Heparan Sulfate Proteoglycan 2 Research Articles

Abstract 5090: Development of HSPG2 targeted glycoengineered antibody-drug conjugates

Abstract Ovarian cancer is associated with poor outcomes and aggressive behaviors. Although surgical removal of tumors and chemotherapy can initially eradicate cancer, many patients eventually experience relapse with chemotherapy-resistant tumors, resulting in poor long-term outcomes and chemotherapy complications. Antibody-drug conjugates (ADCs) are formed by integrating the tumor specificity provided by antibodies (Ab) and small molecule cytotoxicity drugs and can facilitate highly targeted delivery with minimal toxicity to normal tissues. Our group has identified that the overexpression of a novel target, heparan sulfate proteoglycan 2 (HSPG2), is associated with poor prognosis and survival of high grade serous ovarian cancer (HGSOC) patients. We have developed a monoclonal antibody (AM6) that effectively targets HSPG2-expressing tumors. In this study, we used a novel glycoengineering strategy to introduce artificial azide groups at the N-glycan sites of AM6 without affecting its antigen affinity and intracellular localization. The azide groups were then used for site-specific conjugation of the cleavable dibenzyl cyclooctyne - monomethyl auristatin E (DBCO-PAB-MMAE) linker (drug conjugate) to form an AM6 antibody-drug conjugate (AM6 ADCs). Hydrophobic interaction chromatography (HIC) and liquid chromatography/quadrupole time-of-flight/mass spectrometry (LC/Q-TOF/MS) confirmed the successful conjugation of the DBCO-PAB-MMAE to the AM6. In-vitro cytotoxicity studies of AM6 ADCs in OVCAR8 (high HSPG2 expression) and CAOV3 (regular HSPG2 expression) showed the cell line dependent response with lower IC50 in OVCAR8. These results suggest that the effectiveness of AM6 ADCs is proportional to the HSPG2 expression levels of the cells. Future studies will be aimed at the in-vivo evaluation of optimized antibody-drug conjugates. Citation Format: Xiaolei Li, Swayam Prabha. Development of HSPG2 targeted glycoengineered antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5090.

The interaction of endorepellin and neurexin triggers neuroepithelial autophagy and maintains neural tube development

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.

Successful Treatment of Schwartz-Jampel Syndrome with Botulinum Toxin TypeA.

Schwartz-Jampel syndrome (SJS) is a rare autosomal recessive disorder characterized by typical facial dysmorphism, generalized muscle stiffness, joint contracture, and skeletal abnormalities. This condition is caused by mutations in the heparan sulfate proteoglycan2 (HSPG2) gene, which encodes perlecan, a component of the basement membrane. The management of patients with SJS primarily aims to alleviate symptoms related to muscle stiffness. In this report, we describe a male patient with SJS type1A. Trio whole-exome sequencing identified a pathogenic mutation (NM_001291860.1: c.10897C>T; p.Arg3633Ter) and variants of unknown significance (NM_001291860.2: c.413+10G>T). The patient experienced difficulty in opening his eyes and mouth, which significantly limited his daily activities. Botulinum toxinA injection was administered and demonstrated significant clinical improvement after the treatment.

Genome-wide association study identifies genetic variants underlying footrot in Portuguese Merino sheep

