Low Molecular Weight Hyaluronan Research Articles

Bioremodeling of the Forehead and Inferior Temples using Hybrid Cooperative Complexes of High and Low Molecular Weight Hyaluronans (Profhilo®)

Bioremodeling of the Forehead and Inferior Temples using Hybrid Cooperative Complexes of High and Low Molecular Weight Hyaluronans (Profhilo®)

Development of a drying method for proteins based on protein-hyaluronic acid precipitation

This study aims to develop a new method to dry proteins based on protein-hyaluronic acid (HA) precipitation and apply the precipitation-redissolution technique to develop highly concentrated protein formulations. Lysozyme was used as a model protein and HA with various molecular weights (MW) were investigated. Under low ionic strength, low-MW HA (e.g., MW: around 5 K) did not induce lysozyme precipitation. Conversely, high-MW HA (e.g., MW: approximately from 40 K to 1.5 M) precipitated more than 90 % of lysozyme. The dried lysozyme-HA precipitates had moisture levels between 4 % and 5 %. The lysozyme-HA precipitates could be redissolved using PBS (pH 7.4, ionic strength: ∼ 163 mM). The viscosity of the reconstituted solution was dependent on HA MW, e.g., 4 cP for HA40K, and 155 cP for HA1.5 M, suggesting low-MW HA might be a proper excipient for highly concentrated solution formulations for subcutaneous/intraocular injection and high-MW HA may fit for other applications. The tertiary structure of lysozyme after the precipitation-redissolution steps had no significant difference from that of the original lysozyme as confirmed by fluorescence spectroscopy. The denaturation temperature of lysozyme after the precipitation-redissolution steps and that of the original lysozyme were close, indicating they possessed similar thermal stability. The accelerated stability study revealed that lysozyme stored in the dry precipitates was more physically stable than that in the buffer solution. Overall, this new drying technique is suitable for drying proteins and exhibits several benefits such as minimum energy consumption, cost effectiveness, high production yield, and mild processing conditions. In addition, the precipitation-redissolution technique proposed in this study can potentially be used to develop highly concentrated formulations, especially for proteins experiencing poor stability in the liquid state.

High prevalence of CD44 and its ligand low molecular weight hyaluronan in plasma of HNSCC patients: clinical significance.

This study aims to evaluate the role of cancer stem cell marker, CD44, and its ligand HA as potential molecular biomarker for early detection of HNSCC. The expression profile (mRNA/Protein) of CD44 variants were analysed in primary HNSCC lesions and plasma of the patients. Then, prevalence of HA variants was analysed in plasma of the patients. The mRNA expression of CD44 variants, CD44S and CD44v3, were significantly high in both early (stage I/II) and late (stage III/IV) invasive lesions, with predominant expression of CD44v3 in the late-stage lesions. In plasma of HNSCC patients, increased levels of SolCD44, CD44-ICD and unique 62 KD CD44 variants with respect to standard CD44S were seen, in comparison to their prevalence in plasma of normal individuals. The abundance of CD44-ICD and 62 KD variants were significantly high in plasma of late stage HNSCC patients. Interestingly, significantly high level of low molecular weight HA(LMW HA) with respect to high molecular weight HA(HMW HA) was seen in plasma of HNSCC patients irrespective of clinical stages. On the contrary, high HMW HA level in plasma of normal individuals was seen. The high level of LMW HA in plasma of HNSCC patients might be due to combinatorial effect of increased mRNA expression of HA synthesizing enzyme HAS1/2/3 and HA degrading enzyme HYAL1/2, as seen in the primary HNSCC samples. Thus, our data revealed the importance of specific CD44 and HA variants in plasma of HNSCC patients during its development as potential non-invasive molecular biomarker of the disease.

ROS-Mediated Fragmentation Alters the Effects of Hyaluronan on Corneal Epithelial Wound Healing.

