Heparan Sulfate Proteoglycans Research Articles

An integral membrane constitutively active heparanase enhances the tumor infiltration capability of NK cells

ABSTRACT Eradication of cancer cells by the immune system requires extravasation, infiltration and progression of immune cells through the tumor extracellular matrix (ECM). These are also critical determinants for successful adoptive cell immunotherapy of solid tumors. Together with structural proteins, such as collagens and fibronectin, heparan sulfate (HS) proteoglycans are major components of the ECM. Heparanase 1 (HPSE) is the only enzyme known to have endoglycosidase activity that degrades HS. HPSE is expressed at high levels in almost all hematopoietic cells, which suggests that it plays a relevant role in immune cell migration through solid tissues. Besides, tumor cells express also HPSE as a way to facilitate tumor cell resettlement and metastasis. Therefore, an increase in HPSE in the tumor ECM would be detrimental. Here, we analyzed the effects of constitutive expression of an active, membrane-bound HPSE on the ability of human natural killer (NK) cells to infiltrate tumors and eliminate tumor cells. We demonstrate that NK cells expressing a chimeric active form of HPSE on the cell surface as an integral membrane protein, display significantly enhanced infiltration capability into spheroids of various cancer cell lines, as well as into xenograft tumors in immunodeficient mice. As a result, tumor growth was significantly suppressed without causing noticeable side effects. Altogether, our results suggest that a constitutively expressed active HSPE on the surface of immune effector cells enhances their capability to access and eliminate tumor cells. This strategy opens new possibilities for improving adoptive immune treatments using NK cells.

Enhancing intrinsic TGF-β signaling via heparan sulfate glycosaminoglycan regulation to promote mesenchymal stem cell capabilities and chondrogenesis for cartilage repair

Osteoarthritis burdens patients due to the limited regenerative capacity of chondrocytes. Traditional cartilage repair often falls short, necessitating innovative approaches. Mesenchymal stem cells (MSCs) show promise for regeneration. Heparan sulfate glycosaminoglycans (HS-GAGs) regulate cellular functions, making them a target for cartilage repair. This study highlights how Heparinase III (HepIII) cleaves intact HS-GAGs in bone marrow-derived MSCs (BM-MSCs), enhancing their capabilities and specifically promoting chondrogenesis. HepIII-treated BM-MSCs cultured in a hanging drop device for three days, significantly increased cell number and aggregation into a cell sphere with early chondrogenesis. HepIII promoted BM-MSCs toward chondrogenesis, increasing type II collagen, intact HS-GAGs, and sulfated GAG content, while upregulating chondrogenic and heparan sulfate proteoglycan genes. Treatment with the TGF-β inhibitor (SB-431542) in HepIII-treated BM-MSCs demonstrated enhanced intrinsic transforming growth factor-β (TGF-β) signaling and fibronectin expression. This approach also boosted BM-MSC self-renewal, immunosuppressive potential, and modified acetylated histone signatures, offering a cost-effective strategy for cartilage repair by addressing inflammation, metabolic changes, and the high costs of traditional TGF-β methods. From the results, HepIII-treated BM-MSCs show potential for use in combination with other biopolymers as injectable gels to improve cartilage repair in osteoarthritis patients in the near future.

Hippocampal CA2 neurons disproportionately express AAV-delivered genetic cargo.

