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Heparan sulfate proteoglycan 2 (HSPG2) gene encodes the matrix protein Perlecan, and genetic inactivation of this gene creates mice that are embryonic lethal with severe neural tube defects (NTDs). We discovered rare genetic variants of HSPG2 in 10% cases compared to only 4% in controls among a cohort of 369 NTDs. Endorepellin, a peptide cleaved from the domain V of Perlecan, is known to promote angiogenesis and autophagy in endothelial cells. The roles of enderepellin in neurodevelopment remain unclear so far. Our study revealed that endorepellin can migrate to the neuroepithelial cells and then be recognized and bind with the neuroepithelia receptor neurexin in vivo. Through the endocytic pathway, the interaction of endorepellin and neurexin physiologically triggers autophagy and appropriately modulates the differentiation of neural stem cells into neurons as a blocker, which is necessary for normal neural tube closure. We created knock-in (KI) mouse models with human-derived HSPG2 variants, using sperm-like stem cells that had been genetically edited by CRISPR/Cas9. We realized that any HSPG2 variants that affected the function of endorepellin were considered pathogenic causal variants for human NTDs given that the severe NTD phenotypes exhibited by these KI embryos occurred in a significantly higher response frequency compared to wildtype embryos. Our study provides a paradigm for effectively confirming pathogenic mutations in other genetic diseases. Furthermore, we demonstrated that using autophagy inhibitors at a cellular level can repress neuronal differentiation. Therefore, autophagy agonists may prevent NTDs resulting from failed autophagy maintenance and neuronal over-differentiation caused by deleterious endorepellin variants., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Dyssegmental dysplasia (DD) is a severe skeletal dysplasia comprised of two subtypes: lethal Silverman-Handmaker type (DDSH) and nonlethal Rolland-Desbuquois type (DDRD). DDSH is caused by biallelic pathogenic variants in HSPG2 encoding perlecan, whereas the genetic cause of DDRD remains undetermined. Schwartz-Jampel syndrome (SJS) is also caused by biallelic pathogenic variants in HSPG2 and is an allelic disorder of DDSH. In SJS and DDSH, 44 and 8 pathogenic variants have been reported in HSPG2, respectively. Here, we report that five patients with DDRD carried four pathogenic variants in HSPG2: c.9970 G > A (p.G3324R), c.559 C > T (p.R187X), c7006 + 1 G > A, and c.11562 + 2 T > G. Two patients were homozygous for p.G3324R, and three patients were heterozygous for p.G3324R. Haplotype analysis revealed a founder haplotype spanning 85,973 bp shared in the five patients. SJS, DDRD, and DDSH are allelic disorders with pathogenic variants in HSPG2., (© 2024. The Author(s).)

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2 +/- ) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2 +/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure., Competing Interests: Declaration of interests A patent application related to the left ventricle CM differentiation protocol used in this work has been submitted (WO 2020/245612) and is partly licensed to Axol Biosciences. A.S.B. is a beneficiary of this license., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dally ΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dally ΔGAG -specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dally ΔGAG , dlp ΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

SARS-CoV-2 variants evolve to rely more on heparan sulfate (HS) for viral attachment and subsequent infection. In our earlier work, we demonstrated that the Delta variant's spike protein binds more strongly to HS compared to WT SARS-CoV-2, leading to enhanced cell internalization via syndecans (SDCs), a family of transmembrane HS proteoglycans (HSPGs) facilitating the cellular entry of the original strain. Using our previously established ACE2- or SDC-overexpressing cellular models, we now compare the ACE2- and SDC-dependent cellular uptake of heat-inactivated WT SARS-CoV-2 with the Delta and Omicron variants. Internalization studies with inactivated virus particles showed that ACE2 overexpression could not compensate for the loss of HS in Omicron's internalization, suggesting that this variant primarily uses HSPGs to enter cells. Although SDCs increased the internalization of all three viruses, subtle differences could be detected between their SDC isoform preferences. The Delta variant particularly benefitted from SDC1, 2, and 4 overexpression for cellular entry, while SDC4 had the most prominent effect on Omicron internalization. The SDC4 knockdown (KD) in Calu-3 cells reduced the cellular uptake of all three viruses, but the inhibition was the most pronounced for Omicron. The polyanionic heparin also hindered the cellular internalization of all three viruses with a dominant inhibitory effect on Omicron. Omicron's predominant HSPG affinity, combined with its preference for the universally expressed SDC4, might account for its efficient transmission yet reduced pathogenicity.

Background: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence., Methods: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane., Results: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells., Conclusions: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Despite the use of the highly specific anti-HER2 receptor (trastuzumab) therapy, HER2-positive breast cancers account for 20-30% of all breast cancer carcinomas, with HER2 status a challenge to treatment interventions. The heparan sulfate proteoglycans (HSPGs) are prominently expressed in the extracellular matrix (ECM), mediate breast cancer proliferation, development, and metastasis with most studies to date conducted in animal models. This study examined HSPGs in HER2-positive human breast cancer cell lines and their contribution to cancer cell proliferation. The study examined the cells following enhancement (via the addition of heparin) and knockdown (KD; using short interfering RNA, siRNA) of HSPG core proteins. The interaction of HSPG core proteins and AKT signalling molecules was examined to identify any influence of this signalling pathway on cancer cell proliferation. Our findings illustrated the HSPG syndecan-4 (SDC4) core protein significantly regulates cell proliferation with increased BC cell proliferation following heparin addition to cultures and decreased cell number following SDC4 KD. In addition, along with SDC4, significant changes in CK19/AKT signalling were identified as mediators of BC HER2-positive BC cell proliferation. This study provides evidence for a cell growth regulatory axis involving HSPGs/CK19 and AKT that represents a potential molecular target to prevent proliferation of HER2-positive breast cancer cells., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Heparan sulfate proteoglycans (HSPGs) reside in most cells; on their surface, in the pericellular milieu and/or extracellular matrix. In the eye, HSPGs can orchestrate the activity of key signalling molecules found in the ocular environment that promote its development and homeostasis. To date, our understanding of the specific roles played by individual HSPG family members, and the heterogeneity of their associated sulfated HS chains, is in its infancy. The crystalline lens is a relatively simple and well characterised ocular tissue that provides an ideal stage to showcase and model the expression and unique roles of individual HSPGs. Individual HSPG core proteins are differentially localised to eye tissues in a temporal and spatial developmental- and cell-type specific manner, and their loss or functional disruption results in unique phenotypic outcomes for the lens, and other ocular tissues. More recent work has found that different HS sulfation enzymes are also presented in a cell- and tissue-specific manner, and that disruption of these different sulfation patterns affects specific HS-protein interactions. Not surprisingly, these sulfated HS chains have also been reported to be required for lens and eye development, with dysregulation of HS chain structure and function leading to pathogenesis and eye-related phenotypes. In the lens, HSPGs undergo significant and specific changes in expression and function that can drive pathology, or in some cases, promote tissue repair. As master signalling regulators, HSPGs may one day serve as valuable biomarkers, and even as putative targets for the development of novel therapeutics, not only for the eye but for many other systemic pathologies., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists and have no financial disclosures., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