BackgroundOvine footrot caused by Dichelobacter nodosus (D. nodosus) is a contagious disease with serious economic and welfare impacts in sheep production systems worldwide. A better understanding of the host genetic architecture regarding footrot resistance/susceptibility is crucial to develop disease control strategies that efficiently reduce infection and its severity. A genome-wide association study was performed using a customized SNP array (47,779 SNPs in total) to identify genetic variants associated to footrot resistance/susceptibility in two Portuguese native breeds, i.e. Merino Branco and Merino Preto, and a population of crossbred animals. A cohort of 1375 sheep sampled across 17 flocks, located in the Alentejo region (southern Portugal), was included in the analyses.ResultsPhenotypes were scored from 0 (healthy) to 5 (severe footrot) based on visual inspection of feet lesions, following the Modified Egerton System. Using a linear mixed model approach, three SNPs located on chromosome 24 reached genome-wide significance after a Bonferroni correction (p < 0.05). Additionally, six genome-wide suggestive SNPs were identified each on chromosomes 2, 4, 7, 8, 9 and 15. The annotation and KEGG pathway analyses showed that these SNPs are located within regions of candidate genes such as the nonsense mediated mRNA decay associated PI3K related kinase (SMG1) (chromosome 24) and the RALY RNA binding protein like (RALYL) (chromosome 9), both involved in immunity, and the heparan sulfate proteoglycan 2 (HSPG2) (chromosome 2) and the Thrombospodin 1 (THBS1) (chromosome 7) implicated in tissue repair and wound healing processes.ConclusionThis is the first attempt to identify molecular markers associated with footrot in Portuguese Merino sheep. These findings provide relevant information on a likely genetic association underlying footrot resistance/susceptibility and the potential candidate genes affecting this trait. Genetic selection strategies assisted on the information obtained from this study could enhance Merino sheep-breeding programs, in combination with farm management strategies, for a more effective and sustainable long-term solution for footrot control.

Identification of B-cell-related HSPG2 and CDSN as susceptibility loci for Kawasaki disease

Kawasaki disease (KD) is an acute pediatric vasculitis that predominantly affects children under the age of 5 years. To date, genome-wide association studies (GWAS) have identified several KD susceptibility genes (e.g., BLK, CD40, FCGR2A, BCL2L11, and IGHV), which are mainly involved in B cell immunity. In this study, we aimed to identify additional KD susceptibility genes mainly involved in B cell development and functions by analyzing our previous GWAS data and conducting a replication study using new sample. Initially, we selected 30 single nucleotide polymorphisms (SNPs) in B-cell-related genes that were significantly (P < 0.01) associated with KD in our previous GWAS analysis of 247 KD cases with complete type and 1,000 healthy controls. Replication study was performed by genotyping the new 837 KD case samples with Fluidigm system and comparing them with 3,553 control genotypes. Among the 30 candidate SNPs, two were significantly associated with KD (P < 0.001) in the replication study. An even greater association between these SNPs and KD was observed in the combined analysis of GWAS and replication samples: odds ratio (OR) = 1.97 (P = 8.61 × 10−6) for rs2270699 (nonsynonymous SNP: c.10588C > T, p.Arg3530Trp) in the heparan sulfate proteoglycan 2 (HSPG2) gene and OR = 1.28 (P = 1.34 × 10−6) for rs3130992 (intronic SNP) in both the corneodesmosin (CDSN) and psoriasis susceptibility 1 candidate 1 (PSORS1C1) genes. These results suggest that the B-cell-related genes, HSPG2 and CDSN or PSORS1C1, play a role in the development of KD.

Evaluation of Human-Induced Pluripotent Stem Cells Derived from a Patient with Schwartz-Jampel Syndrome Revealed Distinct Hyperexcitability in the Skeletal Muscles.

Schwartz-Jampel syndrome (SJS) is an autosomal recessive disorder caused by loss-of-function mutations in heparan sulfate proteoglycan 2 (HSPG2), which encodes the core basement membrane protein perlecan. Myotonia is a major criterion for the diagnosis of SJS; however, its evaluation is based solely on physical examination and can be challenging in neonates and young children. Furthermore, the pathomechanism underlying SJS-related myotonia is not fully understood, and effective treatments for SJS are limited. Here, we established a cellular model of SJS using patient-derived human-induced pluripotent stem cells. This model exhibited hyper-responsiveness to acetylcholine as a result of abnormalities in the perlecan molecule, which were confirmed via comparison of their calcium imaging with calcium imaging of satellite cells derived from Hspg2-/--Tg mice, which exhibit myotonic symptoms similar to SJS symptoms. Therefore, our results confirm the utility of creating cellular models for investigating SJS and their application in evaluating myotonia in clinical cases, while also providing a useful tool for the future screening of SJS therapies.