A buildup of reactive oxygen species (ROS) occurs in virtually all pathological conditions. Hyaluronan (HA) is a major extracellular matrix component and is susceptible to oxidation by reactive oxygen species (ROS), yet the precise chemical structures of oxidized HA products (oxHA) and their physiological properties remain largely unknown. This study characterized the molecular weight (MW), structures, and physiological properties of oxHA. For this, high-molecular-weight HA (HMWHA) was oxidized using increasing molar ratios of hydrogen peroxide (H2O2) or hypochlorous acid (HOCl). ROS lead to the fragmentation of HA, with the oxHA products produced by HOCl exhibiting an altered chemical structure while those produced by H2O2 do not. HMWHA promotes the viability of human corneal epithelial cells (hTCEpi), while low MWHA (LMWHA), ultra-LMWHA (ULMWHA), and most forms of oxHA do not. HMWHA and LMWHA promote hTCEpi proliferation, while ULMWHA and all forms of oxHA do not. LMWHA and some forms of oxHA promote hTCEpi migration, while HMWHA does not. Finally, all native forms of HA and oxHA produced by HOCl promote in vivo corneal wound healing, while oxHA produced by H2O2 does not. Taken together, our results show that HA fragmentation by ROS can alter the physiological activity of HA by altering its MW and structure.

Injecting hyaluronan in the thoracolumbar fascia: A model study

Hyaluronan (HA) has been recently identified as a key component of the densification of thoracolumbar fascia (TLF), a potential contributor to non-specific lower back pain (LBP) currently treated with manual therapy and systemic or local delivery of anti-inflammatory drugs. The aim of this study was to establish a novel animal model suitable for studying ultrasound-guided intrafascial injection prepared from HA with low and high Mw. Effects of these preparations on the profibrotic switch and mechanical properties of TLF were measured by qPCR and rheology, respectively, while their lubricating properties were evaluated by tribology. Rabbit proved to be a suitable model of TLF physiology due to its manageable size enabling both TLF extraction and in situ intrafascial injection. Surprisingly, the tribology showed that low Mw HA was a better lubricant than the high Mw HA. It was also better suited for intrafascial injection due to its lower injection force and ability to freely spread between TLF layers. No profibrotic effects of either HA preparation in the TLF were observed. The intrafascial application of HA with lower MW into the TLF appears to be a promising way how to increase the gliding of the fascial layers and target the myofascial LBP.

Isolation and identification of hyaluronan-degrading bacteria from Japanese fecal microbiota.

Hyaluronan (HA) is a high-molecular-weight glycosaminoglycan and widely distributed in all connective tissues and organs with diverse biological functions. HA has been increasingly used as dietary supplements targeted to joint and skin health for humans. We here first report isolation of bacteria from human feces that are capable of degrading HA to lower molecular weight HA oligosaccharides (oligo-HAs). The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted feces of healthy Japanese donors were individually incubated in an enrichment medium containing HA, followed by the isolation of candidate strains from streaked HA-containing agar plates and selection of HA-degrading strains by measuring HA using an ELISA. Subsequent genomic and biochemical assays identified the strains as Bacteroides finegoldii, B. caccae, B. thetaiotaomicron, and Fusobacterium mortiferum. Furthermore, our HPLC analysis revealed that the strains degraded HA to oligo-HAs of various lengths. Subsequent quantitative PCR assay targeting the HA degrading bacteria showed that their distribution in the Japanese donors varied. The evidence suggests that dietary HA is degraded by the human gut microbiota with individual variation to oligo-HAs components, which are more absorbable than HA, thereby exerting its beneficial effects.

Hyaluronan regulates sperm-induced inflammatory response by enhancing sperm attachment to bovine endometrial epithelial cells via CD44: in-silico and in-vitro approaches.

Recently, we reported that sperm induce cluster of differentiation 44 (CD44) expression and Toll-like receptor 2 (TLR2)-mediated inflammatory response in bovine uterus. In the present study, we hypothesized that the interaction between CD44 of bovine endometrial epithelial cells (BEECs) and hyaluronan (HA) affects sperm attachment and thereby enhancing TLR2-mediated inflammation. To test our hypothesis, at first, in-silico approaches were employed to define the binding affinity of HA for CD44 and TLR2. Further, an in-vitro experiment using the sperm-BEECs co-culture model was applied to investigate the effect of HA on sperm attachment and inflammatory response. Here, low molecular weight (LMW) HA at different concentrations (0, 0.1, 1, or 10 µg/mL) was incubated with BEECs for 2 h followed by the co-culture without- or with non-capacitated washed sperm (106/ml) for additional 3 h was performed. The present in-silico model clarified that CD44 is a high-affinity receptor for HA. Moreover, TLR2 interactions with HA oligomer (4- and 8-mers) target a different subdomain (h-bonds) compared to TLR2-agonist (PAM3) which targets a central hydrophobic pocket. However, the interaction of LMW HA (32-mers) with TLR2 revealed no stability of HA at any pocket of TLR2. Notably, the immunofluorescence analysis revealed the HA localization in both endometrial stroma and epithelia of ex-vivo endometrial explant. Moreover, ELISA showed significant levels of HA in BEECs culture media. Importantly, BEECs pretreatment with HA prior to sperm exposure increased the number of attached sperm to BEECs, and upregulated the transcriptional levels of pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs in response to sperm. However, BEECs treated with HA only (no sperm exposure) did not show any significant effect on the transcript abundance of pro-inflammatory genes when compared to the non-treated BEECs. Altogether, our findings strongly suggest a possible cross-talk between sperm and endometrial epithelial cells via HA and HA binding receptors (CD44 and TLR2) to induce a pro-inflammatory response in bovine uterus.