With the COVID-19 pandemic continuing, more contagious SARS-CoV-2 variants, including Omicron, have been emerging. The mutations, especially those that occurred on the spike (S) protein receptor-binding domain (RBD), are of significant concern due to their potential capacity to increase viral infectivity, virulence, and breakthrough antibodies' protection. However, the molecular mechanism involved in the pathophysiological change of SARS-CoV-2 mutations remains poorly understood. Here, we summarized 21 RBD mutations and their human angiotensin-converting enzyme 2 (hACE2) and/or neutralizing antibodies' binding characteristics. We found that most RBD mutations, which could increase surface positive charge or polarity, enhanced their hACE2 binding affinity and immune evasion. Based on the dependence of electrostatic interaction of the epitope residue of virus and docking protein (like virus receptors or antibodies) for its invasion, we postulated that the charge and/or polarity changes of novel mutations on the RBD domain of S protein could affect its affinity for the hACE2 and antibodies. Thus, we modeled mutant S trimers and RBD-hACE2 complexes and calculated their electrotactic distribution to study surface charge changes. Meanwhile, we emphasized that heparan sulfate proteoglycans (HSPGs) might play an important role in the hACE2-mediated entry of SARS-CoV-2 into cells. Those hypotheses provide some hints on how SARS-CoV-2 mutations enhance viral fitness and immune evasion, which may indicate potential ways for drug design, next-generation vaccine development, and antibody therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Zhang and Wang.)

Perlecan, a basement membrane-type heparan sulfate proteoglycan, is an important molecule in the functional diversity of organisms because of the diversity of its glycan chains and the multifunctionality of its core proteins. Human diseases associated with perlecan have been identified using gene-deficient mice. Two human diseases related to perlecan have been reported. One is Silverman-Handmaker type dyssegmental dysplasia, resulting from the complete loss of function of the HSPG2 gene that encodes perlecan core protein, which is mapped to chromosome 1p36. The other is Schwartz-Jampel syndrome resulting from the partial loss of function of the HSPG2 gene. Subsequent in vivo and in vitro studies have revealed the organ-specific functions of perlecan, suggesting its involvement in the pathogenesis of various human diseases. In this review, we discuss the role of perlecan in human diseases and summarize our knowledge about perlecan as a future therapeutic target to treat related diseases and for healthy longevity.

The conversion of soluble tau protein to insoluble, hyperphosphorylated neurofibrillary tangles (NFTs) is a major hallmark leading to neuronal death observed in neurodegenerative tauopathies. Unlike NFTs, the involvement of monomeric tau in the progression of tau pathology has been less investigated. Using live-cell confocal microscopy and flow cytometry, we demonstrate that soluble 0N4R monomers were rapidly endocytosed by SH-SY5Y and C6 glioma cells via actin-dependent macropinocytosis. Further, cellular endocytosis of monomeric tau has been demonstrated to be HSPG-dependent, as shown in C6 glial cells with genetic knockouts of xylosyltransferase-1-a key enzyme in HSPG synthesis-with a reduced level of tau uptake. Tau internalization subsequently triggers ERK1/2 activation and therefore, the upregulation of IL-6 and IL-1β. The role of ERK1/2 in regulating the levels of pro-inflammatory gene transcripts was confirmed by inhibiting the MEK-ERK1/2 signaling pathway, which led to the attenuated IL-6 and IL-1β expressions but not that of TNF-α. Moreover, as a key regulator of tau internalization, LRP1 (low-density lipoprotein receptor-related protein 1) levels were downregulated in response to monomeric tau added to C6 cells, while it was upregulated in HSPG-deficient cells, suggesting that the involvement of LRP1 in tau uptake depends on the presence of HSPGs on the cell surface. The subsequent LRP1 knockdown experiment we performed shows that LRP1 deficiency leads to an attenuated propensity for tau uptake and further elevated IL-6 gene expression. Collectively, our data suggest that tau has multiple extracellular binding partners that mediate its internalization through distinct mechanisms. Additionally, this study demonstrates the important role of both HSPGs and LRP1 in regulating cellular immune responses to tau protein monomers, providing a novel target for alleviating the neuroinflammatory environment before the formation of neurofibrillary tangles., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Several classes of heparan sulfate proteoglycan (HSPG) core proteins and all HS biosynthetic/modifying enzymes are evolutionarily conserved from human to Drosophila melanogaster. This genetically tractable model offers highly sophisticated techniques to manipulate gene function in a spatially and temporally controlled manner. Thus, Drosophila genetics has been a powerful system to explore functions of HSPGs in vivo. In this chapter, we will introduce three genetic techniques available in Drosophila: TARGET (temporal and regional gene expression targeting), MARCM (mosaic analysis with a repressible cell marker), and FLP-Out., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Kashin-Beck disease (KBD) is an endemic osteochondropathy. Due to a lack of suitable animal or cellular disease models, the research progress on KBD has been limited. Our goal was to establish the first disease-specific human induced pluripotent stem cell (hiPSC) cellular disease model of KBD, and to explore its etiology and pathogenesis exploiting transcriptome sequencing. HiPSCs were reprogrammed from dermal fibroblasts of two KBD and one healthy control donor via integration-free vectors. Subsequently, hiPSCs were differentiated into chondrocytes through three-week culture. Gene expression profiles in KBD, normal primary chondrocytes, and hiPSC-derived chondrocytes were defined by RNA sequencing. A Venn diagram was constructed to show the number of shared differentially expressed genes (DEGs) between KBD and normal. Gene oncology and Kyoto Encyclopedia of Genes and Genomes annotations were performed, and six DEGs were further validated in other individuals by RT-qPCR. KBD cellular disease models were successfully established by generation of hiPSC lines. Seventeen consistent and significant DEGs present in all compared groups (KBD and normal) were identified. RT-qPCR validation gave consistent results with the sequencing data. Glycosaminoglycan biosynthesis-heparan sulfate/heparin; PPAR signaling pathway; and cell adhesion molecules (CAMs) were identified to be significantly altered in KBD. Differentiated chondrocytes derived from KBD-origin hiPSCs provide the first cellular disease model for etiological studies of KBD. This study also provides new sights into the pathogenesis and etiology of KBD and is likely to inform the development of targeted therapeutics for its treatment., (© 2021 Federation of European Biochemical Societies.)

