Targeting MFAP5 in cancer-associated fibroblasts sensitizes pancreatic cancer to PD-L1-based immunochemotherapy via remodeling the matrix.

Highly desmoplastic and immunosuppressive tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) contributes to tumor progression and resistance to current therapies. Clues targeting the notorious stromal environment have offered hope for improving therapeutic response whereas the underlying mechanism remains unclear. Here, we find that prognostic microfibril associated protein 5 (MFAP5) is involved in activation of cancer-associated fibroblasts (CAFs). Inhibition of MFAP5highCAFs shows synergistic effect with gemcitabine-based chemotherapy and PD-L1-based immunotherapy. Mechanistically, MFAP5 deficiency in CAFs downregulates HAS2 and CXCL10 via MFAP5/RCN2/ERK/STAT1 axis, leading to angiogenesis, hyaluronic acid (HA) and collagens deposition reduction, cytotoxic T cells infiltration, and tumor cells apoptosis. Additionally, in vivo blockade of CXCL10 with AMG487 could partially reverse the pro-tumor effect from MFAP5 overexpression in CAFs and synergize with anti-PD-L1 antibody to enhance the immunotherapeutic effect. Therefore, targeting MFAP5highCAFs might be a potential adjuvant therapy to enhance the immunochemotherapy effect in PDAC via remodeling the desmoplastic and immunosuppressive microenvironment.

Resonant soft X-ray scattering reveals cellulose microfibril spacing in plant primary cell walls.

Cellulose microfibrils are crucial for many of the remarkable mechanical properties of primary cell walls. Nevertheless, many structural features of cellulose microfibril organization in cell walls are not yet fully described. Microscopy techniques provide direct visualization of cell wall organization, and quantification of some aspects of wall microstructure is possible through image processing. Complementary to microscopy techniques, scattering yields structural information in reciprocal space over large sample areas. Using the onion epidermal wall as a model system, we introduce resonant soft X-ray scattering (RSoXS) to directly quantify the average interfibril spacing. Tuning the X-ray energy to the calcium L-edge enhances the contrast between cellulose and pectin due to the localization of calcium ions to homogalacturonan in the pectin matrix. As a consequence, RSoXS profiles reveal an average center-to-center distance between cellulose microfibrils or microfibril bundles of about 20 nm.

Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds.

All cells of terrestrial plants are fortified by walls composed of crystalline cellulose microfibrils and a variety of matrix polymers. Xylans are the second most abundant type of polysaccharides on Earth. Previous studies of Arabidopsis (Arabidopsis thaliana) irregular xylem (irx) mutants, with collapsed xylem vessels and dwarfed stature, highlighted the importance of this cell wall component and revealed multiple players required for its synthesis. Nevertheless, xylan elongation and substitution are complex processes that remain poorly understood. Recently, seed coat epidermal cells were shown to provide an excellent system for deciphering hemicellulose production. Using a coexpression and sequence-based strategy, we predicted several MUCILAGE-RELATED (MUCI) genes that encode glycosyltransferases (GTs) involved in the production of xylan. We now show that MUCI21, a member of an uncharacterized clade of the GT61 family, and IRX14 (GT43 protein) are essential for the synthesis of highly branched xylan in seed coat epidermal cells. Our results reveal that xylan is the most abundant xylose-rich component in Arabidopsis seed mucilage and is required to maintain its architecture. Characterization of muci21 and irx14 single and double mutants indicates that MUCI21 is a Golgi-localized protein that likely facilitates the addition of xylose residues directly to the xylan backbone. These unique branches seem to be necessary for pectin attachment to the seed surface, while the xylan backbone maintains cellulose distribution. Evaluation of muci21 and irx14 alongside mutants that disrupt other wall components suggests that mucilage adherence is maintained by complex interactions between several polymers: cellulose, xylans, pectins, and glycoproteins.

Aspen Tension Wood Fibers Contain ß-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). ß-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. ß-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, ß-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high ß-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

Pectin methylesterase and pectin remodelling differ in the fibre walls of two gossypium species with very different fibre properties.