Characterization of Vascular Patterns Associated with Endothelial Glycocalyx Damage in Early- and Late-Onset Preeclampsia.

This paper provides an assessment of molecular and functional changes in blood vessels, and a description of vascular patterns during preeclampsia (PE). Patients with normal pregnancy, and pregnancy complicated by PE at earlier (20-34 weeks) and later terms (≥34 weeks) underwent a 24 h monitoring of blood pressure, central hemodynamics, arterial stiffness, and myocardial function. The blood levels of the structural components of endothelial glycocalyx (eGC): syndecan-1 (SDC 1), heparan sulfate proteoglycan 2 (HSPG2), and hyaluronic acid (HA) were determined. In early-onset PE, the vascular pattern comprised changes in all structural components of eGCs, including transmembrane proteoglycans levels, and severe disorders of central hemodynamics, arterial stiffness, and myocardial changes, probably leading to more severe course of PE and the formation of morphological grounds for cardiovascular disorders. The vascular pattern in late-onset PE, including changes in HA levels, central hemodynamics, and myocardial function, may be a signal of potential cardiovascular disorder. PE may change adaptive hemodynamic responses to a pathological reaction affecting both arterial elasticity and the left ventricular myocardium, with its subsequent hypertrophy and decompensation, leading to a delayed development of cardiovascular disorders after PE. Further clinical studies of these indicators will possibly identify predictors of PE and long-term consequences of the disease.

HSPG2 Mutation Association with Immune Checkpoint Inhibitor Outcome in Melanoma and Non-Small Cell Lung Cancer.

Simple SummaryImmune checkpoint inhibitors (ICIs) markedly improve the survival benefits of advanced melanoma and non-small cell lung cancer (NSCLC). Nevertheless, only a subset of patients could benefit from such a therapy. Novel and effective clinical biomarkers are needed to assess ICI treatment efficacy. Heparan sulfate proteoglycan 2 (HSPG2) is frequently mutated in melanoma and NSCLC. In this study, we comprehensively integrated the pretreatment somatic mutational profiles and clinical information of both tumors and observed that HSPG2 mutations were associated with favorable tumor immunogenicity and immunotherapeutic efficacy. Our study provides a potential clinical molecular biomarker for evaluating ICI therapy responses.Immune checkpoint inhibitors (ICIs) markedly promote the survival outcome of advanced melanoma and non-small cell lung cancer (NSCLC). Clinically, favorable ICI treatment efficacy is noticed only in a smaller proportion of patients. Heparan sulfate proteoglycan 2 (HSPG2) frequently mutates in both tumors. Herein, we aim to investigate the immunotherapeutic and immunological roles of HSPG2 mutations in melanoma and NSCLC. A total of 631 melanoma samples and 109 NSCLC samples with both somatic mutational profiles and clinical immunotherapy data were curated. In addition, by using The Cancer Genome Atlas data, genomic and immunological traits behind HSPG2 mutations were elucidated. Melanoma patients with HSPG2 mutations had a markedly extended ICI outcome than other patients. An association between HSPG2 mutations and the improved outcome was further confirmed in NSCLC. In addition, an elevated ICI response rate was presented in HSPG2-mutated NSCLC patients (81.8% vs. 29.7%, p = 0.002). Subsequent analyses revealed that HSPG2-mutated patients had a favorable abundance of response immunocytes, an inferior abundance of suppression immunocytes, enhanced mutational burden, and interferon response-relevant signaling pathways. We uncovered that HSPG2 mutations were predictive of a better ICI response and associated with preferable immunogenicity, which may be considered as a genomic determinant to customize biotherapy strategies.

DAL-1/4.1B promotes the uptake of exosomes in lung cancer cells via Heparan Sulfate Proteoglycan 2 (HSPG2).

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.

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits theretention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.