DAL-1/4.1B is frequently absent in lung cancer tissues, which is significantly related to the occurrence and development of lung cancer. In this research, we found that DAL-1/4.1B affected the uptake of exosomes by lung cancer cells. When the expression of DAL-1/4.1B increased and decreased, the ability of exosome uptake enhanced and attenuated correspondingly. And we found that when cells were treated with different vesicles uptake inhibitors (chlorpromazine, methyl-β-cyclodextrin (MβCD), cytochalasin D, chloroquine and heparin) and heparinase (HSPE), only heparin and HSPE counteracted the uptake enhancement effect caused by DAL-1/4.1B. Therefore, we speculated that DAL-1/4.1B might promote the uptake of exosomes through the heparan sulfate proteoglycans (HSPGs) pathway. After screening the expression of HSPGs and HSPE in H292 cells, the expression of heparan sulfate proteoglycan 2 (HSPG2) increased with overexpression of DAL-1/4.1B and decreased with knockdown of DAL-1/4.1B. Meanwhile, exosome uptake decreased with HSPG2 knockdown in H292 and DAL-1/4.1B-overexpressing H292 cells. Moreover, knockdown of DAL-1/4.1B and HSPG2 in lung cancer A549 cells resulted in a similar decrease in exosome uptake, and the expression of HSPG2 was also decreased with DAL-1/4.1B knockdown. These results indicated that HSPG2 directly affected the uptake of exosomes, while DAL-1/4.1B positively affected the expression of HSPG2. Therefore, DAL-1/4.1B may promote cellular adhesion and inhibit migration in cancer cells., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Genetic studies using a model organism, Drosophila melanogaster, have been contributing to elucidating the in vivo functions of heparan sulfate proteoglycans (HSPGs). On the other hand, biochemical analysis of Drosophila glycosaminoglycans (GAGs) has been limited, mainly due to the insufficient amount of the material obtained from the animal. Recently, a novel in vitro system has been developed by establishing mutant cell lines for heparan sulfate (HS)-modifying enzyme genes. Metabolic radiolabeling of GAGs allows us to assess uncharacterized features of Drosophila GAGs and the effects of the mutations on HS structures and function. The novel in vitro system will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The arrangement of lens cells is regulated by ocular growth factors. Although the effects of these inductive molecules on lens cell behavior (proliferation, survival, and fiber differentiation) are well-characterized, the precise mechanisms underlying the regulation of growth factor-mediated signaling in lens remains elusive. Increasing evidence highlights the importance of heparan sulfate proteoglycans (HSPGs) for the signaling regulation of growth factors; however, the identity of the different lens HSPGs and the specific roles they play in lens biology are still unknown., Methods: Semiquantitative real-time (RT)-PCR and immunolabeling were used to characterize the spatial distribution of all known HSPG core proteins and their associated glycosaminoglycans (heparan and chondroitin sulfate) in the postnatal rat lens. Fibroblast growth factor (FGF)-2-treated lens epithelial explants, cultured in the presence of Surfen (an inhibitor of heparan sulfate [HS]-growth factor binding interactions) were used to investigate the requirement for HS in FGF-2-induced proliferation, fiber differentiation, and ERK1/2-signaling., Results: The lens expresses all HSPGs. These HSPGs are differentially localized to distinct functional regions of the lens. In vitro, inhibition of HS-sulfation with Surfen blocked FGF-2-mediated ERK1/2-signaling associated with lens epithelial cell proliferation and fiber differentiation, highlighting that these cellular processes are dependent on HS., Conclusions: These findings support a requirement for HSPGs in FGF-2 driven lens cell proliferation and fiber differentiation. The identification of specific HSPG core proteins in key functional lens regions, and the divergent expression patterns of closely related HSPGs, suggests that different HSPGs may differentially regulate growth factor signaling networks leading to specific biological events involved in lens growth and maintenance.

Background: Heparan sulfate proteoglycan (HSPG) expression is found in many animal tissues and regulates growth factor signaling such as of Fibroblast growth factors (Fgf), Wingless/Int (Wnt) and Hedgehog (HH). Glypicans, which are GPI (glycosylphosphatidylinositol)-anchored proteins, and transmembrane-anchored syndecans represent two major HSPG protein families whose involvement in development and disease has been demonstrated. Their participation in regenerative processes both of the central nervous system and of regenerating limbs is well documented. However, whether HSPG are expressed in regenerating zebrafish fins, is currently unknown., Results: Here, we carried out a systematic screen of glypican and syndecan mRNA expression in regenerating zebrafish fins during the outgrowth phase. We find that 8 of the 10 zebrafish glypicans and the three known zebrafish syndecans show specific expression at 3 days post amputation. Expression is found in different domains of the regenerate, including the distal and lateral basal layers of the wound epidermis, the distal most blastema and more proximal blastema regions., Conclusions: HSPG expression is prevalent in regenerating zebrafish fins. Further research is needed to delineate the function of glypican and syndecan action during zebrafish fin regeneration., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association of Anatomists.)

The Drosophila female germline stem cell (GSC) niche provides an excellent model for understanding the stem cell niche in vivo. The GSC niche is composed of stromal cells that provide growth factors for the maintenance of GSCs and the associated extracellular matrix (ECM). Although the function of stromal cells/growth factors has been well studied, the function of the ECM in the GSC niche is largely unknown. In this study, we investigated the function of syndecan and perlecan, molecules of the heparan sulfate proteoglycan (HSPG) family, as the main constituents of the ECM. We found that both of these genes were expressed in niche stromal cells, and knockdown of them in stromal cells decreased GSC number, indicating that these genes are important niche components. Interestingly, our genetic analysis revealed that the effects of syndecan and perlecan on the maintenance of GSC were distinct. While the knockdown of perlecan in the GSC niche increased the number of cystoblasts, a phenotype suggestive of delayed differentiation of GSCs, the same was not true in the context of syndecan. Notably, the overexpression of syndecan and perlecan did not cause an expansion of the GSC niche, opposing the results reported in the context of glypican, another HSPG gene. Altogether, our data suggest that HSPG genes contribute to the maintenance of GSCs through multiple mechanisms, such as the control of signal transduction, and ligand distribution/stabilization. Therefore, our study paves the way for a deeper understanding of the ECM functions in the stem cell niche., (© 2021 Japanese Society of Developmental Biologists.)