Hippocampal area CA2 is unique in many ways, largely based on the complement of genes expressed there. We and others have observed that CA2 neurons exhibit a uniquely robust tropism for adeno-associated viruses (AAVs) of multiple serotypes and variants. In this study, we aimed to systematically investigate the propensity for AAV tropism toward CA2 across a wide range of AAV serotypes and variants, injected either intrahippocampally or systemically, including AAV1, 2, 5, 6, 8, 9, DJ, PHP.B, PHP.eB, and CAP-B10. We found that most serotypes and variants produced disproportionally high expression of AAV-delivered genetic material in hippocampal area CA2, although two serotypes (AAV6 and DJ) did not. In an effort to understand the mechanism(s) behind this observation, we considered perineuronal nets (PNNs) that ensheathe CA2 pyramidal cells and, among other functions, buffer diffusion of ions and molecules. We hypothesized that PNNs might attract AAV particles and maintain them in close proximity to CA2 neurons, thereby increasing exposure to AAV particles. However, genetic deletion of PNNs from CA2 had no effect on AAV transduction. Next, we next considered the AAV binding factors and receptors known to contribute to AAV transduction. We found that the AAV receptor (AAVR), which is critical to transduction, is abundantly expressed in CA2, and knockout of AAVR nearly abolished expression of AAV-delivered material by all serotypes tested. Additionally, we found CA2 enrichment of several cell-surface glycan receptors that AAV particles attach to before interacting with AAVR, including heparan sulfate proteoglycans, N-linked sialic acid and N-linked galactose. Indeed, CA2 showed the highest expression of AAVR and the investigated glycan receptors within the hippocampus. We conclude that CA2 neurons are endowed with multiple factors that make it highly susceptible to AAV transduction, particularly to the systemically available PHP variants, including CAP-B10. Given the curved structure of hippocampus and the relatively small size of CA2, systemic delivery of engineered PHP or CAP variants could all but eliminate the need for intrahippocampal AAV injections, particularly when injecting recombinase-dependent AAVs into animals that express recombinases in CA2.

The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling.

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue- specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine-knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.

Feasibility assessment of using cRISPR-Cas9 to improve the infiltration of CAR-T cells in solid tumors

Abstract. Chimeric antigen receptor T-cell immunotherapy (CAR-T) has been developing for decades, CAR-T is playing an increasingly important role in tumor treatment. However, because fibroblasts (CAFs) in solid tumors secrete proteins and glycans to form ECM, CAR-T is faced with the challenge of improving tumor invasion. To this end, a new scheme was put forth to alter CAR T cells such that they release heparinase (HPSE) to break down heparan sulfate proteoglycan (HSPG), which is covered on the outermost layer of the cancerous cells by CAF and released. In order to solve the above problems, CRISPR-Cas9 was proposed to modify CAR T to enhance the secretion of HPSE. Although this method has the advantage of broad spectrum, it still has certain defects. The matrix characteristics of different solid tumors and the subpopulations and regulatory mechanisms of HPSE need to be further studied. The off-target effects of CRISPR-Cas9 gene editing technology and the high cost and time-consuming problems of flow cytometry also limit its application. It is anticipated to enhance cell infiltration in solid tumors, boost CAR-T therapy's effectiveness in treating solid tumors, and advance the field of CAR-T therapy.

The potential of lactoferrin as antiviral and immune-modulating agent in viral infectious diseases.

Emerging infectious diseases are caused by unpredictable viruses with the dangerous potential to trigger global pandemics. These viruses typically initiate infection by utilizing the anionic structures of host cell surface receptors to gain entry. Lactoferrin (Lf) is a multifunctional glycoprotein with multiple properties such as antiviral, anti-inflammatory and antioxidant activities. Due to its cationic structure, Lf naturally interacts with certain host cell receptors, such as heparan sulfate proteoglycans, as well as viral particles and other receptors that are targeted by viruses. Therefore, Lf may interfere with virus-host cell interactions by acting as a receptor competitor for viruses. Herein we summarize studies in which this competition was investigated with SARS-CoV-2, Zika, Dengue, Hepatitis and Influenza viruses in vitro. These studies have demonstrated not only Lf's competitive properties, but also its potential intracellular impact on host cells, such as enhancing cell survival and reducing infection efficiency by inhibiting certain viral enzymes. In addition, the immunomodulatory effect of Lf is highlighted, as it can influence the activity of specific immune cells and regulate cytokine release, thereby enhancing the host's response to viral infections. Collectively, these properties promote the potential of Lf as a promising candidate for research in viral infectious diseases.

Excess Weight Impairs Oocyte Quality, as Reflected by mtDNA and BMP-15.

