного моніторингу конформаційних змін в молекулі BSA. Конформаційні зміни ініціювали сечовиною в концентраціях до 8 М. Барвник Sq1 поглинає і випромінює у водному середовищі при 635 нм і 645 нм, а барвник Sq2 – відповідно, 654 нм і 670 нм. Встановлено, що ковалентне зв’язування барвників Sq1 і Sq2 з BSA, а також утворення їх комплексів з BSA приводять до довгохвильового зсуву смуг поглинання і флуоресценції, та зростання квантового виходу флуоресценції. Розгортання молекули BSA зменшує інтенсивність флуоресценції у комплексі барвника Sq2 з BSA в три рази, а зміна інтенсивності флуоресценції барвника Sq1 у кон’югаті з BSA складає лише 20%. У той же час, ефективність переносу енергії (FRET) з барвника-донора Sq1, кон’югованого з BSA, на некон’югований барвник-акцептор Sq2 зменшується при конформаційних змінах в BSA значно більше, приблизно в 10 разів. Таким чином, метод FRET є найбільш чутливим і може бути запропонований для моніторингу конформаційних змін в молекулах BSA

THE IMPACT OF CONFORMATIONAL CHANGES IN ALBUMIN MOLECULE (BSA) ON THE SPECTRAL PROPERTIES OF SQUARAINE AND DICYANOMETHYLENE SQUARAINE DYES

The work was to develop a highly sensitive, fluorescence based method to monitor conformational changes in proteins. With this aim, the influence of unfolding of bovine serum albumin (BSA) molecules on the spectral properties of squaraine dye Sq1 and dicyanomethylene squaraine dye Sq2 was investigated. The conformational changes were initiated by urea in the concentration of up to 8 M. The following three intensity-based methods of using these dyes were evaluated: (i) dye Sq1 was covalently bound to BSA; (ii) dye Sq1 was noncovalently bound to BSA; and (iii) dye Sq2 was noncovalently bound to BSA. The change in the fluorescence intensity of these dyes upon the BSA unfolding was measured. An additional, fourth method was based on the measurements of the change in the Förster Resonance Energy Transfer (FRET) between Sq1 (donor) covalently bound to BSA and Sq2 (acceptor) able to noncovalent binding with BSA. The change in the FRET was due to the BSA unfolding initiated by urea. The absorption and emission maxima measured in water were 635 / 645 nm for dye Sq1 and 654 / 670 nm for Sq2. Addition of 7–8 M urea to aqueous solutions of dyes Sq1 and Sq2 increased their fluorescence intensities by factor of 1.5 and 2.3 while the quantum yield increased by factor of 1.8 and 1.9, respectively. The absorption intensity of Sq1 in the presence of urea had a tendency to decrease while for Sq2 it increased by factor of 1.3. The formation of complexes of dyes Sq1 and Sq2 with BSA as well as the covalent conjugation of Sq1 with BSA caused the red-shift in the absorption and emission maxima and an increase in the quantum yields. Unfolding of BSA molecule resulted in a blue-shift of the absorption and emission bands and a 3-fold decrease in the fluorescence intensity of Sq2–BSA complex. Unfolding of protein in Sq1–BSA conjugate had almost no effect on the position and shape of the absorption and emission bands but decreased the fluorescence intensity by about 20%. At the same time, according to the fourth method the efficiency of FRET from Sq1 conjugated with BSA, to non-conjugated Sq2 exhibited much more pronounced decrease (~10 times) upon BSA unfolding. Thus, the FRET based approach was found to be more sensitive compared to the intensity based methods and therefore can changes in BSA

Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression

BACKGROUND ; AIMS: Integrin alphavbeta6 is highly expressed on certain activated epithelia, where it mediates attachment to fibronectin and serves as coreceptor for the activation of latent transforming growth factor (TGF)-beta1. Because its role in liver fibrosis is unknown, we studied alphavbeta6 function in vitro and explored the antifibrotic potential of the specific alphavbeta6 antagonist EMD527040. METHODS: Experimental liver fibrosis was studied in rats after bile duct ligation (BDL) and in Mdr2(abcb4)(-/-) mice. Different doses of EMD527040 were given to rats from week 2 to 6 after BDL and to Mdr2(-/-) mice from week 4 to 8. Liver collagen was quantified, and expression of alphavbeta6 and fibrosis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. alphavbeta6-expressing cells, bile duct proliferation, and apoptosis were assessed histologically. The effect of EMD527040 on cholangiocyte adhesion, proliferation, apoptosis, and TGF-beta1 activation was studied in vitro. RESULTS: alphavbeta6 was highly expressed on proliferating bile duct epithelia in fibrosis, with 100-fold increased transcript levels in advanced fibrosis. EMD527040 attenuated bile ductular proliferation and peribiliary collagen deposition by 40%-50%, induced down-regulation of fibrogenic and up-regulation of fibrolytic genes, and improved liver architecture and function. In vitro alphavbeta6 inhibition reduced activated cholangiocyte proliferation, their adhesion to fibronectin, and endogenous activation of TGF-beta1 by 50% but did not affect bile duct apoptosis. CONCLUSIONS: Integrin alphavbeta6 is strongly up-regulated in proliferating bile duct epithelia and drives fibrogenesis via adhesion to fibronectin and auto/paracrine TGF-beta1 activation. Pharmacologic inhibition of alphavbeta6 potently inhibits the progression of primary and secondary biliary fibrosis