Increasing the healthy/unhealthy fatty acid (FA) ratio in meat is one of the urgent tasks required to address consumer concerns. However, the regulatory mechanisms ultimately resulting in FA profiles vary among animals and remain largely unknown. In this study, using ~1.2 Tb high-quality RNA-Seq-based transcriptomic data of 188 samples from four key metabolic tissues (rumen, liver, muscle, and backfat) together with the contents of 49 FAs in backfat, the molecular regulatory mechanisms of these tissues contributing to FA formation in cattle were explored. Using this large dataset, the alternative splicing (AS) events, one of the transcriptional regulatory mechanisms in four tissues were identified. The highly conserved and absent AS events were detected in rumen tissue, which may contribute to its functional differences compared with the other three tissues. In addition, the healthy/unhealthy FA ratio related AS events, differential expressed (DE) genes, co-expressed genes, and their functions in four tissues were analysed. Eight key genes were identified from the integrated analysis of DE, co-expressed, and AS genes between animals with high and low healthy/unhealthy FA ratios. This study provides an applicable pipeline for AS events based on comprehensive RNA-Seq analysis and improves our understanding of the regulatory mechanism of FAs in beef cattle.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor., Competing Interests: Competing interest statement: The technology described in the manuscript has been licensed to a start-up company., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: The basement membrane (BM) provides mechanical shaping of tissues during morphogenesis. The Drosophila BM proteoglycan Perlecan is vital for this process in the wing imaginal disc. This function is thought to be fostered by the heparan sulfate chains attached to the domain I of vertebrate Perlecan. However, this domain is not present in Drosophila, and the source of Perlecan for the wing imaginal disc BM remains unclear. Here, we tackle these two issues., Results: In silico analysis shows that Drosophila Perlecan holds a domain I. Moreover, by combining in situ hybridization of Perlecan mRNA and protein staining, together with tissue-specific Perlecan depletion, we find that there is an autonomous and a non-autonomous source for Perlecan deposition in the wing imaginal disc BM. We further show that both sources cooperate for correct distribution of Perlecan in the wing imaginal disc and morphogenesis of this tissue., Conclusions: These results show that Perlecan is fully conserved in Drosophila, providing a valuable in vivo model system to study its role in BM function. The existence of two different sources for Perlecan incorporation in the wing imaginal disc BM raises the possibility that inter-organ communication mediated at the level of the BM is involved in organogenesis., (© 2020 American Association of Anatomists.)

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C-C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Heparan sulfate proteoglycans (HSPGs) are components of the cell surface and extracellular matrix, which bear long polysaccharides called heparan sulfate (HS) attached to the core proteins. HSPGs interact with a variety of ligand proteins through the HS chains, and mutations in HSPG-related genes influence many biological processes and cause various diseases. In particular, recent findings from vertebrate and invertebrate studies have raised the importance of glycosylphosphatidylinositol-anchored HSPGs, glypicans, as central players in the development and functions of synapses. Glypicans are important components of the synapse-organizing protein complexes and serve as ligands for leucine-rich repeat transmembrane neuronal proteins (LRRTMs), leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (RPTPs), and G-protein-coupled receptor 158 (GPR158), regulating synapse formation. Many of these interactions are mediated by the HS chains of glypicans. Neurexins (Nrxs) are also synthesized as HSPGs and bind to some ligands in common with glypicans through HS chains. Therefore, glypicans and Nrxs may act competitively at the synapses. Furthermore, glypicans regulate the postsynaptic expression levels of ionotropic glutamate receptors, controlling the electrophysiological properties and non-canonical BMP signaling of synapses. Dysfunctions of glypicans lead to failures in neuronal network formation, malfunction of synapses, and abnormal behaviors that are characteristic of neurodevelopmental disorders. Recent human genetics revealed that glypicans and HS are associated with autism spectrum disorder, neuroticism, and schizophrenia. In this review, we introduce the studies showing the roles of glypicans and HS in synapse formation, neural plasticity, and neurological disorders, especially focusing on the mouse and Drosophila as potential models for human diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kamimura and Maeda.)

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis results in poor yields of recombinant human bone morphogenetic proteins (rhBMPs) from CHO cell cultures. Upon incubation of rhBMP-2 and rhBMP-7 with CHO cells at 37 °C, both rhBMP-2 and rhBMP-7 bound to the cell surface HSPGs in CHO cells, but only rhBMP-2 was actively internalized into CHO cells. Cell surface HSPGs were found to serve as the main receptor for rhBMP-2 internalization. It was also found that the cell surface HSPG-mediated endocytosis of rhBMP-2 occurred through both the clathrin- and caveolin-dependent pathways. Blockage of rhBMP-2 internalization by the addition of structural analogs of HSPGs such as dextran sulfate (DS) and heparin dramatically increased rhBMP-2 production in recombinant CHO (rCHO) cell cultures. Compared to the control cultures, addition of DS (1.0 g/L) and heparin (0.2 g/L) resulted in a 22.0- and 19.0-fold increase in the maximum rhBMP-2 concentration, respectively. In contrast, the production of rhBMP-7, which was not internalized into the rCHO cells, did not dramatically increase upon addition of DS and heparin. Taken together, rhBMPs have a different fate in terms of HSPG-mediated internalization in CHO cells. HSPG-mediated endocytosis of each rhBMP should be understood individually to increase the rhBMP yield in rCHO cell cultures.

Subversion of heparan sulfate proteoglycans (HSPGs) is thought to be a common virulence mechanism shared by many microbial pathogens. The prevailing assumption is that pathogens co-opt HSPGs as cell surface attachment receptors or as inhibitors of innate host defense. However, there are few data that clearly support this idea in vivo We found that deletion of syndecan-1 (Sdc1), a major cell surface HSPG of epithelial cells, causes a gain of function in a mouse model of scarified corneal infection, where Sdc1 -/- corneas were significantly less susceptible to Streptococcus pneumoniae infection. Administration of excess Sdc1 ectodomains significantly inhibited S. pneumoniae corneal infection, suggesting that Sdc1 promotes infection as a cell surface attachment receptor. However, S. pneumoniae did not interact with Sdc1 and Sdc1 was shed upon S. pneumoniae infection, indicating that Sdc1 does not directly support S. pneumoniae adhesion. Instead, Sdc1 promoted S. pneumoniae adhesion by driving the assembly of fibronectin (FN) fibrils in the corneal basement membrane to which S. pneumoniae attaches when infecting injured corneas. S. pneumoniae specifically bound to corneal FN via PavA, and PavA deletion significantly attenuated S. pneumoniae virulence in the cornea. Excess Sdc1 ectodomains inhibited S. pneumoniae corneal infection by binding to the Hep II domain and interfering with S. pneumoniae PavA binding to FN. These findings reveal a previously unknown virulence mechanism of S. pneumoniae where key extracellular matrix (ECM) interactions and structures that are essential for host cell homeostasis are exploited for bacterial pathogenesis. IMPORTANCE Bacterial pathogens have evolved several ingenious mechanisms to subvert host cell biology for their pathogenesis. Bacterial attachment to the host ECM establishes a niche to grow and is considered one of the critical steps of infection. This pathogenic mechanism entails coordinated assembly of the ECM by the host to form the ECM structure and organization that are specifically recognized by bacteria for their adhesion. We serendipitously discovered that epithelial Sdc1 facilitates the assembly of FN fibrils in the corneal basement membrane and that this normal biological function of Sdc1 has detrimental consequences for the host in S. pneumoniae corneal infection. Our studies suggest that bacterial subversion of the host ECM is more complex than previously appreciated., (Copyright © 2020 Jinno et al.)