This prospective, case-control study evaluated the impact of obesity on oocyte quality based on mtDNA expression in cumulus cells (CC), and on bone morphogenetic protein 15 (BMP-15) and heparan sulfate proteoglycan 2 (HSPG2) in follicular fluid (FF). It included women 18 to <40 years of age, divided according to BMI < 24.9 (Group 1, n = 28) and BMI > 25 (Group 2, n = 22). Demographics, treatment, and pregnancy outcomes were compared. The mtDNA in CC, BMP-15, HSPG2, the lipid profile, the hormonal profile, and C-reactive protein were evaluated in FF and in blood samples. The BMP-15 levels in FF and the mitochondrial DNA in CC were higher in Group 1 (38.8 ± 32.5 vs. 14.3 ± 10.8 ng/mL; p = 0.001 and 1.10 ± 0.3 vs. 0.87 ± 0.18-fold change; p = 0.016, respectively) than in Group 2. High-density lipoprotein levels in blood and FF were higher in Group 1 (62 ± 18 vs. 50 ± 12 mg/dL; p = 0.015 and 34 ± 26 vs. 20.9 ± 7.2 mg/dL; p = 0.05, respectively). Group 2 had higher blood C-reactive protein (7.1 ± 5.4 vs. 3.4 ± 4.3 mg/L; p = 0.015), FF (5.2 ± 3.8 vs. 1.5 ± 1.6 mg/L; p = 0.002) and low-density lipoprotein levels (91 ± 27 vs. 71 ± 22 mg/dL; p = 0.008) vs. Group 1. Group 1 demonstrated a trend toward a better clinical pregnancy rate (47.8% vs. 28.6%: p = 0.31) and frozen embryo transfer rate (69.2% vs. 53.8; p = 0.69). Higher BMI resulted in lower BMP-15 levels and reduced mtDNA expression, which reflect decreased oocyte quality in overweight women.

Downregulation of Genes for Skeletal Muscle Extracellular Matrix Components by Cisplatin.

The extracellular matrix (ECM) in skeletal muscle is involved in a variety of physiological functions beyond the mechanical support of muscle tissue, nerves, and blood vessels; however, the role of the ECM in skeletal muscle remains unclear. There is little information regarding changes in the expression of factors comprising the ECM during cisplatin-induced muscle atrophy. In the present study, we examined the changes in gene expressions for skeletal muscle extracellular matrix components in skeletal muscle during cisplatin-induced muscle atrophy. Intraperitoneal administration of cisplatin caused muscle atrophy in mice and during this cisplatin-induced muscle atrophy, the expression of many procollagen genes (Col1a1, Col1a2, Col3a1, Col4a1, Col5a1, and Col5a2), elastin (Eln), fibronectin (Fn1), Laminin (Lama1, Lama2, and Lamb1) decorin (Dcn), heparan sulphate proteoglycans (Hspg2) and integrin (Itgb1) constituting the ECM was suppressed. Additional studies are needed to elucidate the pathological significance and mechanisms of reduced gene expression of ECM components associated with cisplatin-induced muscle atrophy.

Decellularized extracellular matrix from bovine ovarian tissue maintains the protein composition of the native matrisome

Recent approaches of regenerative reproductive medicine investigate the decellularized extracellular matrix to develop a transplantable engineered ovary (TEO). However, a full proteomic analysis is not usually performed after the decellularization process to evaluate the preservation of the extracellular matrix (ECM). In this study, the ECM of the bovine ovarian cortex was analyzed before and after decellularization using mass spectrometry and bioinformatics. A total of 155 matrisome proteins were identified in the native ECM of the bovine ovarian cortex, with 145 matrisome proteins detected in the decellularized ECM. After decellularization, only 10 matrisome proteins were lost, and notably, none belonged to the category of reproductive biological processes. Histology and histochemistry analyses were employed to assess the general morphology of both native and decellularized ECM, allowing for the identification of the most abundant ECM proteins. Moreover, our study highlighted collagen type VI alpha 3 and heparan sulfate proteoglycan 2 as the most abundant components in the bovine ovarian ECM, mirroring the composition observed in the human ovary. These findings enhance our understanding of the composition of both native and decellularized ECM, with the potential implications for the development of a TEO. SignificanceThe significance of the present study lies on the possibility of advancing towards developing a bioengineered ovary, which is the ultimate strategy to regain fertility in women. The results demonstrate that the decellularized extracellular matrix of the bovine ovary maintains the protein composition of the native matrisome, using a recently described decellularization protocol. The decellularized matrix may serve as scaffolding for seeding ovarian stromal cells and follicles to create a bioengineered ovary, and as closer its composition is to the native matrix the better. Also, comparing the bovine ovarian matrisome, which was described for the first time here, with the human ovarian matrisome, we could see a great similarity, suggesting that the bovine ovary decellularized matrix may serve as a model for developing a human bioengineered ovary.