Characterization of the Molecular Weight of Hyaluronan in Eye Products Using a Novel Method of Size Exclusion High-Pressure Liquid Chromatography.

Hyaluronan (HA) exists in two forms, high molecular weight HA (HMWHA) and low molecular weight HA (LMWHA), which have distinct physiological functions. Therefore it is imperative to know the form of HA within pharmaceutical products, including eye products. This study developed an accurate, sensitive, and quantitative method to characterize the form of HA in eye products. Thereafter, the effects of the HA-containing eye products on corneal wound healing were investigated. The MW distributions and concentrations of HA in over the counter eye products were determined by size exclusion chromatography (SEC) high-pressure liquid chromatography (HPLC). The effects of the eye products containing HA on corneal wound healing were characterized both in vitro and in vivo using the scratch assay and the debridement wound model, respectively. The concentrations and MWs of HA were successfully determined within a range of 0.014 to 0.25 mg/mL using SEC HPLC. The concentrations of HA in the ophthalmic products varied from 0.14 to 4.0 mg/mL and the MWs varied from ∼100 kDa to >2500 kDa. All but one HA-containing eye product had an inhibitory effect on corneal wound healing, whereas pure HA promoted corneal wound healing. A novel SEC-HPLC method was developed for quantifying and characterizing the MW of HA in eye products. Although HA promoted corneal wound healing, HA-containing eye products inhibited corneal wound healing, likely caused by preservatives. SEC-HPLC could be implemented as a routine method for determining the form of HA in commercially available ophthalmic products.

Hyaluronan and Reactive Oxygen Species Signaling-Novel Cues from the Matrix?

Hyaluronan (HA) is a naturally occurring non-sulfated glycosaminoglycan (GAG) localized to the cell surface and the tissue extracellular matrix (ECM). It is composed of disaccharides containing glucuronic acid and N-acetylglucosamine, is synthesized by the HA synthase (HAS) enzymes and is degraded by hyaluronidase (HYAL) or reactive oxygen and nitrogen species (ROS/RNS) actions. HA is deposited as a high molecular weight (HMW) polymer and degraded to low molecular weight (LMW) fragments and oligosaccharides. HA affects biological functions by interacting with HA-binding proteins (hyaladherins). HMW HA is anti-inflammatory, immunosuppressive, and antiangiogenic, whereas LMW HA has pro-inflammatory, pro-angiogenetic, and oncogenic effects. ROS/RNS naturally degrade HMW HA, albeit at enhanced levels during tissue injury and inflammatory processes. Thus, the degradation of endothelial glycocalyx HA by increased ROS challenges vascular integrity and can initiate several disease progressions. Conversely, HA exerts a vital role in wound healing through ROS-mediated HA modifications, which affect the innate immune system. The normal turnover of HA protects against matrix rigidification. Insufficient turnover leads to increased tissue rigidity, leading to tissue dysfunction. Both endogenous and exogenous HMW HA have a scavenging capacity against ROS. The interactions of ROS/RNS with HA are more complex than presently perceived and present an important research topic.

Hyaluronan breakdown by snake venom hyaluronidases: From toxins delivery to immunopathology.

