Screening core genes for minimal change disease based on bioinformatics and machine learning approaches.

Based on bioinformatics and machine learning methods, we conducted a study to screen the core genes of minimal change disease (MCD) and further explore its pathogenesis. First, we obtained the chip data sets GSE108113 and GSE200828 from the Gene Expression Comprehensive Database (GEO), which contained MCD information. We then used R software to analyze the gene chip data and performed functional enrichment analysis. Subsequently, we employed Cytoscape to screen the core genes and utilized machine learning algorithms (random forest and LASSO regression) to accurately identify them. To validate and analyze the core genes, we conducted immunohistochemistry (IHC) and gene set enrichment analysis (GSEA). Our results revealed a total of 394 highly expressed differential genes. Enrichment analysis indicated that these genes are primarily involved in T cell differentiation and p13k-akt signaling pathway of immune response. We identified NOTCH1, TP53, GATA3, and TGF-ß1 as the core genes. IHC staining demonstrated significant differences in the expression of these four core genes between the normal group and the MCD group. Furthermore, GSEA suggested that their up-regulation may be closely associated with the pathological changes in MCD kidneys, particularly in the glycosaminoglycans signaling pathway. In conclusion, our study highlights NOTCH1, TP53, GATA3, and TGF-ß1 as the core genes in MCD and emphasizes the close relationship between glycosaminoglycans and pathogenesis of MCD.

Microgels With Electrostatically Controlled Molecular Affinity to Direct Morphogenesis.

Concentration gradients of soluble signaling molecules-morphogens-determine the cellular organization in tissue development. Morphogen-releasing microgels have shown potential to recapitulate this principle in engineered tissue constructs, however, with limited control over the molecular cues in space and time. Inspired by the functionality of sulfated glycosaminoglycans (sGAGs) in morphogen signaling in vivo, a library of sGAG-based microgels is developed and designated as µGel Units to Instruct Development (µGUIDEs). Adjustment of the microgel's sGAG sulfation patterns and concentration enabled the programming of electrostatic affinities that control the release of morphogens. Based on computational analyses of molecular transport processes, µGUIDEs provided unprecedented precision in the spatiotemporal modulation of vascular endothelial growth factor (VEGF) gradients in a microgel-in-gel vasculogenesis model and kidney organoid cultures. The versatile approach offers new options for creating morphogen signaling centers to advance the understanding of tissue and organ development.

Natural history of valve disease in patients with mucopolysaccharidosis II and the impact of enzyme replacement therapy.

Mucopolysaccharidosis II (MPS II, Hunter syndrome) is a rare, X-linked lysosomal storage disease caused by reduced activity of iduronate-2-sulfatase (I2S), with subsequent cellular accumulation of the glycosaminoglycans (GAGs), heparan sulfate, and dermatan sulfate (DS). DS is a major component of the extracellular matrix of heart valves, which can be affected in MPS II. We investigated the natural history of valve disease in MPS II and the impact of long-term intravenous enzyme replacement therapy (ERT) with recombinant I2S (idursulfase). In total, 604 cardiac examinations were assessed from serial follow-up of 80 male patients (49 neuronopathic). Valve disease was classified according to standard practice from hemodynamic features evident from echocardiography. The natural history group comprised 48 patients (up to 14.8 years of follow-up; median, 2.6 years; 24 patients started ERT during the study); 56 patients were treated (up to 14.2 years of follow-up; median, 6.2 years). Lifetime GAG burden (calculated from urinary GAG measurements) correlated significantly with the degree of valve disease. Onset of moderate-to-severe valve disease was significantly delayed in treated (median age at onset, 29.1 ± 2 [95% CI: 25.2-32.9] years; Kaplan-Meier estimation) versus untreated patients (17.6 ± 1 [95% Cl: 15.8-19.4] years; p < 0.0001). Cox regression modeling found that long-term ERT reduced the probability of developing severe valve disease (χ2, 32.736; significant after 5 years of ERT). Overall, this study found that valve disease severity in MPS II correlates with GAG burden and that progression is delayed by long-term ERT.

Implementation of Newborn Screening for Mucopolysaccharidosis Type IVA and Long-Term Monitoring in Taiwan.