Background/purpose: Allergen-specific immunotherapy (SIT) is now considered curative to allergic diseases such as asthma. Mechanistically, our previous work showed DNA hypermethylation of cytokine genes, in T-helper cells, in allergic asthmatic children treated with allergen-SIT. In this study, we extended to work to assess possible changes in the DNA methylomes of peripheral blood mononuclear cells (PBMCs), isolated from mite allergen-SIT asthmatic children, to explore further the underlying methylation changes., Methods: Thirteen allergic asthmatic children who received Der p-SIT, 12 non-SIT allergic asthmatic controls, and 12 healthy controls were enrolled. Bisulfite-converted DNA from Der p-stimulated PBMCs was analyzed using Human Methylation 450 k BeadChip. Pyrosequencing and quantitative real-time PCR were used to validate the DNA methylation levels and the gene expression of individual samples., Results: We identified 108 significantly differentially methylated regions (DMRs) unique to Der p-treated PBMCs, with 53 probes linked to demethylated DMRs, and 55 probes linked to methylated DMRs. Three associated genes (BCL6, HSPG2, and HSP90AA1), of selected DMRs, were subjected to bisulfite pyrosequencing. Of these, BCL6 showed significant hypomethylation, while HSPG2 and HSP90AA1 were hypermethylated in SIT group, compared to the AA group. Furthermore, SIT group had significantly higher gene expression of BCL6 and lower gene expression of HSPG2. KEGG pathway analysis further revealed DMR genes involved in ECM-receptor interactions, asthma, and antigen processing and presentation pathways., Conclusions: Several DNA regions showed DNA methylation altered by Der p specific immunotherapy, indicating desensitization-associated methylomes. Genes belonging to these SIT-altered pathways may represent therapeutic targets for better clinical management of asthma., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interests., (Copyright © 2020. Published by Elsevier B.V.)

This study reviewed some new aspects of the modular proteoglycan perlecan, a colossal proteoglycan with a 467 kDa core protein and five distinct functional domains. Perlecan is a heparan sulphate proteoglycan that transiently displays native CS sulphation motifs 4-C-3 and 7-D-4 during tissue morphogenesis these are expressed by progenitor cell populations during tissue development. Perlecan is susceptible to fragmentation by proteases during tissue development and in pathological tissues particularly in domains IV and V. The fragmentation pattern of domain IV has been suggested as a means of grading prostate cancer. Domain V of perlecan is of interest due to its interactive properties with integrin α5β1 that promotes pericyte migration enhancing PDGF-BB-induced phosphorylation of PDGFRβ, Src homology region 2 domain-containing phosphatase-2, and focal adhesion kinase supporting the repair of the blood brain barrier following ischaemic stroke. Fragments of domain V can also interact with α2β1 integrin disrupting tube formation by endothelial cells. LG1-LG2, LG3 fragments can antagonise VEGFR2, and α2β1 integrin interactions preventing angiogenesis by endothelial cells. These domain V fragments are of interest as potential anti-tumour agents. Perlecan attached to the luminal surfaces of endothelial cells in blood vessels acts as a flow sensor that signals back to endothelial and smooth muscle cells to regulate vascular tone and blood pressure. Perlecan also acts as a flow sensor in the lacuno-canalicular space regulating osteocytes and bone homeostasis. Along with its biomechanical regulatory properties in cartilaginous tissues this further extends the functional repertoire of this amazingly diverse functional proteoglycan., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Alterations in the composition of the extracellular matrix (ECM) critically regulate the cellular responses in tissue repair, remodeling, and fibrosis. After injury, proteolytic degradation of ECM generates bioactive ECM fragments, named matricryptins, exposing cryptic sites with actions distinct from the parent molecule. Matricryptins contribute to the regulation of inflammatory, reparative, and fibrogenic cascades through effects on several different cell types both in acute and chronic settings. Fibroblasts play a major role in matricryptin generation not only as the main cellular source of ECM proteins, but also as producers of matrix-degrading proteases. Moreover, several matricryptins exert fibrogenic or reparative actions by modulating fibroblast phenotype and function. This review manuscript focuses on the mechanisms of matricyptin generation in injured and remodeling tissues with an emphasis on fibroblast-matricryptin interactions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Marfan syndrome (MFS) is a connective tissue disease caused by variants in the FBN1 gene. Nevertheless, other genes influence the manifestations of the disease, characterized by high clinical variability even within families. We mapped modifier loci for cardiovascular and skeletal manifestations in the mg∆ loxPneo mouse model for MFS and the synthenic loci in the human genome. Corroborating our findings, one of those loci was identified also as a modifier locus in MFS patients. Here, we investigate the HSPG2 gene, located in this region, as a candidate modifier gene for MFS. We show a correlation between Fbn1 and Hspg2 expression in spinal column and aorta in non-isogenic mg∆ loxPneo mice. Moreover, we show that mice with severe phenotypes present lower expression of Hspg2 than those mildly affected. Thus, we propose that HSPG2 is a strong candidate modifier gene for MFS and its role in modulating disease severity should be investigated in patients.

Hemolytic uremic syndrome (HUS) is a severe renal disease that is often preceded by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). The exact mechanism of Stx-mediated inflammation on human glomerular microvascular endothelial cells (HGMVECs) during HUS is still not well understood. In this study, we investigated the effect of Stx1 on the gene expression of proteins involved in leucocyte-mediated and complement-mediated inflammation. Our results showed that Stx1 enhances the mRNA and protein expression of heparan sulfate proteoglycan (HSPG) syndecan-4 in HGMVECs pre-stimulated with tumor necrosis factor α (TNFα). CD44 was upregulated on mRNA but not on protein level; no effect on the mRNA expression of other tested HSPGs glypican-1 and betaglycan was observed. Furthermore, Stx1 upregulated the mRNA, cell surface expression, and supernatant levels of the intercellular adhesion molecule-1 (ICAM-1) in HGMVECs. Interestingly, no effect on the protein levels of alternative pathway (AP) components was observed, although C3 mRNA was upregulated. All observed effects were much stronger in HGMVECs than in human umbilical endothelial cells (HUVECs), a common model cell type used in endothelial studies. Our results provide new insights into the role of Stx1 in the pathogenesis of HUS. Possibilities to target the overexpression of syndecan-4 and ICAM-1 for STEC-HUS therapy should be investigated in future studies.