Both chebulagic acid and punicalagin inhibit respiratory syncytial virus entry via multi-targeting glycoprotein and fusion protein.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections, with no currently available small-molecule drugs that are both safe and effective. A major obstacle in antiviral drug development is the rapid emergence of drug-resistant viral strains. Targeting multiple viral compounds may help mitigate the development of resistance. Herein, we conducted a drug screening using the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to identify compounds that simultaneously target the RSV fusion (F) protein, glycoprotein (G), and the host heparan sulfate proteoglycans (HSPGs). From this screening, 10 candidate compounds were identified for their ability to interact with all three targets. Among these 10 candidates, chebulagic acid (CHLA) and punicalagin (PUG) demonstrated the most potent inhibition of RSV replication. In vitro dose-response assays confirmed the antiviral efficacy of CHLA (IC50: 0.07864 µM) and PUG (IC50: 0.08065 µM). Further experiments revealed both CHLA and PUG disrupt RSV attachment and membrane fusion by targeting the RSV-F and G proteins, rather than HSPG. Notably, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with docking assays predicting their binding sites at cysteines 176 and 182. Additionally, CHLA enhanced the conformational stability of the RSV-F protein before fusion. In an in vivo study, both CHLA and PUG were shown to alleviate RSV-induced pulmonary pathology by reducing viral titers, mitigating lung injury, and suppressing the inflammatory responses in the lungs. Our findings suggest that CHLA and PUG hold potential as therapeutic agents for RSV infection.IMPORTANCEA significant challenge in developing anti-respiratory syncytial virus (RSV) agents is the rapid emergence of resistant viral strains. Designing drugs that target multiple viral components can effectively reduce the likelihood of developing resistant strains. In this study, we screened compounds from the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to simultaneously targe the RSV fusion (F) protein, glycoprotein (G), and host heparan sulfate proteoglycans (HSPGs). Our findings revealed that chebulagic acid (CHLA) and punicalagin (PUG) significantly inhibited RSV replication both in vitro and in vivo and interacted with all three targets. Both CHLA and PUG were able to disrupt RSV attachment and membrane fusion. Mechanistically, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with CHLA also enhancing the conformational stability of the RSV-F protein before fusion. In conclusion, our study suggests that CHLA and PUG hold promise as therapeutic agents against RSV infection.

Identification of HSPG2 as a bladder pro-tumor protein through NID1/AKT signaling

PurposeHeparan sulfate proteoglycans (HSPGs) are complex molecules found on the cell membrane and within the extracellular matrix, increasingly recognized for their role in tumor progression. This study aimed to investigate the involvement of Heparan sulfate proteoglycan 2 (HSPG2) in the progression of bladder cancer.MethodsWe identified HSPG2 as a promoter of bladder tumor progression using single-cell RNA sequencing and transcriptome analysis of sequencing data from seven patient samples obtained from the Gene Expression Omnibus (GEO) database (GSE135337). Transcript profiles of 28 normal tissues and 407 bladder tumor tissues were analyzed for HSPG2 expression and survival outcomes using the Sanger tools and cBioPortal databases. HSPG2-overexpressing T24 and Biu-87 cell lines were generated, and cell proliferation and migration were assessed using CCK-8 and Transwell assays. Western blotting and immunostaining were performed to evaluate the activation of Nidogen-1 (NID1)/protein kinase B (AKT) signaling. Mouse models with patient-derived tumor organoids (HSPG2high and HSPG2low) were established to assess anticancer effects.ResultsOur results demonstrated a marked upregulation of HSPG2 in malignant bladder tumors, which correlated significantly with poor patient prognosis. HSPG2 overexpression consistently enhanced bladder tumor cell proliferation and conferred chemotherapy resistance, as shown in both in vitro and in vivo experiments. Mechanistically, HSPG2 upregulated NID1 expression, leading to the activation of the AKT pro-survival signaling pathway and promoting sustained tumor growth in bladder cancer.ConclusionThis study highlights the critical pro-tumor role of HSPG2/NID1/AKT signaling in bladder cancer and suggests its potential as a therapeutic target in clinical treatment.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Heparin treatment is associated with a delayed diagnosis of Alzheimer’s dementia in electronic health records from two large United States health systems