BACKGROUND: Mucopolysaccharidosis IVA (MPS IVA) is a rare lysosomal storage disorder arising from a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). METHODS: From September 2019 to October 2023, a total of 264,843 Taiwanese newborns underwent screening for MPS IVA using dried blood spots and tandem mass spectrometry. RESULTS: Among the 95 referred infants, nine (9%) were confirmed to have MPS IVA (Group 1), 18 (19%) were highly suspected to have MPS IVA (Group 2), 61 (64%) were identified as heterozygotes of MPS IVA (Group 3), and seven (7%) were determined not to have MPS IVA (Group 4). A total of 34 different GALNS (HGNC:4122) gene variants were identified through our MPS IVA newborn screening program. The most prevalent variant was c.857C>T p.(Thr286Met), found in 33 cases (29%), followed by c.953T>G p.(Met318Arg) in 22 cases (19%). Intravenous enzyme replacement therapy (ERT) was initiated in five patients at ages ranging from 0.3 to 1.7 years. The estimated incidence of MPS IVA in this screening program was 3.4 per 100,000 live births. CONCLUSIONS: Due to the progressive nature of MPS IVA, an early diagnosis facilitated by newborn screening and prompt initiation of ERT before irreversible organ damage occurs may result in improved clinical outcomes.

The diagnosis and management of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II) is a rare X-linked recessive inherited lysosomal storage disease. With pathogenic variants of the IDS gene, the activity of iduronate-2-sulfatase (IDS) is reduced or lost, causing the inability to degrade glycosaminoglycans (GAGs) in cells and influencing cell function, eventually resulting in multisystemic manifestations, such as a coarse face, dysostosis multiplex, recurrent respiratory tract infections, and hernias. Diagnosing MPS II requires a combination of clinical manifestations, imaging examinations, urinary GAGs screening, enzyme activity, and genetic testing. Currently, symptomatic treatment is the main therapeutic approach. Owing to economic and drug availability issues, only a minority of patients opt for enzyme replacement therapy or hematopoietic stem cell transplantation. The limited awareness of the disease, the lack of widespread detection technology, and uneven economic development contribute to the high rates of misdiagnosis and missed diagnosis in China.

Effects of age, elastin density, and glycosaminoglycan accumulation on the delamination strength of human thoracic and abdominal aortas.

Aortic dissection is a life-threatening condition caused by layer separation. Despite extensive research, the relationship between the aortic wall's structural integrity and dissection risk remains unclear. Glycosaminoglycan (GAG) accumulation and elastin loss are suspected to play significant roles. We investigated how age-related changes in aortic structure affect dissection susceptibility. Peeling tests were performed on longitudinal and circumferential thoracic (TA) and abdominal aortic (AA) strips from 35 donors aged 13-76 years (mean 38 ± 15 years, 34 % female). GAG, elastin, collagen, and smooth muscle cell (SMC) contents were assessed using bidirectional histology. Young TAs resisted longitudinal peeling better than circumferential, with delamination strengths of 65.4 mN/mm and 44.2 mN/mm, respectively. Delamination strength decreased with age in both directions, more rapidly longitudinally, equalizing at ∼20-25 mN/mm in older TAs. Delamination strength in AAs was 22 % higher than in TAs. No sex differences were observed. GAG density increased, while elastin density decreased by 2.5 % and 4 % per decade, respectively. Collagen density did not change with age, while SMC density decreased circumferentially. GAGs partially mediated the reduction in longitudinal delamination strength due to aging, while circumferential strength reduction was not mediated by changes in either GAG or elastin densities. This study explains why aortic dissections are more common in TAs, especially in older individuals, and why they typically propagate spirally. TAs exhibit lower delamination strength compared to AAs and experience strength reduction with age, a phenomenon linked to increased GAG accumulation and elastin loss. These findings enhance our understanding of the pathophysiological mechanisms behind aortic dissection. STATEMENT OF SIGNIFICANCE: This work explores the age-dependent relationships between delamination strength in human aortas and wall structural content. We investigated 35 human aortas from donors aged 13 to 76 years, providing new insights into the biomechanical and histological factors that influence aortic dissection risk. Our findings elucidate how variations in elastin, glycosaminoglycan, collagen, and smooth muscle cell densities impact the structural integrity of the aorta, contributing significantly to the understanding of aortic dissection mechanisms.