Pectin, a major component of the primary cell walls of dicot plants, is synthesized in Golgi, secreted into the wall as methylesters and subsequently de-esterified by pectin methylesterase (PME). Pectin remodelling by PMEs is known to be important in regulating cell expansion in plants, but has been poorly studied in cotton. In this study, genome-wide analysis showed that PMEs are a large multi-gene family (81 genes) in diploid cotton (Gossypium raimondii), an expansion over the 66 in Arabidopsis and suggests the evolution of new functions in cotton. Relatively few PME genes are expressed highly in fibres based on EST abundance and the five most abundant in fibres were cloned and sequenced from two cotton species. Their significant sequence differences and their stage-specific expression in fibres within a species suggest sub-specialisation during fibre development. We determined the transcript abundance of the five fibre PMEs, total PME enzyme activity, pectin content and extent of de-methylesterification of the pectin in fibre walls of the two cotton species over the first 25-30 days of fibre growth. There was a higher transcript abundance of fibre-PMEs and a higher total PME enzyme activity in G. barbadense (Gb) than in G. hirsutum (Gh) fibres, particularly during late fibre elongation. Total pectin was high, but de-esterified pectin was low during fibre elongation (5-12 dpa) in both Gh and Gb. De-esterified pectin levels rose thereafter when total PME activity increased and this occurred earlier in Gb fibres resulting in a lower degree of esterification in Gb fibres between 17 and 22 dpa. Gb fibres are finer and longer than those of Gh, so differences in pectin remodelling during the transition to wall thickening may be an important factor in influencing final fibre diameter and length, two key quality attributes of cotton fibres.

The cell wall of the Arabidopsis pollen tube--spatial distribution, recycling, and network formation of polysaccharides.

The pollen tube is a cellular protuberance formed by the pollen grain, or male gametophyte, in flowering plants. Its principal metabolic activity is the synthesis and assembly of cell wall material, which must be precisely coordinated to sustain the characteristic rapid growth rate and to ensure geometrically correct and efficient cellular morphogenesis. Unlike other model species, the cell wall of the Arabidopsis (Arabidopsis thaliana) pollen tube has not been described in detail. We used immunohistochemistry and quantitative image analysis to provide a detailed profile of the spatial distribution of the major cell wall polymers composing the Arabidopsis pollen tube cell wall. Comparison with predictions made by a mechanical model for pollen tube growth revealed the importance of pectin deesterification in determining the cell diameter. Scanning electron microscopy demonstrated that cellulose microfibrils are oriented in near longitudinal orientation in the Arabidopsis pollen tube cell wall, consistent with a linear arrangement of cellulose synthase CESA6 in the plasma membrane. The cellulose label was also found inside cytoplasmic vesicles and might originate from an early activation of cellulose synthases prior to their insertion into the plasma membrane or from recycling of short cellulose polymers by endocytosis. A series of strategic enzymatic treatments also suggests that pectins, cellulose, and callose are highly cross linked to each other.

[Elastin and microfibrils in vascular development and ageing: complementary or opposite roles?]. / Rôle de l'élastine et des microfibrilles de la paroi artérielle au cours du développement et du vieillissement: complémentarité ou opposition?

Large arteries allow the vascular system to be more than a simple route in which the blood circulates within the organism. The elastic fibers present in the wall endow these vessels with elasticity and are responsible for the smoothing of the blood pressure and flow, which are delivered discontinuously by the heart. This function is very important to ensure appropriate hemodynamics. Elastic fibers are composed of elastin (90%) and fibrillin-rich microfibrils (10%) which provide the vessels with elasticity and are also signals able to bind to relatively specific cell membrane receptors. Stimulation of the high affinity elastin receptor by elastin peptides or tropoelastin--the elastin precursor--triggers an increase in intracellular free calcium in vascular cells, especially endothelial cells, associated with attachment, migration or proliferation. Similar effects of the stimulation of endothelial cells by microfibrils or fibrillin-1 fragments, which bind to integrins, have been demonstrated. This dual function--mechanical and in signaling--makes the elastic fibers an important actor of the development and ageing processes taking place in blood vessels. An alteration of the elastin (Eln) or fibrillin (Fbn) gene products leads to severe genetic pathologies of the cardiovascular system, such as supravalvular aortic stenosis, or Williams Beuren syndrome--in which elastin deficiency induces aortic stenoses--or Marfan syndrome, in which on the contrary fibrillin-1 deficiency promotes the appearance of aortic aneurysms. Genetically-engineered mouse models of these pathologies (such as Eln+/- mice and Fbn-1+/mgΔ mice, Eln+/-Fbn-1+/- mice) have permitted a better understanding of the pathogenesis of these syndromes. In particular, it has been shown that elastin and fibrillin-1 roles can be complementary in some aspects, while they can be opposed in some other situations. For instance, the double heterozygosity in elastin and fibrillin-1 leads to increased arterial wall stress--compared to the level induced by one of these two deficiencies alone--while the decrease in diameter induced by Eln deficiency is partly compensated by an additional deficiency in Fbn-1. Also, it is now clear that early modifications of elastin or fibrillin-1 availability can alter the normal signaling action of these proteins and lead to long term modifications of the vascular physiology and ageing processes.