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2 C1532Y - Neo Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2 C1532Y - Neo cartilage in vitro , suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Ocken et al.)

Heparan sulfate proteoglycan 2 (HSPG2), also known as perlecan, is a large multi-domain extracellular matrix proteoglycan, which contributes to the invasion, metastasis and angiogenesis of solid tumor. However, very little is known about the effect of HSPG2 on acute myeloid leukemia (AML). This study aims to investigate the prognostic value of the HSPG2 gene in terms of overall survival and leukemia-free survival in patients with AML. Bone marrow mononuclear cells (BMMCs) from 4 AML patients and 3 healthy controls were processed for RNA-Sequencing (RNA-seq). The mRNA expression level of HSPG2 in BMMCs and CD34 + hematopoietic stem/progenitor cells (HSPC) obtained from enrolled participants and human leukemic cell lines was detected by RT-qPCR. Then the correlations between the expression of HSPG2 and a variety of important clinical parameters, such as median white blood cell (WBC) count and bone marrow (BM) blasts, were further analyzed. The expression level of HSPG2 was significantly upregulated in AML patients at the time of diagnosis, downregulated after complete remission and then elevated again at relapse. Moreover, HSPG2 expression was associated with median WBC count (P < 0.001), median hemoglobin (P = 0.02), median platelet count (P = 0.001), and BM blasts (P < 0.001) in AML patients. Patients with high HSPG2 expression had both worse overall survival (OS) (P = 0.001) and poorer leukemia-free survival (LFS) (P = 0.047). In the multivariate analysis model, HSPG2 was identified as an independent prognostic biomarker of AML. Taken together, these results indicate that HSPG2 overexpression was associated with poor prognosis in AML patients, and may be a prognostic biomarker and therapeutic target of AML.

R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs., Competing Interests: RD, Pv, IJ, TM, GP, JM, DL, JC, MH, CS, MM, AL, RR No competing interests declared

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, influences various signaling pathways in endothelial cells by binding to VEGFR2. In this study, we discovered that soluble endorepellin activates the canonical stress signaling pathway consisting of PERK, eIF2α, ATF4, and GADD45α. Specifically, endorepellin evoked transient activation of VEGFR2, which, in turn, phosphorylated PERK at Thr 980 Subsequently, PERK phosphorylated eIF2α at Ser 51 , upregulating its downstream effector proteins ATF4 and GADD45α. RNAi-mediated knockdown of PERK or eIF2α abrogated the endorepellin-mediated up-regulation of GADD45α, the ultimate effector protein of this stress signaling cascade. To functionally validate these findings, we utilized an ex vivo model of angiogenesis. Exposure of the aortic rings embedded in 3D fibrillar collagen to recombinant endorepellin for 2-4 h activated PERK and induced GADD45α vis à vis vehicle-treated counterparts. Similar effects were obtained with the established cellular stress inducer tunicamycin. Notably, chronic exposure of aortic rings to endorepellin for 7-9 days markedly suppressed vessel sprouting, an angiostatic effect that was rescued by blocking PERK kinase activity. Our findings unravel a mechanism by which an extracellular matrix protein evokes stress signaling in endothelial cells, which leads to angiostasis., (© 2020 Kapoor et al.)

Previous studies have reported that heparan sulfate proteoglycans (HSPGs) promote amyloid-beta peptide and tau fibrillization in Alzheimer disease (AD) and provide resistance against proteolytic breakdown. We compared the expression levels of 17 HSPG core proteins in 18 AD cases and 6 controls. RT-PCR was used to analyze transcription levels. Immunohistochemistry was performed to localize HSPGs in the brain tissue. We detected expression of all HSPG genes investigated. SDC1, GPC3, and CD44v3 showed the lowest levels of expression, while SDC3 and GPC1 showed the highest. Remarkably, SDC4 and SRGN were overexpressed in most of the areas analyzed. Immunohistochemistry revealed the presence of both SDC4 and SRGN mostly associated with tau and amyloid-β pathology throughout the AD brains. In conclusion, in view of the involvement of HSPGs in AD pathology, especially SDC4 and SRGN, there would seem to be a relationship between the regulation of core protein expression and the pathological features suggesting HSPGs are potential inducers of the disease., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Localised breast cancer can be cured by surgery and adjuvant treatments, but mortality remains high as some tumours metastasize early. Perlecan is a basement membrane (BM) protein involved in tumour development and progression. Here, mRNA and protein expression of perlecan, and mRNA expression of matrix degrading enzymes were studied in normal breast and invasive breast cancer, and correlated to prognostic risk factors, in particular oestrogen status. Moreover, plasma levels of perlecan were measured in patients with breast cancer and compared with controls. mRNA data was extracted from the Cancer Genome Atlas database. Perlecan protein expression was visualized using immunofluorescence and plasma levels measured by ELISA assay. Perlecan mRNA levels were twice as high in normal breast compared with breast cancer tissue. A strong correlation was found between mRNA expression of perlecan and several matrix-degrading enzymes in oestrogen receptor positive (ER+) tumours. Perlecan protein was localized to both epithelial and vascular BMs, but absent in the stroma in normal breast. In breast cancer, the expression of perlecan in epithelial BM was fragmented or completely lost, with a marked upregulation of perlecan expression in the stroma. Significantly higher levels of perlecan were found in plasma of ER+ patients when compared with ER- patients. This study shows that perlecan expression and degradation in breast cancer may be linked to the ER status of the tumour.

Purpose: The study investigated potential correlations between the expression levels of ADAMTS1 and HSPG2 in cumulus cells (CCs) and controlled ovarian hyperstimulation (COH) outcomes., Methods: RT-PCR was used to determine ADAMTS1 and HSPG2 mRNA levels in mice CCs at different timepoints (0, 4, 8, 12, and 16 h) after human chorionic gonadotropin (hCG) injection, and in CCs after RNAi treatment. Women with polycystic ovary syndrome (PCOS) (n = 45) and normal ovulatory controls (n = 103) undergoing IVF/ICSI were recruited. Relative ADAMTS1 and HSPG2 mRNA levels were measured by RT-PCR. Moreover, correlations of ADAMTS1 and HSPG2 levels with COH outcomes were analyzed., Results: At different timepoints after hCG treatment, ADAMTS1 mRNA had the highest level at 12 h, whereas HSPG2 showed opposite profiles to ADAMTS1 with the lowest level at 12 h. HSPG2 expression was upregulated after ADAMTS1 RNAi treatment The PCOS group had higher HSPG2 and lower ADAMTS1 expression levels than controls. In normal ovulatory women (control group), a higher expression of ADAMTS1 and lower expression of HSPG2 were associated with more mature oocytes, transplantable embryos, and good quality embryos, whereas higher transplantable embryo rates and good quality embryo rates were obtained only with lower HSPG2 expression. ROC curves showed the co-measurement of ADAMTS1 and HSPG2 had a better predictive power than separate analyses., Conclusion: The dynamic profiles of ADAMTS1 and HSPG2 were inversely correlated in CCs. In PCOS and normal ovulatory patients, higher ADAMTS1 and lower HSPG2 expression levels in CCs were related to better COH outcomes.