Snake venom enzymes have a broad range of molecular targets in plasma, tissues, and cells, among which hyaluronan (HA) is outstanding. HA is encountered in the extracellular matrix of diverse tissues and in the bloodstream, and its different chemical configurations dictate the diverse morphophysiological processes in which it participates. Hyaluronidases are highlighted among the enzymes involved in HA metabolism. This enzyme has been detected along the phylogenetic tree, suggesting that hyaluronidases exert multiple biological effects on different organisms. Hyaluronidases have been described in tissues, blood and snake venoms. Snake venom hyaluronidases (SVHYA) contribute to tissue destruction in envenomations and are called spreading factors since their action potentiates venom toxin delivery. Interestingly, SVHYA are clustered in Enzyme Class 3.2.1.35 together with mammalian hyaluronidases (HYAL). Both HYAL and SVHYA of Class 3.2.1.35 act upon HA, generating low molecular weight HA fragments (LMW-HA). LMW-HA generated by HYAL becomes a damage-associated molecular pattern that is recognized by Toll-like receptors 2 and 4, triggering cell signaling cascades culminating in innate and adaptive immune responses that are characterized by lipid mediator generation, interleukin production, chemokine upregulation, dendritic cell activation and T cell proliferation. In this review, aspects of the structures and functions of HA and hyaluronidases in both snake venoms and mammals are presented, and their activities are compared. In addition, the potential immunopathological consequences of HA degradation products generated after snakebite envenoming and their use as adjuvant to enhance venom toxin immunogenicity for antivenom production as well as envenomation prognostic biomarker are also discussed.

Impact of hyaluronan size on localization and solubility of the extracellular matrix in the mouse brain.

Hyaluronan (HA) is a central component of the extracellular matrix (ECM) in the brain and plays a pivotal role in neural development and plasticity. Brain HA exists in 2 distinct forms of the ECM: the diffuse ECM, which is soluble in saline and detergents, and the condensed ECM, which forms aggregates, such as perineuronal nets (PNNs). Although the physiological functions of HA significantly differ depending on its size, size differences in HA have not yet been examined in the 2 ECM types, which is partly because of the lack of methods to rapidly and accurately measure the molecular weight (MW) of HA. In this study, we established a simple method to simultaneously assess the MW of HA in multiple crude biological samples. HA was purified through single-step precipitation from tissue extracts using biotinylated HA-binding protein and streptavidin-coupled magnetic beads, followed by separation on gel electrophoresis. By applying this method to HA in the mouse brain, we revealed that the condensed ECM contained higher MW HA than the diffuse ECM. Higher MW HA and lower MW HA exhibited different spatial distributions: the former was confined to PNNs, whereas the latter was widely present throughout the brain. Furthermore, the limited degradation of HA showed that only higher MW HA was required to form an insoluble HA-aggrecan complex. The present study demonstrated that the MW of HA in the brain strongly correlates with the localization and solubility of the ECM it forms.

Insight into the assembly of lipid-hyaluronan complexes in osteoarthritic conditions.

Interactions between molecules in the synovial fluid and the cartilage surface may play a vital role in the formation of adsorbed films that contribute to the low friction of cartilage boundary lubrication. Osteoarthritis (OA) is the most common degenerative joint disease. Previous studies have shown that in OA-diseased joints, hyaluronan (HA) not only breaks down resulting in a much lower molecular weight (MW), but also its concentration is reduced ten times. Here, we have investigated the structural changes of lipid-HA complexes as a function of HA concentration and MW to simulate the physiologically relevant conditions that exist in healthy and diseased joints. Small angle neutron scattering and dynamic light scattering were used to determine the structure of HA-lipid vesicles in bulk solution, while a combination of atomic force microscopy and quartz crystal microbalance was applied to study their assembly on a gold surface. We infer a significant influence of both MW and HA concentrations on the structure of HA-lipid complexes in bulk and assembled on a gold surface. Our results suggest that low MW HA cannot form an amorphous layer on the gold surface, which is expected to negatively impact the mechanical integrity and longevity of the boundary layer and could contribute to the increased wear of the cartilage that has been reported in joints diseased with OA.

Hybrid high- and low-molecular weight chains of hyaluronan is an effective modality treatment for increasing female sexual satisfaction: An interventional, randomized-control parallel study.