Functional plant cell wall design revealed by the Raman imaging approach.

Using the Raman imaging approach, the optimization of the plant cell wall design was investigated on the micron level within different tissue types at different positions of a Phormium tenax leaf. Pectin and lignin distribution were visualized and the cellulose microfibril angle (MFA) of the cell walls was determined. A detailed analysis of the Raman spectra extracted from the selected regions, allowed a semi-quantitative comparison of the chemical composition of the investigated tissue types on the micron level. The cell corners of the parenchyma revealed almost pure pectin and the cell wall an amount of 38-49% thereof. Slight lignification was observed in the parenchyma and collenchyma in the top of the leaf and a high variability (7-44%) in the sclerenchyma. In the cell corners and in the cell wall of the sclerenchymatic fibres surrounding the vascular tissue, the highest lignification was observed, which can act as a barrier and protection of the vascular tissue. In the sclerenchyma high variable MFA (4°-40°) was detected, which was related with lignin variability. In the primary cell walls a constant high MFA (57°-58°) was found together with pectin. The different plant cell wall designs on the tissue and microlevel involve changes in chemical composition as well as cellulose microfibril alignment and are discussed and related according to the development and function.

Onion epidermis as a new model to study the control of growth anisotropy in higher plants.

To elucidate the role of cellulose microfibrils in the control of growth anisotropy, a link between their net orientation, in vitro cell wall extensibility, and anisotropic cell expansion was studied during development of the adaxial epidermis of onion (Allium cepa) bulb scales using polarization confocal microscopy, creep tests, and light microscopy. During growth the net cellulose alignment across the whole thickness of the outer epidermal wall changed from transverse through random to longitudinal and back to transverse relative to the bulb axis. Cell wall extension in vitro was always higher transverse than parallel to the net cellulose alignment. The direction of growth anisotropy was perpendicular to the net microfibril orientation and changed during development from longitudinal to transverse to the bulb axis. The correlation between the degree of growth anisotropy and the net cellulose alignment was poor. Thus the net cellulose microfibril orientation across the whole thickness of the outer periclinal epidermis wall defines the direction but not the degree of growth anisotropy. Strips isolated from the epidermis in the directions perpendicular and transverse to a net cellulose orientation can be used as an extensiometric model to prove a protein involvement in the control of growth anisotropy.

Pectins influence microfibril aggregation in celery cell walls: An atomic force microscopy study.

Atomic force microscopy (AFM) was used to image the microfibrilar network of celery parenchyma cell wall material (CWM), before and after each step in the selective extraction of pectins and hemicelluloses. The images collected were subjected to image analysis and the diameters of the cellulose microfibrils were measured following each step in the extraction process. Not only was an increase in the mean size of the microfibrils observed as the pectins were selectively removed, but an increase in the proportion of large to small microfibrils was also observed. This suggests that removal of the pectic matrix not only results in the swelling of existing microfibrils, but also removal of pectins would enable the microfibrils to move closer together within the cell wall, and hence have a greater tendency to self-associate and form aggregates.

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged.

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.

The mechanical function and structure of aortic microfibrils in the lobster Homarus americanus.