Human cytomegalovirus (HCMV) infections are wide-spread among the general population with manifestations ranging from asymptomatic to severe developmental disabilities in newborns and life-threatening illnesses in individuals with a compromised immune system. Nearly all current drugs suffer from one or more limitations, which emphasizes the critical need to develop new approaches and new molecules. We reasoned that a 'poly-pharmacy' approach relying on simultaneous binding to multiple receptors involved in HCMV entry into host cells could pave the way to a more effective therapeutic outcome. This work presents the study of a synthetic, small molecule displaying pleiotropicity of interactions as a competitive antagonist of viral or cell surface receptors including heparan sulfate proteoglycans and heparan sulfate-binding proteins, which play important roles in HCMV entry and spread. Sulfated pentagalloylglucoside (SPGG), a functional mimetic of heparan sulfate, inhibits HCMV entry into human foreskin fibroblasts and neuroepithelioma cells with high potency. At the same time, SPGG exhibits no toxicity at levels as high as 50-fold more than its inhibition potency. Interestingly, cell-ELISA assays showed downregulation in HCMV immediate-early gene 1 and 2 (IE 1&2) expression in presence of SPGG further supporting inhibition of viral entry. Finally, HCMV foci were observed to decrease significantly in the presence of SPGG suggesting impact on viral spread too. Overall, this work offers the first evidence that pleiotropicity, such as demonstrated by SPGG, may offer a new poly-therapeutic approach toward effective inhibition of HCMV., Competing Interests: S.K.M. was employed by Jes Tech, KBR Wyle Laboratories. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Perlecan, a heparan sulfate proteoglycan, acts as a mechanical sensor for bone to detect external loading. Deficiency of perlecan increases the risk of osteoporosis in patients with Schwartz-Jampel Syndrome (SJS) and attenuates loading-induced bone formation in perlecan deficient mice (Hypo). Considering that intracellular calcium [Ca 2+ ] i is an ubiquitous messenger controlling numerous cellular processes including mechanotransduction, we hypothesized that perlecan deficiency impairs bone's calcium signaling in response to loading. To test this, we performed real-time [Ca 2+ ] i imaging on in situ osteocytes of adult murine tibiae under cyclic loading (8N). Relative to wild type (WT), Hypo osteocytes showed decreases in the overall [Ca 2+ ] i response rate (-58%), calcium peaks (-33%), cells with multiple peaks (-53%), peak magnitude (-6.8%), and recovery speed to baseline (-23%). RNA sequencing and pathway analysis of tibiae from mice subjected to one or seven days of unilateral loading demonstrated that perlecan deficiency significantly suppressed the calcium signaling, ECM-receptor interaction, and focal adhesion pathways following repetitive loading. Defects in the endoplasmic reticulum (ER) calcium cycling regulators such as Ryr1/ryanodine receptors and Atp2a1/Serca1 calcium pumps were identified in Hypo bones. Taken together, impaired calcium signaling may contribute to bone's reduced anabolic response to loading, underlying the osteoporosis risk for the SJS patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Perlecan/Hspg2, a large monomeric heparan sulfate proteoglycan, is found in the basement membrane and extracellular matrix, where it acts as a matrix scaffold, growth factor depot, and tissue barrier. Perlecan deficiency leads to skeletal dysplasia in Schwartz-Jampel Syndrome (SJS) and is a risk factor for osteoporosis. In the SJS-mimicking murine model (Hypo), inferior cortical bone quality and impaired mechanotransduction in osteocytes were reported. This study focused on trabecular bone, where perlecan deficiency was hypothesized to result in structural deficit and altered response to disuse and re-loading. We compared the Hypo versus WT trabecular bone in both axial and appendicular skeletons of 8-38-week-old male mice, and observed severe trabecular deficit in Hypo mice, approximately 50% reduction of Tb.BV/TV regardless of skeletal site and animal age. Defects in endochondral ossification (e.g., accelerated mineralization), increases in osteoclast activity, and altered differentiation of bone progenitor cells in marrow contributed to the Hypo phenotype. The Hypo trabecular bone deteriorated further under three-week hindlimb suspension as did the WT. Re-ambulation partially recovered the lost trabecular bone in Hypo, but not in WT mice. The novel finding that low-impact loading could counter detrimental disuse effects in the perlecan-deficient skeleton suggests a strategy to maintain skeletal health in SJS patients., Competing Interests: The authors declare no conflict of interests.

Cyclin B is a ubiquitous regulatory molecule and has been implicated in mitosis and meiosis in oocytes. Phenomenon that differ in the length of cyclin B 3'UTR in crustacean has attracted much attention, although molecular details are poorly understood. The study of 3'UTR-interacting proteins could yield much information in translational regulation and the mRNA localization process. Previous studies on crayfish suggested that the 3'UTR (1300 bp) probably contains the potential regulatory sequence/motifs such as CPEs and K-box et al. In present study, using pull-down assay coupled with mass spectrometry approach allowing us to explore the potential proteins associated with the 3'UTR. We finally identified four candidate proteins including Hspg 2, Vtg, eef1a and Tuba1a, which annotated as significant roles involved in cell differentiation, lipid transporter activity, and meiotic cell cycle process. The preliminary results will contribute to the advance in understanding the translational activation of cyclin B in oocyte maturation regulation in crustacean., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Planar cell polarity (PCP) plays crucial roles in developmental processes such as gastrulation, neural tube closure and hearing. Wnt pathway mutants are often classified as PCP mutants due to similarities between their phenotypes. Here, we show that in the zebrafish lateral line, disruptions of the PCP and Wnt pathways have differential effects on hair cell orientations. While mutations in the PCP genes vangl2 and scrib cause random orientations of hair cells, mutations in wnt11f1, gpc4 and fzd7a/b induce hair cells to adopt a concentric pattern. This concentric pattern is not caused by defects in PCP but is due to misaligned support cells. The molecular basis of the support cell defect is unknown but we demonstrate that the PCP and Wnt pathways work in parallel to establish proper hair cell orientation. Consequently, hair cell orientation defects are not solely explained by defects in PCP signaling, and some hair cell phenotypes warrant re-evaluation.