The effect of enzymatic GAG degradation on transverse shear properties of porcine cornea.

The structural integrity of cornea depends on properties of its extracellular matrix, mainly a mixture of collagen fibers and soluble proteoglycans (PGs). PGs are macromolecules of negatively charged sulphated glycosaminoglycans (GAGs) covalently attached to a protein core. GAGs appear as bridges between adjacent collagen fibers and could facilitate force transfer between them. Furthermore, GAGs are responsible for corneal hydration by attracting and maintaining water molecules into the extracellular matrix. Based on these observations, GAGs are expected to be essential for biomechanical properties of cornea. The primary objective of the present study was to determine the effects of GAGs on shear properties of cornea. For this purpose, GAGs were enzymatically removed from porcine corneal disks by keratanase II enzyme. After confirming the successful removal of GAGs by histochemical methods, torsional rheometry was performed to characterize the shear stiffness of GAG-depleted samples as a function of axial strain. It was found that the shear modulus of all samples was a function of applied shear strain and compressive strain. Beyond the range of linear viscoelastic response, the average complex shear modulus decreased with increasing the shear strain. Increasing the axial strain from 0% to 40% significantly increased the average complex shear modulus of corneal disks in all groups. Finally, the enzyme treatment with keratanase II enzyme significantly decreased the shear stiffness. The experimental measurements were discussed in terms of microstructural and compositional properties of corneal extracellular matrix and it was concluded that GAGs play a significant role in defining shear properties of cornea.

Natural Regenerative Hydrogels for Wound Healing.

Regenerative hydrogels from natural polymers have come forth as auspicious materials for use in regenerative medicine, with interest attributed to their intrinsic biodegradability, biocompatibility, and ability to reassemble the extracellular matrix. This review covers the latest advances in regenerative hydrogels used for wound healing, focusing on their chemical composition, cross-linking mechanisms, and functional properties. Key carbohydrate polymers, including alginate, chitosan, hyaluronic acid, and polysaccharide gums, including agarose, carrageenan, and xanthan gum, are discussed in terms of their sources, chemical structures and specific properties suitable for regenerative applications. The review further explores the categorization of hydrogels based on ionic charge, response to physiological stimuli (i.e., pH, temperature) and particularized roles in wound tissue self-healing. Various methods of cross-linking used to enhance the mechanical and biological performance of these hydrogels are also examined. By highlighting recent innovations and ongoing challenges, this article intends to give a detailed understanding of natural hydrogels and their potential to revolutionize regenerative medicine and improve patient healing outcomes.

Mucopolysaccharidosis-Plus Syndrome: Is This a Type of Mucopolysaccharidosis or a Separate Kind of Metabolic Disease?

Several years ago, dozens of cases were described in patients with symptoms very similar to mucopolysaccharidosis (MPS). This new disease entity was described as mucopolysaccharidosis-plus syndrome (MPSPS). The name of the disease indicates that in addition to the typical symptoms of conventional MPS, patients develop other features such as congenital heart defects and kidney and hematopoietic system disorders. The symptoms are highly advanced, and patients usually do not survive past the second year of life. MPSPS is inherited in an autosomal recessive manner and is caused by a homozygous-specific mutation in the gene encoding the VPS33A protein. To date, it has been described in 41 patients. Patients with MPSPS exhibited excessive excretion of glycosaminoglycans (GAGs) in the urine and exceptionally high levels of heparan sulfate in the plasma, but the accumulation of substrates is not caused by a decrease in the activity of any lysosomal enzymes. Here, we discuss the pathomechanisms and symptoms of MPSPS, comparing them to those of MPS. Moreover, we asked the question whether MPSPS should be classified as a type of MPS or a separate disease, as contrary to 'classical' MPS types, despite GAG accumulation, no defects in lysosomal enzymes responsible for degradation of these compounds could be detected in MPSPS. The molecular mechanism of the appearance of GAG accumulation in MPSPS is suggested on the basis of results available in the literature.