Subclinical infection can be an initiator of inflammaging leading to degenerative disk disease: evidence from host-defense response mechanisms

There is considerable controversy on the role of genetics, mechanical and environmental factors, and, recently, on subclinical infection in triggering inflammaging leading to disk degeneration. The present study investigated sequential molecular events in the host, analyzing proteome level changes that will reveal triggering factors of inflammaging and degeneration. Ten MRI normal disks (ND) from braindead organ donors and 17 degenerated disks (DD) from surgery were subjected to in-gel-based label-free ESI-LC-MS/MS analysis. Bacterial-responsive host-defense response proteins/pathways leading to Inflammaging were identified and compared between ND and DD. Out of the 263 well-established host-defense response proteins (HDRPs), 243 proteins were identified, and 64 abundantly expressed HDRPs were analyzed further. Among the 21 HDRPs common to both ND and DD, complement factor 3 (C3) and heparan sulfate proteoglycan 2 (HSPG2) were significantly upregulated, and lysozyme (LYZ), superoxide dismutase 3 (SOD3), phospholipase-A2 (PLA2G2A), and tissue inhibitor of metalloproteinases 3 (TIMP-3) were downregulated in DD. Forty-two specific HDRPs mainly, complement proteins, apolipoproteins, and antimicrobial proteins involved in the complement cascade, neutrophil degranulation, and oxidative-stress regulation pathways representing an ongoing host response to subclinical infection and uncontrolled inflammation were identified in DD. Protein-Protein interaction analysis revealed cross talk between most of the expressed HDRPs, adding evidence to bacterial presence and stimulation of these defense pathways. The predominance of HDRPs involved in complement cascades, neutrophil degranulation, and oxidative-stress regulation indicated an ongoing infection mediated inflammatory process in DD. Our study has documented increasing evidence for bacteria's role in triggering the innate immune system leading to chronic inflammation and degenerative disk disease.

Fibrinogen in the glioblastoma microenvironment contributes to the invasiveness of brain tumor-initiating cells.

Glioblastomas (GBMs) are highly aggressive, recurrent, and lethal brain tumors that are maintained via brain tumor‐initiating cells (BTICs). The aggressiveness of BTICs may be dependent on the extracellular matrix (ECM) molecules that are highly enriched within the GBM microenvironment. Here, we investigated the expression of ECM molecules in GBM patients by mining the transcriptomic databases and also staining human GBM specimens. RNA levels for fibronectin, brevican, versican, heparan sulfate proteoglycan 2 (HSPG2), and several laminins were high in GBMs compared to normal brain, and this was corroborated by immunohistochemistry. While fibrinogen transcript was at normal level in GBM, its protein immunoreactivity was prominent within GBM tissues. These ECM molecules in tumor specimens were in proximity to, and surrounding BTICs. In culture, fibronectin and pan‐laminin induced the adhesion of BTICs onto the plastic substratum. However, fibrinogen increased the size of the BTIC spheres by facilitating the adhesive property, motility, and invasiveness of BTICs. These features of elevated invasiveness were corroborated in resected GBM specimens by the close proximity of fibrinogen with matrix metalloproteinase (MMP)‐2 and‐9, which are proteases implicated in metastasis. Moreover, the effect of fibrinogen‐induced invasiveness was attenuated in BTICs where MMP‐2 and ‐9 have been inhibited with siRNAs or pharmacological inhibitors. Our results implicate fibrinogen in GBM as a mediator of the invasive properties of BTICs, and as a target for therapy to reduce BTIC tumorigenecity.

A Novel Pathogenic HSPG2 Mutation in Schwartz-Jampel Syndrome.