AbstractRecent studies suggest that heparan sulfate proteoglycans (HSPG) contribute to the predisposition to, protection from, and potential treatment and prevention of Alzheimer’s disease (AD). Here, we used electronic health records (EHR) from two different health systems to examine whether heparin therapy was associated with a delayed diagnosis of AD dementia. Longitudinal EHR data from 15,183 patients from the Mount Sinai Health System (MSHS) and 6207 patients from Columbia University Medical Center (CUMC) were used in separate survival analyses to compare those who did or did not receive heparin therapy, had a least 5 years of observation, were at least 65 years old by their last visit, and had subsequent diagnostic code or drug treatment evidence of possible AD dementia. Analyses controlled for age, sex, comorbidities, follow-up duration and number of inpatient visits. Heparin therapy was associated with significant delays in age of clinical diagnosis of AD dementia, including +1.0 years in the MSMS cohort (P &lt; 0.001) and +1.0 years in the CUMC cohort (P &lt; 0.001). While additional studies are needed, this study supports the potential roles of heparin-like drugs and HSPGs in the protection from and prevention of AD dementia.

Cancer-Associated Fibroblasts Expressing Sulfatase 1 Facilitate VEGFA-Dependent Microenvironmental Remodeling to Support Colorectal Cancer.

Tumor stroma plays a critical role in fostering tumor progression and metastasis. Cancer-associated fibroblasts (CAF) are a major component of the tumor stroma. Identifying the key molecular determinants for the protumor properties of CAFs could enable the development of more effective treatment strategies. In this study, through analyses of single-cell sequencing data, we identified a population of CAFs expressing high levels of sulfatase 1 (SULF1), which was associated with poor prognosis in patients with colorectal cancer. Colorectal cancer models using mice with conditional SULF1 knockout in fibroblasts revealed the tumor-supportive function of SULF1+ CAFs. Mechanistically, SULF1+ CAFs enhanced the release of VEGFA from heparan sulfate proteoglycan. The increased bioavailability of VEGFA initiated the deposition of extracellular matrix and enhanced angiogenesis. In addition, intestinal microbiota-produced butyrate suppressed SULF1 expression in CAFs through its histone deacetylase (HDAC) inhibitory activity. The insufficient butyrate production in patients with colorectal cancer increased the abundance of SULF1+ CAFs, thereby promoting tumor progression. Importantly, tumor growth inhibition by HDAC was dependent on SULF1 expression in CAFs, and patients with colorectal cancer with more SULF1+ CAFs were more responsive to treatment with the HDAC inhibitor chidamide. Collectively, these findings unveil the critical role of SULF1+ CAFs in colorectal cancer and provide a strategy to stratify patients with colorectal cancer for HDAC inhibitor treatment. Significance: SULF1+ cancer-associated fibroblasts play a tumor-promoting role in colorectal cancer by stimulating extracellular matrix deposition and angiogenesis and can serve as a biomarker for the therapeutic response to HDAC inhibitors in patients.