Psychological, behavioral, hormonal, surgical, and psychopharmacologic approaches are the only acknowledged treatments for Female Sexual Dysfunction (FSD). The study is conducted to assess the efficacy of hybrid cooperative complexes of high and low molecular weight hyaluronan (hybrid H-HA/L-HA) in treating females with varying of sexual dysfunction and to assess the female genital self-image scale (FGSIS), female sexual function index (FSFI), and dermatology life quality index (DLQI) before and after treatment. Sixty female patients were divided into two groups. The study group enrolled 30 female patients injected with hybrid H-HA/L-HA, while the control group enrolled 30 female patients injected with saline. Patients were recruited from the clinic searching for medical advice. Controls were selected from close associates of the cases who were attending with the patients or healthy escorts of dermatology patients attending the dermatology outpatient clinic. We assessed socio-demographic, clinical evaluation, the (FGSIS), (FSFI), and (DLQI) before and after treatment. The first assessment was conducted at the first visit, and the second assessment was conducted after 1 month of the second injection. Significant increase in the frequency of sexual intercourse/week in the study group after the first and second injection sessions compared to the controls was observed (p < 0.05). There was statistically significant amelioration in desire, arousal, lubrication, orgasm, satisfaction domains, and total score of the FSFI (p ≤ 0.05). The study demonstrated significant increasing differences in all domains of the FGSIS (p ≤ 0.05). The symptoms and feelings, leisure, personal relationships, and total scores were significantly higher post first and second injection of (hybrid H-HA/L-HA) sessions compared to the controls (p < 0.05). The (hybrid H-HA/L-HA) injection for rejuvenating the genital area appears to be a safe and effective way to enhance female genital self-image, sexuality, and quality of life with high levels of satisfaction as a minimally invasive method.

Low molecular weight hyaluronan inhibits lung epithelial ion channels by activating the calcium-sensing receptor

Herein, we tested the hypothesis that low molecular weight hyaluronan (LMW-HA) inhibits lung epithelial ions transport in-vivo, ex-vivo, and in-vitro by activating the calcium-sensing receptor (CaSR). Twenty-four hours post intranasal instillation of 50–150 µg/ml LMW-HA to C57BL/6 mice, there was a 75% inhibition of alveolar fluid clearance (AFC), a threefold increase in the epithelial lining fluid (ELF) depth, and a 20% increase in lung wet/dry (W/D) ratio. Incubation of human and mouse precision cut lung slices with 150 µg/ml LMW-HA reduced the activity and the open probability (Po) of epithelial sodium channel (ENaC) in alveolar epithelial type 2 (ATII) cells, and in mouse tracheal epithelial cells (MTEC) monolayers as early as 4 h. The Cl− current through cystic fibrosis transmembrane conductance regulator (CFTR) and the activity of Na,K-ATPase were both inhibited by more than 66% at 24 h. The inhibitory effects of LMW-HA on ion channels were reversed by 1 µM NPS-2143, or 150 µg/ml high molecular weight hyaluronan (HMW-HA). In HEK-293 cells expressing the calcium-sensitive Cl− channel TMEM16-A, CaSR was required for the activation of the Cl− current by LMW-HA. This is the first demonstration of lung ions and water transport inhibition by LMW-HA, and its mediation through the activation of CaSR.

The Effect of Exposure to Escherichia coli with Low Molecular Weight Hyaluronan, High Molecular Weight Hyaluronan, Hyaluronidase in the Cervix Premature Pregnant Model of Balb/C Mice

Introduction: Infection is the biggest cause of preterm labor, about 25-40%, and ascending infection through the vagina is the most common route of infection. This study aims to evaluate the level of Low Molecular Weight Hyaluronan (LMWHA), level of High Molecular Weight Hyaluronan (HMWHA), and the level of the enzyme hyaluronidase in Balb/c premature labor due to infection with Escherichia coli model mice. Methods: This study used an actual experimental design carried out in the laboratory in vivo on balb/c mice exposure to Escherichia coli with a randomized post-test only with a control group design. In the premature model of exposure to Escherichia coli balb/c mice on the cervix of 109 CFU/ml, cervical samples were taken and the levels of LMWHA, HMWHA, and hyaluronidase were measured using spectrophotometry. This study was divided into 2 groups. Control group and treatment group. Results: Low Molecular Weight Hyaluronan (LMWHA) and of the hyaluronidase enzyme levels in premature Balb/C cervical mice exposed to E. coli were higher than in Balb/C cervical mice. High Molecular Weight Hyaluronan (HMWHA) levels in premature Balb/C cervical mice exposed to E. coli were lower Conclusion: In the cervix of the infected by E. coli mice model, LMWHA and hyaluronidase enzymes increased and HMWHA decreased.