Schwartz–Jampel syndrome is a rare autosomal recessive disease caused by mutation in the heparan sulfate proteoglycan 2 (HSPG2) gene. Its cardinal symptoms are skeletal dysplasia and neuromuscular hyperactivity. Herein, we identified a new pathogenic mutation site (NM_005529.6:c.1125C>G; p.Cys375Trp) of HSPG2 leading to Schwartz–Jampel syndrome by whole-exome sequencing. This mutation carried by the asymptomatic parents was previously registered in a single-nucleotide polymorphism database of the National Institutes of Health as a coding sequence variant rs543805444. The pathogenic nature of this missense mutation was demonstrated by in silico pathogenicity assessment, clinical presentations, and cellular function of primary fibroblast derived from patients. Various in silico software applications predicted the mutation to be pathogenic [Sorting Intolerant From Tolerant (SIFT), 0; Polyphen-2, 1; CADD (Combined Annotation Dependent Depletion), 23.7; MutationTaster, 1; DANN (deleterious annotation of genetic variants using neural networks); 0.9]. Needle electromyography revealed extensive complex repetitive discharges and multiple polyphasic motor unit action potentials in axial and limb muscles at rest. Short exercise test for myotonia showed Fournier pattern I. At cellular levels, mutant primary fibroblasts had reduced levels of secreted perlecan and impaired migration ability but normal capability of proliferation. Patients with this mutation showed more neuromuscular instability and relatively mild skeletal abnormality comparing with previously reported cases.

The Association between Genes Polymorphisms of Heparan Sulfate Proteoglycan 2 (HSPG2) and Chondroitin Sulfate Proteoglycan 2 (CSPG2) and Intracranial Aneurysm Susceptibility: A Meta-Analysis

We aimed to investigate whether the polymorphisms of gene heparan sulfate proteoglycan 2 (HSPG2) and chondroitin sulfate proteoglycan 2 (CSPG2) are associated with increased risk of intracranial aneurysms (IAs) susceptibility. The Cochrane Library, Medline, PubMed, and Embase databases were carefully searched for potential researches before Mar 30, 2018. The title, abstract, and full text were assessed to determine whether the paper was suitable for inclusion. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were presented to assess the associations between CSPG2 and HSPG2 polymorphisms and IAs susceptibility. We enrolled 7 papers, 4 matched single nucleotide polymorphisms (SNPs) of CSPG2 (rs173686, rs251124) or HSPG2 (rs173686, rs251124), and a total of 8651 participations (3674 with IAs and 4977 for control). For the rs251124 polymorphism of CSPG2, the quantitative synthesis from 5 studies showed significant difference in the gene allele comparison of T vs. C (OR, 1.129; 95% CI, 1.029, 1.238; P=0.01). Similar results were found for rs3767137 of HSPG2 (A vs. G, OR, 0.842, 95% CI=0.759-0.935, P=0.001). However, for the rs173686 polymorphism of CSPG2 and rs7556412 polymorphism of HSPG2, no significant difference was found (P=0.259 and P=0.474, respectively). The SNPs rs251124 of CSPG2 and rs3767137 of HSPG2 had statistically significant associations with IAs susceptibility. The minor allele T of rs251124 demonstrated a harmful effect but the minor allele A of rs3767137 demonstrated a protective role with regard to the risk of IAs. However, no such associations were found in the SNPs rs173686 of CSPG2 and rs7556412 of HSPG2.

Three Novel HSPG2 Mutations Causing Schwartz-Jampel Syndrome

Schwartz-Jampel syndrome (SJS) is a rare autosomal recessive hereditary disorder characterized by the triad of myotonia, facial dysmorphisms, and skeletal deformities. Less than 150 cases have been reported in the medical literature. SJS is caused by mutations in the gene heparan sulfate proteoglycan 2 (HSPG2) located on chromosome 1p34-36.1 which encodes perlecan, a major component of basement membranes. Here we report three novel mutations in a 6-year-old girl.

HSPG2 overexpression independently predicts poor survival in patients with acute myeloid leukemia

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.