SULF1 expression is increased and promotes fibrosis through the TGF-β1/SMAD pathway in idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology. Despite the increasing global incidence and poor prognosis, the exact pathogenic mechanisms remain elusive. Currently, effective therapeutic targets and treatment methods for this disease are still lacking. This study tried to explore the pathogenic mechanisms of IPF. We found elevated expression of SULF1 in lung tissues of IPF patients compared to normal control lung tissues. SULF1 is an enzyme that modifies heparan sulfate chains of heparan sulfate proteoglycans, playing a critical role in biological regulation. However, the effect of SULF1 in pulmonary fibrosis remains incompletely understood. Our study aimed to investigate the impact and mechanisms of SULF1 in fibrosis.MethodsWe collected lung specimens from IPF patients for transcriptome sequencing. Validation of SULF1 expression in IPF patients was performed using Western blotting and RT-qPCR on lung tissues. ELISA experiments were employed to detect SULF1 concentrations in IPF patient plasma and TGF-β1 levels in cell culture supernatants. We used lentiviral delivery of SULF1 shRNA to knock down SULF1 in HFL1 cells, evaluating its effects on fibroblast secretion, activation, proliferation, migration, and invasion capabilities. Furthermore, we employed Co-Immunoprecipitation (Co-IP) to investigate the regulatory mechanisms involved.ResultsThrough bioinformatic analysis of IPF transcriptomic sequencing data (HTIPF) and datasets GSE24206, and GSE53845, we identified SULF1 may potentially play a crucial role in IPF. Subsequently, we verified that SULF1 was upregulated in IPF and predominantly increased in fibroblasts. Furthermore, SULF1 expression was induced in HFL1 cells following exposure to TGF-β1. Knockdown of SULF1 suppressed fibroblast secretion, activation, proliferation, migration, and invasion under both TGF-β1-driven and non-TGF-β1-driven conditions. We found that SULF1 catalyzes the release of TGF-β1 bound to TGFβRIII, thereby activating the TGF-β1/SMAD pathway to promote fibrosis. Additionally, TGF-β1 induces SULF1 expression through the TGF-β1/SMAD pathway, suggesting a potential positive feedback loop between SULF1 and the TGF-β1/SMAD pathway.ConclusionsOur findings reveal that SULF1 promotes fibrosis through the TGF-β1/SMAD pathway in pulmonary fibrosis. Targeting SULF1 may offer a promising therapeutic strategy against IPF.

Targeting heparanase/heparan sulfate proteoglycans by non‐anticoagulant heparins: Opportunities for innovative therapeutic approaches in sarcomas

AbstractSarcomas are a heterogeneous group of aggressive mesenchymal malignancies. They account for 1% of all tumors in the general population and 15–20% in pediatric age and young adults. Despite differences in histology and pathobiology, the diverse types of sarcomas are traditionally managed with a common multi‐modal approach including surgery, radiotherapy, and aggressive polychemotherapy. Unfortunately, the prognosis for advanced or recurrent disease remains poor. Moreover, the disease rarity and a high cellular, molecular, and genetic/epigenetic heterogeneity make identification of therapeutic targets challenging. Therefore it remains an urgent need to identify effective therapies to improve patients' outcome. Common or peculiar biological motifs including deregulation of growth factor signaling, proangiogenic and promigratory pathways, tumor‐microenviroment interactions, transcriptional and epigenetic machinery, and differentiation program, provide actionable dependencies exploitable for therapeutic intervention. Among these, a deregulated heparan sulfate proteoglycan system due to aberrant expression of key components as well as structural/functional modifications mediated by endosulfatases and the endoβ‐d‐glycosidase heparanase, is emerging as a crucial player in tumor growth and progression and as a valuable therapeutic target across different sarcoma subtypes. In preclinical studies, non‐anticoagulant heparins have been shown to counteract the growth and metastatic dissemination of various sarcoma models according to their heparan sulfate mimetic and anti‐heparanase activities. Heparin derivatives also improved the anti‐sarcoma efficacy of molecularly targeted agents and cytotoxic drugs. In this minireview, we summarize the current knowledge about the interplay between the heparanase/heparan sulfate proteoglycan system and pathways involved in sarcomagenesis and disease progression. We illustrate the current understanding of the mechanisms of action of non‐anticoagulant heparins and the contribution of their anti‐heparanase, anti‐receptor tyrosine kinase, and likely immunomodulatory activities to their anti‐sarcoma effects. Finally, we discuss a few aspects worthy of exploration highlighting how elucidation of mechanisms underpinning antitumor activities of non‐anticoagulant heparin derivatives in the different sarcoma biological contexts may suggest new vulnerabilities and therapeutic approaches.