Structural Insights into Endostatin-Heparan Sulfate Interactions Using Modeling Approaches.

Glycosaminoglycans (GAGs) play a key role in a variety of biological processes in the extracellular matrix (ECM) via interactions with their protein targets. Due to their high flexibility, periodicity and electrostatics-driven interactions, GAG-containing complexes are very challenging to characterize both experimentally and in silico. In this study, we, for the first time, systematically analyzed the interactions of endostatin, a proteolytic fragment of collagen XVIII known to be anti-angiogenic and anti-tumoral, with heparin (HP) and representative heparan sulfate (HS) oligosaccharides of various lengths, sequences and sulfation patterns. We first used conventional molecular docking and a docking approach based on a repulsive scaling-replica exchange molecular dynamics technique, as well as unbiased molecular dynamic simulations, to obtain dynamically stable GAG binding poses. Then, the corresponding free energies of binding were calculated and the amino acid residues that contribute the most to GAG binding were identified. We also investigated the potential influence of Zn2+ on endostatin-HP complexes using computational approaches. These data provide new atomistic details of the molecular mechanism of HP's binding to endostatin, which will contribute to a better understanding of its interplay with proteoglycans at the cell surface and in the extracellular matrix.

Extracellular Matrix Components and Mechanosensing Pathways in Health and Disease.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are essential components of the extracellular matrix (ECM) with pivotal roles in cellular mechanosensing pathways. GAGs, such as heparan sulfate (HS) and chondroitin sulfate (CS), interact with various cell surface receptors, including integrins and receptor tyrosine kinases, to modulate cellular responses to mechanical stimuli. PGs, comprising a core protein with covalently attached GAG chains, serve as dynamic regulators of tissue mechanics and cell behavior, thereby playing a crucial role in maintaining tissue homeostasis. Dysregulation of GAG/PG-mediated mechanosensing pathways is implicated in numerous pathological conditions, including cancer and inflammation. Understanding the intricate mechanisms by which GAGs and PGs modulate cellular responses to mechanical forces holds promise for developing novel therapeutic strategies targeting mechanotransduction pathways in disease. This comprehensive overview underscores the importance of GAGs and PGs as key mediators of mechanosensing in maintaining tissue homeostasis and their potential as therapeutic targets for mitigating mechano-driven pathologies, focusing on cancer and inflammation.

Human Serum, Following Absorption of Fish Cartilage Hydrolysate, Promotes Dermal Fibroblast Healing through Anti-Inflammatory and Immunomodulatory Proteins.

Background/Objectives: Marine collagen peptides (MCPs) and glycosaminoglycans (GAGs) have been described as potential wound-healing (WH) agents. Fish cartilage hydrolysate (FCH) is a natural active food ingredient obtained from enzymatic hydrolysis which combines MCPs and GAGs. Recently, the clinical benefits of FCH supplementation for the skin, as well as its mode of action, have been demonstrated. Some of the highlighted mechanisms are common to the WH process. The aim of the study is therefore to investigate the influence of FCH supplementation on the skin healing processes and the underlying mechanisms. Methods: To this end, an ex vivo clinical approach, which takes into account the clinical digestive course of nutrients, coupled with primary cell culture on human dermal fibroblasts (HDFs) and ultra-deep proteomic analysis, was performed. The effects of human serum enriched in circulating metabolites resulting from FCH ingestion (FCH-enriched serum) were assessed on HDF WH via an in vitro scratch wound assay and on the HDF proteome via diaPASEF (Data Independent Acquisition-Parallel Accumulation Serial Fragmentation) proteomic analysis. Results: Results showed that FCH-enriched human serum accelerated wound closure. In support, proteins with anti-inflammatory and immunomodulatory properties and proteins prone to promote hydration and ECM stability showed increased expression in HDFs after exposure to FCH-enriched serum. Conclusions: Taken together, these data provide valuable new insights into the mechanisms that may contribute to FCH's beneficial impact on human skin functionality by supporting WH. Further studies are needed to reinforce these preliminary data and investigate the anti-inflammatory and immunomodulatory properties of FCH.