Preeclampsia and phototherapy

Preeclampsia is a leading cause of maternal morbidity and mortality worldwide, complicating 2–8% of pregnancies, and is recognized as an independent risk factor for CVD by the ACC/AHA. The etiology of preeclampsia is poorly understood. However, the role of placental extracellular matrix (pECM) and extravillous trophoblast in placental blood vessel formation and preeclampsia is understood.The objective is to identify phenotypical changes in extravillous trophoblast (HTR8/SVneo) in a preeclampsia microenvironment using healthy and preeclamptic patient placental samples vs Dahl S rats (SS) as an animal model of superimposed preeclampsia and explore a novel therapeutic modality restoring trophoblastic phenotype by phototherapy using a 670nm light (NIR).Placentas from rats and patients were decellularized and characterized using pECM markers galectin‐3 and heparan sulfate proteoglycan 2 (HSP2) by western blot and immunofluorescence. pECM was used as substrate for HTR8/SVneo. Proliferation, migration, and apoptosis assays were used to determine cell behavior in the placental microenvironment. Immunofluorescence for placental growth factor (PLGF) and transforming growth factor (TGFβ) was performed on HTR8/SVneo cultured on both pECM. NIR was applied to HTR8/SVneo cultured on patient and rat pECM with 4J/cm2 intensity.In patient samples galectin‐3 expression was reduced by 34.21%±13.16 in preeclamptic PE compared to control patient pECM. HSP2 was reduced by 14.75%±6.11 in PE compared to control patient pECM. In rats galectin‐3 expression was reduced to 51.58%±3.78 in SS compared to control rat pECM. HSP2 was reduced to 45.15%± 0.63 in SS compared to control rat pECM.Migration of HTR8/SVneo on pECM (patient: control 11.08±0.82, PE 6.45±0.88; rats: control 3.87±0.96, SS 4.42±1.18) and proliferation of HTR8/SVneo on pECM were decreased in patients (# of cells X 1000) control: 59.125±6262.9, PE:67.125±7706.5 and rat control 5.16±0.79, SS 4.25±0.6 samples. Proliferation and migration were restored by NIR treatment: proliferation patient: control 101.375±2075.4; PE 106.875±4870.7, migration rat: control 12.58±7.06, SS 5.11±2.55. An increased number of apoptotic HTR8/SVneo was observed in preeclamptic pECM compared to healthy pECM: patient control 2.4±0.24, PE 3±0.4 and rat apoptotic cells control 5.2±0.89; SS 7.4±1.23). NIR decreased the number of apoptotic cells, patient:1.2 ±0.58, PE 1.4±0.5; rat control 3.9±0.43, SS 5.4±0.73.PLGF and TGFβ were downregulated in HTR8/SVneo when cultured on preeclamptic pECM from patients compared to healthy pECM and unchanged in rats. Fluorescence Intensity of PLGF in patient pECM; control 102.12±14.91, PE 68.77±9.73, TGFβ control 88.86±2.51, PE 63.59±4.83 and rats; PLGF control 65.18±0.79, SS 61.09±0.68; TGFβ control 60.78±0.48, SS 61.47 ± 0.45. The decrease was reversed by NIR treatment in patients; PLGF control 165.05±12.32, PE 175.14±12.48, TGFβ control 74.99±7.17, PE 91.03±2.27; rat PLGF control 66.58±0.99, SS 65.57±1.52, TGFβ control 66.67±1.58, SS 63.82±1.53).Extracellular matrix is altered in preeclampsia, and HTR8/SVneo trophoblasts respond similarly to altered matrix in preeclamptic placentas from patients and rats. 670nm light application restored the phenotype of HTR8/SVneo on preeclamptic pECM in patients and rats suggesting NIR as a therapeutic.

Impaired testicular signaling of vitamin A and vitamin K contributes to the aberrant composition of the extracellular matrix in idiopathic germ cell aplasia