A highly sensitive sandwich-type electrochemical sensor for detection glypican-3 based on H-rGO-CMC@Pt NPs and aptamers

Glypican-3 (GPC3), which is a surface heparan sulfate proteoglycan, has been widely known the ideal biomarker for diagnosis and treatment of hepatocellular carcinoma (HCC). Accurate and sensitive detection of serum GPC3 level is an important and challenging task. In this paper, a sandwich electrochemical aptasensor was designed to specifically detect serum GPC3 level through aptamer-target-aptamer recognition. A GPC3 aptamer (GPC3Apt) immobilized on the surface of gold nanoparticles@reduced graphene oxide modified screen-printed electrode (Au NPs@rGO/SPCE) was used as the capture probe. Hemin-reduced graphene oxide-carboxymethyl chitosan@platinum nanoparticles (H-rGO-CMC@Pt NPs), with good mimicking peroxidases activity, were labeled with amination GPC3 aptamer, which was employed as the signal probe. Once the target GPC3 was anchored on the surface of GPC3Apt/Au NPs@rGO/SPCE, the H-rGO-CMC@Pt NPs-GPC3Apt was further specifically bound to the GPC3, forming the aptamer-target-aptamer sandwich structure, which can catalyze the oxidation of hydroquinone (HQ) to benzoquinone (BQ) and lead the oxidation current of HQ recorded by DPV to changing. The linear relationship between the current signal of oxidation of HQ and GPC3 concentration range of 0.0001–3.0 µg/mL and 3.0–60.0 µg/mL, with a low detection limit (LOD) of 0.0685 ng/mL. Additionally, the electrochemical aptasensor was successfully applied for the detection of GPC3 in human serum samples with the recovery of 99.95–104.06 % and relative standard deviation (RSD) of 1.31–5.22 %. Thus, the sandwich aptasensor could provide a new strategy for detection of serum GPC3 level and improve the early diagnosis rate of HCC.

Abstract C019: A novel SDC1-targeted therapeutic antibody inhibits macropinocytosis and induces anti-tumor immunity in pancreatic cancer

Abstract Given that the mutated KRAS oncogene (KRAS*) dominates the genetic landscape of pancreatic ductal adenocarcinoma (PDAC), the development of KRAS* inhibitors represents a significant breakthrough in PDAC therapy. However, emerging evidence suggests that PDAC may eventually overcome its dependency on KRAS* and develop resistance to KRAS* inhibitor treatment. Our laboratory is dedicated to uncovering novel, accessible therapeutic targets that govern the mechanisms essential for KRAS*- driven PDAC cell survival. Our recent work has identified Syndecan1 (SDC1), a major heparan sulfate proteoglycan, as a critical mediator of KRAS* activity, promoting nutrient salvaging and fueling cancer cell growth, even in cases where PDAC cells have evaded KRAS* targeted therapy. Our finding positions SDC1 as a promising new therapeutic target for PDAC therapy. In our pursuit of new therapeutic approaches that can directly and specifically target SDC1 activity, we have successfully developed a monoclonal antibody against SDC1 (22B mAb), which exhibits remarkable sensitivity and specificity for human SDC1. Our results indicate that 22B mAb exerts substantial inhibition of PDAC cell growth in vitro and desirable tumor inhibitory effects in vivo in murine PDAC tumors and human PDAC cell-derived xenograft tumors. Interrogation of mechanism of actions further demonstrates that 22B elicits both direct effect in inhibiting macropinocytosis to block nutrient salvage of PDAC cells and indirect effect in inducing antibody-dependent cellular cytotoxicity (ADCC) to eliminate tumor cells. Notably, our 22B mAb synergizes with KRAS* inhibitors, chemotherapy, or immunotherapy, resulting in significantly enhanced therapeutic effects. In summary, our 22B mAb represents the first in class of anti-SDC1 antibody that not only directly targets the PDAC cells but also elicits immune cell-mediated killing. Additionally, our antibody holds the potential for developing new anti-PDAC therapeutic strategies, including antibody drug conjugates with cytotoxic or radionuclides payloads, for SDC1 targeted therapy/theranostics in PDAC. Thus, our newly developed 22B therapeutic antibody provides an alternate strategy for PDAC treatment and opens up new research horizons in therapeutic targeting of SDC1 in other malignancies characterized by aberrant SDC1 expression. Citation Format: Zecheng Yang, Madelaine S Theardy, Shuaitong Chen, Yongkun Wei, Mitsunobu Takeda, Xiaofei Wang, Jun Yao, Jennifer Li, Jangho Park, Yangxi Zheng, Long T Vien, Laura Bover, Haoqiang Ying, Wantong Yao. A novel SDC1-targeted therapeutic antibody inhibits macropinocytosis and induces anti-tumor immunity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C019.