Genetic Variance in Heparan Sulfation Is Associated With Salt Sensitivity.

BACKGROUND: High heritability of salt sensitivity suggests an essential role for genetics in the relationship between sodium intake and blood pressure (BP). The role of glycosaminoglycan genes, which are crucial for salinity tolerance, remains to be elucidated. METHODS: Interactions between 54 126 variants in 130 glycosaminoglycan genes and daily sodium excretion on BP were explored in 20 420 EPIC-Norfolk (European Prospective Investigation Into Cancer in Norfolk) subjects. The UK Biobank (n=414 132) and the multiethnic HELIUS study (Healthy Life in an Urban Setting; n=2239) were used for validation. Afterward, the urinary glycosaminoglycan composition was studied in HELIUS participants (n=57) stratified by genotype and upon dietary sodium loading in a time-controlled crossover intervention study (n=12). RESULTS: rs2892799 in NDST3 (heparan sulfate N-deacetylase/N-sulfotransferase 3) showed the strongest interaction with sodium on mean arterial pressure (false discovery rate 0.03), with higher mean arterial pressure for the C allele in high sodium conditions. Also, rs9654628 in HS3ST5 (heparan sulfate-glucosamine 3-sulfotransferase 5) showed an interaction with sodium on systolic BP (false discovery rate 0.03). These interactions were multiethnically validated. Stratifying for the rs2892799 genotype showed higher urinary expression of N-sulfated heparan sulfate epitope D0S0 for the T allele. Conversely, upon dietary sodium loading, urinary D0S0 expression was higher in participants with stable BP after sodium loading, and sodium-induced effects on this epitope were opposite in individuals with and without BP response to sodium. CONCLUSIONS: The C allele of rs2892799 in NDST3 exhibits higher BP in high sodium conditions when compared with low sodium conditions, whereas no differences were detected for the T allele. Concomitantly, both alleles demonstrate distinct expressions of D0S0, which, in turn, correlates with sodium-mediated BP elevation. These findings underscore the potential significance of genetic glycosaminoglycan variation in human BP regulation.

Simultaneous co-assembly of collagen and glycosaminoglycans to build a biomimetic extracellular matrix for bone regeneration.

Glycosaminoglycans (GAGs), also known as shape modules, are considered junctions that help define the shape of collagen matrix and further promote mineralization during osteogenesis. Many attempts have been made to immobilize GAGs on assembled collagen to modify the latter's surface state. However, it remains unclear how GAGs spontaneously identify collagen molecules during fibrillogenesis in vivo. Understanding the relationship between GAGs and collagen from both the bone physiology and materials science perspectives is of fundamental interest. Here, we introduced hyaluronic acid (HA, a main member of GAGs) during collagen self-assembly, in a process called modification cooperating with self-assembly (MCS). The molecular docking and morphological studies revealed that HA can help define collagen monomer deposition and thus promote fibrillogenesis through steric hindrance or by directly forming hydrogen bonds. Meanwhile, HA acts as a templating chaperone (TC) to increase the local mineral concentration within intrafibrillar channels but does not initiate nucleation, thus improving the crystallinity of formed apatite. The scaffolds synthesized through MCS model significantly improved the physicochemical stability and mechanical strength of collagen-based scaffolds. The optimized scaffolds promoted in-situ osteogenesis by stimulating the osteogenic differentiation of bone mesenchymal stem cells, either in an osteogenic medium, or after implantation into critical calvarial defects. This study provides novel insights towards evolving engineering scaffolds from inert supports to functional substitutes.

Discriminating Benign from Malignant Lung Diseases Using Plasma Glycosaminoglycans and Cell-Free DNA.