In recent years, there have been significant advances in the treatment of breast cancer resulting in remarkably high survival rates. However, treatment options for metastatic triple negative breast cancer (TNBC) are quite limited due to a lack of identifiable, unique markers. Using a phage display-based whole cell biopanning procedure, we developed two human antibodies that bind to tumor cells with a metastatic TNBC phenotype. Our studies further identified domain 1 of HSPG2 (perlecan) protein as the cognate cell surface antigen bound by the antibody. Immunohistochemistry studies utilizing patient tissue samples revealed significant cell surface expression of HSPG2 in both primary tumors and metastatic lesions. Further, higher HSPG2 expression correlated with poor survival in TNBC. The affinity-matured antibody inhibited the growth of triple negative MDA-MB-231 tumors to a greater extent in nude mice than in NSG mice, pointing to the potential role of natural killer cell-mediated antibody-dependent cell cytotoxicity. This mechanism of action was confirmed through in vitro assays using mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). These results suggest that HSPG2 is a promising target in metastatic TNBC and HSPG2-targeted antibodies could represent a potentially novel class of targeted therapeutics for TNBC.

Objective:  To analyze the effects of estrogen alone or in combination with progestogens and tibolone (TIB) on the expression of the extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), of perlecan, and of heparanase (HPSE) of the vascular walls of the carotid arteries., Methods:  A total of 30 250-day-old ovariectomized Wistar rats were orally treated for 5 weeks with: a) 1 mg/kg of estradiol benzoate (EB); b) EB + 0.2 mg/kg of medroxyprogesterone acetate (MPA); c) EB + 0.2mg/kg of norethisterone acetate (NETA); d) EB + 2 mg/kg of dydrogesterone (DI); e) 1 mg/kg of TIB; f) placebo (CTR). Following treatment, the expression of mRNA for MMP-2, MMP-9, and HPSE was analyzed by real-time polymerase chain-reaction (PCR), and the expression of MMP-2, of MMP-9, of tissue inhibitor of metalloproteinase 2 (TIMP-2), and of perlecan was quantified by immunohistochemistry in the carotid arteries., Results:  The groups showed significant differences on mRNA HPSE expression ( p  = 0.048), which was higher in the EB, EB + MPA, and TIB groups. There was no statistically significant difference in mRNA MMP-2 or MMP-9 expression. The immunohistochemical expression of MMP-2, of TIMP-2, of MMP-9, of HPSE, and of perlecan showed no differences between groups., Conclusion:  Estradiol alone or associated with MPA and TIB treatment can increase mRNA HSPE expression of the walls of the carotid arteries in ovariectomized rats., Competing Interests: The authors have no conflicts of interests to declare., (Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.)

The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis.

Introduction: Previous research found that diabetes mellitus capable to aggravate osteoarthritis disease. In brief, the hyperglycemia condition in diabetes mellitus has an impact on protein glycation of all joint components, including molecule, such as perlecan. The protein expression of perlecan reflects the presence amount of perlecan in the matrix of articular cartilage. However, the impact of hyperglycemia on articular perlecan has not been explained. Moreover, the role of perlecan as a mechanotransducer for chondrocytes in type 1 Diabetes mellitus remains unclear., Aim: This research aims to analyze the effect of hyperglycemia in type 1 Diabetes mellitus to the mRNA level and protein expression of perlecan., Methods: Thirty-five adult male rats were divided into seven groups, such as three groups of rat model with anterior cruciate ligament transection (ACLT) at right knee (ACLT1, ACLT2, ACLT3); three groups of rats with ACLT at right knee which followed by Streptozotocin injection for diabetic mice model (DM1, DM2, DM3); and control group (N). Rat sacrificed at the third week, fourth week, and sixth week after two months of maintenance. The mRNA level and protein expression were analyzed by using PCR and Western blot. All of data was analyzed by ANOVA., Results: Protein expression of perlecan in ACLT mice with diabetes mellitus (DM1, DM2, DM3 group) was gradually decreased according to the increased hyperglycemia duration. Whilst, protein expression of perlecan within ACLT mice without diabetes mellitus (ACLT1, ACLT2, ACLT3 group) was increased. The similar result also demonstrated by the mRNA level of perlecan. Group of DM1, DM2, DM3 exhibited decreased mRNA level of perlecan over the hyperglycemia duration. While, ACLT1, ACLT2, and ACLT3 group had a gradually increased of perlecan mRNA level., Conclusion: Hyperglycemia on osteoarthritic condition decreased mRNA level and protein expression of perlecan which increased the severity of osteoarthritis disease.

The extracellular matrix (ECM) of blood vessels, which is composed of both the vascular basement membrane (BM) and the interstitial ECM is identified as a crucial component of the vasculature. We here focus on the unique molecular composition and scaffolding of the capillary ECM, which provides structural support to blood vessels and regulates properties of endothelial cells and pericytes. The major components of the BM are collagen IV, laminins, heparan sulfate proteoglycans and nidogen and also associated proteins such as collagen XVIII and fibronectin. Their organization and scaffolding in the BM is required for proper capillary morphogenesis and maintenance of vascular homeostasis. The BM also regulates vascular mechanosensing. A better understanding of the mechanical and structural properties of the vascular BM and interstitial ECM therefore opens new perspectives to control physiological and pathological angiogenesis and vascular homeostasis. The overall aim of this review is to explain how ECM scaffolding influences angiogenesis and capillary integrity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Heparan sulfate (HS) regulates diverse cell signalling events in intervertebral disc development and homeostasis. The aim of the present study was to investigate the effect of ablation of perlecan HS/CS on murine intervertebral disc development. Genetic models carrying mutations in genes encoding HS biosynthetic enzymes have identified multiple roles for HS in tissue homeostasis. In the present study, we utilised an Hspg2 exon 3 null HS/CS-deficient mouse to assess the role of perlecan HS in disc cell regulation. HS makes many important contributions to growth factor sequestration, stabilisation/delivery, and activation of receptors directing cellular proliferation, differentiation, and assembly of extracellular matrix. Perlecan HS/CS-mediated interactions promote extracellular matrix assembly/stabilisation and tissue functional properties, and thus, removal of perlecan HS/CS should affect extracellular matrix function and homeostasis. Hspg2 exon 3 null intervertebral discs accumulated significantly greater glycosaminoglycan in the nucleus pulposus, annulus fibrosus, and vertebral growth plates than C57BL/6 wild-type (WT) I intervertebral discs. Proliferation of intervertebral disc progenitor cells was significantly higher in Hspg2 exon 3 null intervertebral discs, and these cells became hypertrophic by 12 weeks of age and were prominent in the vertebral growth plates but had a disorganised organisation. C57BL/6 WT vertebral growth plates contained regular columnar growth plate chondrocytes. Exostosis-like, ectopic bone formation occurred in Hspg2 exon 3 null intervertebral discs, and differences were evident in disc cell maturation and in matrix deposition in this genotype, indicating that perlecan HS/CS chains had cell and matrix interactive properties which repressively maintained tissue homeostasis in the adult intervertebral disc., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)