Engineered AAV capsid transport mutants overcome transduction deficiencies in the aged CNS

Adeno-associated virus-based gene therapy has enjoyed great successes over the last decade, with FDA approved therapeutics and a robust clinical pipeline. Nonetheless, barriers to successful translation remain. For example, advanced age is associated with impaired brain transduction, with the diminution of infectivity depending on anatomical region and capsid. Given that CNS gene transfer is often associated with neurodegenerative diseases where age is the chief risk-factor, we sought to better understand the causes of this impediment. We assessed two AAV variants hypothesized to overcome factors negatively impacting transduction in the aged brain; specifically, changes in extracellular and cell-surface glycans, and intracellular transport. We evaluated a heparin sulfate proteoglycan null variant with or without mutations enhancing intracellular transport. Vectors were injected into the striatum of young-adult or aged rats to address whether improving extracellular diffusion, removing glycan receptor dependence, or improving intracellular transport are important factors in transducing the aged brain. We found that, regardless of the viral capsid, there was a reduction in many of our metrics of transduction in the aged brain. However, the transport mutant was less sensitive to age, suggesting that changes in the cellular transport of AAV capsids is a key factor in age-related transduction deficiency.

Differential regulation of heparan sulfate biosynthesis in fibroblasts cocultured with normal vs. cancerous prostate cells.

Heparan sulfate proteoglycans (HSPGs) regulate a wide range of biological activities in both physiological and pathological conditions. Altered expression or deregulated function of HSPGs and their heparan sulfate (HS) chains significantly contribute to carcinogenesis as well and crucially depends on the functioning of the complex system of HS biosynthetic/modifying enzymes termed as "GAGosome". Here, we aimed at investigating the expression profile of the system in a cell culture model of stroma-epithelial crosstalk and searching for transcription factors potentially related to the regulation of expression of the genes involved. Coculture of BjTERT-fibroblasts with normal PNT2 human prostate epithelial cells resulted in significant downregulation (2-4-fold) of transcriptional activity of HS metabolism-involved genes (EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE) in both cell types, whereas coculture with prostate cancer cells (LNCaP, PC3, DU145) demonstrated no significant interchanges. Human Transcription Factor RT2 Profiler PCR array and manual RT-PCR verification supposed FOS, MYC, E2F, SRF, NR3C1 as potential candidates for regulation and/or coordination of HS biosynthesis. Taken together, transcriptional activity of HS biosynthetic system in normal fibroblasts and prostate epithelial cells during their coculture might be controlled by their intercellular communication, reflecting of adaptation of these cells to each other. The regulation is attenuated or abrogated if normal fibroblasts interact with prostate cancer cells making the cancer cells independent of the limiting effects of fibroblasts, thus contributing to possibility of unlimited growth and progression. Overall, these data demonstrate an ability of cell-cell interactions to affect transcriptional activity of HS biosynthesis-involved genes.