We aimed to investigate the use of free glycosaminoglycan profiles (GAGomes) and cfDNA in plasma to differentiate between lung cancer and benign lung disease, in a cohort of 113 patients initially suspected of lung cancer. GAGomes were analyzed in all samples using the MIRAM® Free Glycosaminoglycan Kit with ultra-high-performance liquid chromatography and electrospray ionization triple quadrupole mass spectrometry. In a subset of samples, cfDNA concentration and NGS-data was available. We detected two GAGome features, 0S chondroitin sulfate (CS), and 4S CS, with cancer-specific changes. Based on the observed GAGome changes, we devised a model to predict lung cancer. The model, named the GAGome score, could detect lung cancer with 41.2% sensitivity (95% CI: 9.2-54.2%) at 96.4% specificity (95% CI: 95.2-100.0%, n = 113). When we combined the GAGome score with a cfDNA-based model, the sensitivity increased from 42.6% (95% CI: 31.7-60.6%, cfDNA alone) to 70.5% (95% CI: 57.4-81.5%) at 95% specificity (95% CI: 75.1-100%, n = 74). Notably, the combined GAGome and cfDNA testing improved the sensitivity, compared to cfDNA alone, especially in ASCL stage I (55.6% vs 11.1%). Our findings show that plasma GAGome profiles can enhance cfDNA testing performance, highlighting the applicability of a multiomics approach in lung cancer diagnostics.

Urinary Free Glycosaminoglycans Identify Adults at High Risk of Developing Early-stage High-grade Bladder Cancer.

Background and objective: Screening for bladder cancer (BCa) could reduce mortality via early detection of early-stage high-grade (Ta/T1 N0 M0 grade 2-3) disease. Noninvasive biomarkers could aid in screening, but current markers lack the specificity required. The urinary free glycosaminoglycan profile (GAGome) is a promising biomarker for early detection of BCa metabolism. Methods: In a prospective case-control development study, we included patients with BCa or no evidence of disease (NED) and measured the urinary GAGome. We then developed a score to predict the probability of BCa using GAGome features that correlated with BCa versus NED according to Bayesian regression. Next, in a retrospective, population-based, case-control study, we included adults from the Lifelines Cohort Study who were presumed healthy at baseline. All cases with BCa confirmed in the cancer registry by the 2-yr or 6-yr study visit were matched to randomly selected control subjects. We developed a reference logistic regression model using age and sex to predict BCa at 7 yr after baseline. We then added the GAGome score to the model and assessed model improvement using the likelihood ratio test. We dichotomized outputs for the reference model and saturated model (reference + GAGome score) into high-risk versus low-risk categories using a 99% specificity cutoff and estimated the sensitivity for association with BCa at 7 yr. Key findings and limitations: We prospectively included 51 individuals with BCa and 38 with NED and observed alterations in three GAGome features compatible with BCa. We developed a score that discriminated BCa with an area under the receiver operating characteristic curve of 0.77 (95% confidence interval [CI] 0.67-0.87). We retrospectively selected a cohort of 1088 presumed healthy adults (median age 48 yr, 56% females), of whom 48 had developed BCa by 7 yr after baseline (median time to diagnosis 1.4 yr). The GAGome score was an independent predictor of BCa at 7 yr when added to the reference model (p < 0.001). The sensitivity for BCa at 7 yr for high-risk subjects was 31% (95% CI 20-43%) using the saturated model and 17% (95% CI 4.7-29%) using the reference model at 99% specificity (95% CI 98-99%). Conclusions and clinical implications: The urinary free GAGome is specifically altered in BCa and can be used for noninvasive identification of adults at high risk of developing BCa, independent of age and sex. This information could be useful for the design of risk-stratified targeted screening programs for BCa. Patient summary: We tested whether measurement of a class of sugars called glycosaminoglycans (GAGs) in urine could be used for early detection of bladder cancer. Our results show that GAG levels in urine can distinguish people at high risk of developing bladder cancer within 7 years, even if they are healthy at the time of the urine sampling.

Novel simultaneous analysis of 18 types of glycosaminoglycan-derived disaccharides using 4-aminobenzoic acid ethyl ester derivatization by HPLC with fluorescence detection.