Improving the Mechanical Properties of Damaged Hair Using Low-Molecular Weight Hyaluronate.

Chemical treatments of hair such as dyeing, perming and bleaching could cause mechanical damage to the hair, which weakens the hair fibers and makes the hair break more easily. In this work, hyaluronate (HA) with different molecular weight (MW) was investigated for its effects on restoring the mechanical properties of damaged hair. It was found that low-MW HA (average MW~42 k) could significantly improve the mechanical properties, specifically the elastic modulus, of overbleached hair. The fluorescent-labeling experiments verified that the low-MW HA was able to penetrate into the cortex of the hair fiber, while high-MW HA was hindered. Fourier transform infrared spectrometry (FT-IR) results implied the formation of additional intermolecular hydrogen bonds in the HA-treated hair. Thermos gravimetric analysis (TGA) indicated that the HA-treated hair exhibited decreased content of loosely bonded water, and differential scanning calorimetry (DSC) characterizations suggested stronger water bonding inside the HA-treated hair, which could alleviate the weakening effect of loosely bonded water on the hydrogen bond networks within keratin. Therefore, the improved elastic modulus and mechanical strength of the HA-treated hair could be attributed to the enhanced formation of hydrogen bond networks within keratin. This study illustrates the capability of low-MW HA in hair damage repair, implying an enormous potential for other moisturizers to be used in hair care products.

Hybrid cooperative complexes of high and low molecular weight hyaluronans (96 mg/3 mL) for the treatment of skin laxity of the inner arm and abdomen

article: Hybrid cooperative complexes of high and low molecular weight hyaluronans (96 mg/3 mL) for the treatment of skin laxity of the inner arm and abdomen - Gazzetta Medica Italiana - Archivio per le Scienze Mediche 2022 July-August;181(7-8):487-95 - Minerva Medica - Journals

Key Role of Hyaluronan Metabolism for the Development of Brain Metastases in Triple-Negative Breast Cancer.

Breast cancer (BC) is the second-most common cause of brain metastases (BM) and BCBM patients have a reduced quality of life and a poor prognosis. Hyaluronan (HA), and in particular the hyaluronidase Hyal-1, has been already linked to the development of BCBM, and therefore presents an interesting opportunity to develop new effective therapeutic options. HA metabolism was further discovered by the CRISPR/Cas9-mediated knockout of HYAL1 and the shRNA-mediated down-regulation of HA-receptor CD44 in the brain-seeking triple-negative breast cancer (TNBC) cell line MDA-MB-231-BR. Therefore, the impact of Hyal-1 on adhesion, disruption, and invasion through the brain endothelium, both in vitro and in vivo, was studied. Our analysis points out a key role of Hyal-1 and low-molecular-weight HA (LMW-HA) in the formation of a pericellular HA-coat in BC cells, which in turn promotes tumor cell adhesion, disruption, and migration through the brain endothelium in vitro as well as the extent of BM in vivo. CD44 knockdown in MDA-MB-231-BR significantly reduced the pericellular HA-coat on these cells, and, consequently, tumor cell adhesion and invasion through the brain endothelium. Thus, the interaction between Hyal-1-generated LMW-HA fragments and the HA-receptor CD44 might represent a potential target for future therapeutic options in BC patients with a high risk of cerebral metastases formation.

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

A Mechanoelastic Glimpse on Hyaluronan-Coated Extracellular Vesicles.

Cancer cells secrete extracellular vesicles (EVs) covered with a carbohydrate polymer, hyaluronan (HA), linked to tumor malignancy. Herein, we have unravelled the contour lengths of HA on a single cancer cell-derived EV surface using single-molecule force spectroscopy (SMFS), which divulges the presence of low molecular weight HA (LMW-HA < 200 kDa). We also discovered that these LMW-HA-EVs are significantly more elastic than the normal cell-derived EVs. This intrinsic elasticity of cancer EVs could be directly allied to the LMW-HA abundance and associated labile water network on EV surface as revealed by correlative SMFS, hydration dynamics with fluorescence spectroscopy, and molecular dynamics simulations. This method emerges as a molecular biosensor of the cancer microenvironment.