To study pathogenic features of the somatic testicular microenvironment associated with idiopathic germ cell aplasia. Cross-sectional study. Tertiary referral center for reproductive medicine. Testicular specimens from men with idiopathic nonobstructive azoospermia (iNOA) prospectively submitted to microdissection testicular sperm extraction. Of 20 specimens used for histology, 10 were also available for proteomic analysis. Primary Sertoli cells with normal karyotype and phenotype were also used. Patients with iNOA were dichotomized according to a positive versus negative sperm retrieval at microdissection testicular sperm extraction, and on the isolated extracellular matrix (ECM) the proteomic analysis was performed. Proteomic analysis of the ECM from testicular specimens with positive versus negative sperm retrieval. Gene ontology enrichment was used to identify upstream regulators based on the 11 deregulated ECM proteins, which were validated by immunohistochemistry and quantitative polymerase chain reaction. Continuous variables were expressed as medians and interquartile range. Germ cell aplasia was characterized by an increased signaling of the retinoic acid in Sertoli cells and associated with decreased expression of the basal membrane markers nidogen-2 and heparan sulfate proteoglycan-2. Decreased levels of the interstitial matrisome-associated factor IX and its regulator VKORC1 were, instead, coupled with decreased signaling of vitamin K in Leydig cells. An altered expression of a further eight ECM proteins was also found, including laminin-4 and laminin-5. Peripheral levels of the two vitamins were within the reference range in the two cohorts of iNOA men. We identified the pathogenetic signature of the somatic human testicular microenvironment, providing two vitamin-related mechanistic insights related to the molecular determinants of the idiopathic germ cell aplasia.

Modular Proteoglycan Perlecan/HSPG2: Mutations, Phenotypes, and Functions.

Heparan sulfate proteoglycan 2 (HSPG2) is an essential, highly conserved gene whose expression influences many developmental processes including the formation of the heart and brain. The gene is widely expressed throughout the musculoskeletal system including cartilage, bone marrow and skeletal muscle. The HSPG2 gene product, perlecan is a multifunctional proteoglycan that preserves the integrity of extracellular matrices, patrols tissue borders, and controls various signaling pathways affecting cellular phenotype. Given HSPG2’s expression pattern and its role in so many fundamental processes, it is not surprising that relatively few gene mutations have been identified in viable organisms. Mutations to the perlecan gene are rare, with effects ranging from a relatively mild condition to a more severe and perinatally lethal form. This review will summarize the important studies characterizing mutations and variants of HSPG2 and discuss how these genomic modifications affect expression, function and phenotype. Additionally, this review will describe the clinical findings of reported HSPG2 mutations and their observed phenotypes. Finally, the evolutionary aspects that link gene integrity to function are discussed, including key findings from both in vivo animal studies and in vitro systems. We also hope to facilitate discussion about perlecan/HSPG2 and its role in normal physiology, to explain how mutation can lead to pathology, and to point out how this information can suggest pathways for future mechanistic studies.

Increased HSPG2 expression independently predicts poor survival in patients with oligoastrocytoma and oligodendroglioma.

Perlecan, which is also called heparan sulfate proteoglycan 2 (HSPG2), is a protein encoded by the HSPG2 gene that maps to 1p36.12 in the human genome. In this study, we assessed the independent prognostic value of HSPG2 in terms of overall survival (OS) and recurrence-free survival (RFS) in patients with LGG. A retrospective study was conducted by using data in the Cancer Genome Atlas-Low Grade Glioma (TCGA-LGG). Increased HSPG2 expression was an independent prognostic indicator of poor OS in oligoastrocytoma (HR: 1.644, 95% CI: 1.116-2.423, p = 0.012) and in oligodendroglioma (HR: 1.459, 95% CI: 1.138-1.871, p = 0.003). In addition, increased HSPG2 expression independently predicted poor RFS in oligodendroglioma (HR: 1.402, 95% CI: 1.110-1.770, p = 0.005). Furthermore, we observed that high HSPG2 expression was associated with significantly shorter OS and RFS in oligodendroglioma, no matter the patients received radiotherapy or not. Using copy number alterations (CNAs) and DNA methylation data in TCGA-LGG, we found that DNA copy deletion was generally associated with decreased HSPG2 expression. Regression analysis suggested a weak negative correlation between HSPG2 expression and HSPG2 DNA methylation (Pearson's r = -0.388). Increased HSPG2 expression could independently predict poor OS in oligoastrocytoma and oligodendroglioma and also independently predicted poor RFS in oligodendroglioma. Its expression is modulated by both DNA copy number and DNA methylation in oligodendroglioma.