Glycosaminoglycans (GAGs), including hyaluronic acid (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), heparan sulfate (HS)/heparin (HP), and keratan sulfate (KS), play pivotal roles in living organisms. Generally, GAGs are analyzed after enzymatic digestion into unsaturated or saturated disaccharides. Due to high structural similarity between disaccharides, however, separation during analysis is challenging. Additionally, little is known about the structures of GAGs and their functional relationships. Elucidating the function of GAGs requires highly sensitive quantitative analytical methods. We developed a method for the simultaneous analysis of 18 types of disaccharides derived from HA (1 type), CS/DS (7 types), HS/HP (8 types), and KS (2 types) potentially detectable in analyses of human urine. The simple method involves HPLC separation with fluorescence detection following derivatization of GAG-derived disaccharides using 4-aminobenzoic acid ethyl ester (ABEE) as a pre-labeling agent and 2-picoline borane as a reductant. The ABEE derivatization reaction can be performed under aqueous conditions, and excess derivatization reagents can be easily, rapidly, and safely removed. This method enables highly sensitive simultaneous analysis of the 18 abovementioned types of GAG-derived disaccharides using HPLC with fluorescence detection in small amounts of urine (1 mL) in a single run. The versatile method described here could be applied to the analysis of GAGs in other biological samples.

Heparan Sulfate-Collagen Surface Multilayers Support Serum-Free Microcarrier Culture of Mesenchymal Stem Cells.

The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (µCs) cultures. Additionally, rHS surfaces reduced ß-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.

Rational design of glycosaminoglycan binding cyclic peptides using cPEPmatch.

Cyclic peptides present a robust platform for drug design, offering high specificity and stability due to their conformationally constrained structures. In this study, we introduce an updated version of the Cyclic Peptide Matching program (cPEPmatch) tailored for the identification of cyclic peptides capable of mimicking protein-glycosaminoglycan (GAG) binding sites. We focused on engineering cyclic peptides to replicate the GAG-binding affinity of antithrombin III (ATIII), a protein that plays a crucial role in modulating anticoagulation through interaction with the GAG heparin. By integrating computational and experimental methods, we successfully identified a cyclic peptide binder with promising potential for future optimization. MD simulations and MM-GBSA calculations were used to assess binding efficacy, supplemented by umbrella sampling to approximate free energy landscapes. The binding specificity was further validated through NMR and ITC experiments. Our findings demonstrate that the computationally designed cyclic peptides effectively target GAGs, suggesting their potential as novel therapeutic agents. This study advances our understanding of peptide-GAG interactions and lays the groundwork for future development of cyclic peptide-based therapeutics.

Evaluation of Chondral Defect Repair Using Human Fibronectin Adhesion Assay-Derived Chondroprogenitors Suspended in Lyophilized Fetal Collagen Scaffold: An Ex Vivo Osteochondral Unit Model Study.

Introduction: Chondral defect repair is challenging due to a scarcity of reparative cells and the need to fill a large surface area, compounded by the absence of self-healing mechanisms. Fibronectin adhesion assay-derived chondroprogenitors (FAA-CPs) have emerged as a promising alternative with enhanced chondrogenic ability and reduced hypertrophy. De-cellularized bio-scaffolds are reported to act as extracellular matrix, mimicking the structural and functional characteristics of native tissue, thereby facilitating cell attachment and differentiation. This study primarily assessed the synergistic effect of FAA-CPs suspended in fetal cartilage-derived collagen-containing scaffolds in repairing chondral defects. Methodology: The de-cellularized and lyophilized fetal collagen was prepared from the tibio-femoral joint of a 36 + 4-week gestational age fetus. FAA-CPs were isolated from osteoarthritic cartilage samples (n = 3) and characterized. In ex vivo analysis, FAA-CPs at a density of 1 × 106 cells were suspended in the lyophilized scaffold and placed into the chondral defects created in the Osteochondral Units and harvested on the 35th day for histological examination. Results: The lyophilized scaffold of de-cellularized fetal cartilage with FAA-CPs demonstrated effective healing of the critical size chondral defect. This was evidenced by a uniform distribution of cells, a well-organized collagen-fibrillar network, complete filling of the defect with alignment to the surface, and favorable integration with the adjacent cartilage. However, these effects were less pronounced in the plain scaffold control group and no demonstrable repair observed in the empty defect group. Conclusion: This study suggests the synergistic potential of FAA-CPs and collagen scaffold for chondral repair which needs to be further explored for clinical therapy. Supplementary Information: The online version contains supplementary material available at 10.1007/s43465-024-01192-6.