Marfan syndrome, a connective tissue disorder affecting the cardiovascular system, is caused by mutations of fibrillin-based microfibrils. These mutations often affect the calcium-binding domains, resulting in structural changes to the proteins. It is hypothesized that these Ca+2 binding sites regulate the structure and mechanical properties of the microfibrils. The mechanical properties of fresh and extracted lobster aortic rings in calcium solutions (1, 13 and 30 mM Ca+2) were measured. Samples underwent amino acid compositional analysis. Antibodies were produced against the material comprising extracted aortic rings. The ultrastructure of strained and unstrained samples was examined using transmission electron microscopy. Calcium level altered the tangent modulus of fresh vessels. These rings were significantly stiffer when tested at 30 mM Ca+2 compared to rings tested at 1 mM Ca+2. Amino acid comparisons between extracted samples, porcine and human fibrillin showed compositional similarity. Immunohistochemical analysis showed that antibodies produced against the material in extracted samples localized to the known microfibrillar elements in the lobster aorta and cross-reacted with fibrillin microfibrils of mammalian ciliary zonules. Ultrastructurally, vessels incubated in low calcium solutions showed diffuse interbead regions while those incubated in physiological or high calcium solutions showed interbead regions with more defined lateral edges.

Secondary cell-wall assembly in flax phloem fibres: role of galactans.

Non-lignified fibre cells (named gelatinous fibres) are present in tension wood and the stems of fibre crops (such as flax and hemp). These cells develop a very thick S2 layer within the secondary cell wall, which is characterised by (1) cellulose microfibrils largely parallel to the longitudinal axis of the cell, and (2) a high proportion of galactose-containing polymers among the non-cellulosic polysaccharides. In this review, we focus on the role of these polymers in the assembly of gelatinous fibres of flax. At the different stages of fibre development, we analyse in detail data based on sugar composition, linkages of pectic polymers, and immunolocalisation of the beta-(1-->4)-galactans. These data indicate that high molecular-mass gelatinous galactans accumulate in specialised Golgi-derived vesicles during fibre cell-wall thickening. They consist of RG-I-like polymers with side chains of beta-(1-->4)-linked galactose. Most of them are short, but there are also long chains containing up to 28 galactosyl residues. At fibre maturity, two types of cross-linked galactans are identified, a C-L structure that resembles the part of soluble galactan with long side chains and a C-S structure with short chains. Different possibilities for soluble galactan to give rise to C-L and C-S are analysed. In addition, we discuss the prospect for the soluble galactan in preventing the newly formed cellulose chains from completing immediate crystallisation. This leads to a hypothesis that firstly the secretion of soluble galactans plays a role in the axial orientation of cellulose microfibrils, and secondly the remodelling and cross-linking of pectic galactans are linked to the dehydration and the assembly of S2 layer.

Mechanical properties of primary plant cell wall analogues.

Mechanical effects of turgor pressure on cell walls were simulated by deforming cell wall analogues based on Acetobacter xylinus cellulose under equi-biaxial tension. This experimental set-up, with associated modelling, allowed quantitative information to be obtained on cellulose alone and in composites with pectin and/or xyloglucan. Cellulose was the main load-bearing component, pectin and xyloglucan leading to a decrease in modulus when incorporated. The cellulose-only system could be regarded as an essentially linear elastic material with a modulus ranging from 200 to 500 MPa. Pectin incorporation modified extensibility properties of the system by topology/architecture changes of cellulose fibril assemblies, but the cellulose/pectin composites could still be described as a linear elastic material with a modulus ranging from 120 to 250 MPa. The xyloglucan/cellulose composite could not be modelled as a linear elastic material. Introducing xyloglucan into a cellulose network or a cellulose/pectin composite led to very compliant materials characterised by time-dependent creep behaviour. Modulus values obtained for the composite materials were compared with mechanical data found for plant-derived systems. After comparing bi-axial and uni-axial behaviour of the different composites, structural models were proposed to explain the role of each polysaccharide in determining the mechanical properties of these plant primary cell wall analogues.

In vitro versus in vivo cellulose microfibrils from plant primary wall synthases: structural differences.

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1-->3)-beta-d-glucan (callose). The taurocholate extracts, which did not require the addition of Mg(2+), were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV(I), a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.