Lumican, a Multifunctional Cell Instructive Biomarker Proteoglycan Has Novel Roles as a Marker of the Hypercoagulative State of Long Covid Disease.

This study has reviewed the many roles of lumican as a biomarker of tissue pathology in health and disease. Lumican is a structure regulatory proteoglycan of collagen-rich tissues, with cell instructive properties through interactions with a number of cell surface receptors in tissue repair, thereby regulating cell proliferation, differentiation, inflammation and the innate and humoral immune systems to combat infection. The exponential increase in publications in the last decade dealing with lumican testify to its role as a pleiotropic biomarker regulatory protein. Recent findings show lumican has novel roles as a biomarker of the hypercoagulative state that occurs in SARS CoV-2 infections; thus, it may also prove useful in the delineation of the complex tissue changes that characterize COVID-19 disease. Lumican may be useful as a prognostic and diagnostic biomarker of long COVID disease and its sequelae.

Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons.

Radial neuronal migration is a key neurodevelopmental event for proper cortical laminar organization. The multipolar-to-bipolar transition, a critical step in establishing neuronal polarity during radial migration, occurs in the subplate/intermediate zone (SP/IZ), a distinct region of the embryonic cerebral cortex. It has been known that the extracellular matrix (ECM) molecules are enriched in the SP/IZ. However, the molecular constitution and functions of the ECM formed in this region remain poorly understood. Here, we identified neurocan (NCAN) as a major chondroitin sulfate proteoglycan in the mouse SP/IZ. NCAN binds to both radial glial-cell-derived tenascin-C (TNC) and hyaluronan (HA), a large linear polysaccharide, forming a ternary complex of NCAN, TNC, and HA in the SP/IZ. Developing cortical neurons make contact with the ternary complex during migration. The enzymatic or genetic disruption of the ternary complex impairs radial migration by suppressing the multipolar-to-bipolar transition. Furthermore, both TNC and NCAN promoted the morphological maturation of cortical neurons in vitro. The present results provide evidence for the cooperative role of neuron- and radial glial-cell-derived ECM molecules in cortical development.

Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 14 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism, reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated an overall milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, and some had alternative symptomatologies with rational biological links to SMC3. Analyses of tumor and model system transcriptomic data and epigenetic data in a subset of cases suggest that SMC3 pLoF variants reduce SMC3 expression but do not strongly support clustering with functional genomic signatures of typical CdLS. Our finding of substantial population-scale LoF intolerance in concert with variable growth and developmental features in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multilayered genomic data paired with careful phenotyping.

Chondroitin sulfate modification of CSPG4 regulates the maintenance and differentiation of glioma-initiating cells via integrin-associated signaling.

Glioma stem cell/glioma-initiating cell (GIC) and their niches are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanisms of GIC maintenance/differentiation, we performed a unique integrated proteogenomics utilizing GIC clones established from patient tumors having the potential to develop glioblastoma. After the integration and extraction of the transcriptomics/proteomics data, we found that chondroitin sulfate proteoglycan 4 (CSPG4) and its glycobiosynthetic enzymes were significantly upregulated in GICs. Glyco-quantitative PCR array revealed that chondroitin sulfate (CS) biosynthetic enzymes, such as xylosyltransferase 1 (XYLT1) and carbohydrate sulfotransferase 11, were significantly downregulated during serum-induced GIC differentiation. Simultaneously, the CS modification on CSPG4 was characteristically decreased during the differentiation and also downregulated by XYLT1 knockdown. Notably, the CS degradation on CSPG4 by ChondroitinaseABC treatment dramatically induced GIC differentiation, which was significantly inhibited by the addition of CS. GIC growth and differentiation ability were significantly suppressed by CSPG4 knockdown, suggesting that CS-CSPG4 is an important factor in GIC maintenance/differentiation. To understand the molecular function of CS-CSPG4, we analyzed its associating proteins in GICs and found that CSPG4, but not CS-CSPG4, interacts with integrin αV during GIC differentiation. This event sequentially upregulates integrin-extracellular signal-regulated kinase signaling, which can be inhibited by cyclic-RGD (Arg-Gly-Asp) integrin αV inhibitor. These results indicate that CS-CSPG4 regulates the GIC microenvironment for GIC maintenance/differentiation via the CS moiety, which controls integrin signaling. This study demonstrates a novel function of CS on CSPG4 as a niche factor, so-called "glyco-niche" for GICs, and suggests that CS-CSPG4 could be a potential target for malignant glioma.

[Genetic analysis of a fetus with Cornelia de Lange syndrome due to variant of SMC3 gene].

OBJECTIVE: To explore the genetic basis for a fetus featuring oligodactyly. METHODS: A fetus with hand deformity identified by ultrasound at the Maternal and Child Health Care Hospital of Hubei Province on October 20, 2018 was selected as the study subject. Clinical information and ultrasonographic finding of the pregnant woman were collected. Following elected abortion, umbilical cord and peripheral venous blood samples of the couple were collected for the extraction of genomic DNA. Copy number variation sequencing (CNV-seq) and trio-whole exome sequencing (trio-WES) were carried out. Candidate variants were verified by Sanger sequencing. RESULTS: Ultrasonographic examination at 30+2 weeks of gestation revealed that the fetus had small right hand with absence of 2nd-5th fingers, whilst its left hand had appeared to be normal. By CNV-seq, no pathogenic or likely pathogenic copy number variation (CNV) (≥ 100 Kb) was detected in the fetus. Trio-WES revealed that the fetus had harbored a novel heterozygous c.3298G>A (p.Val1100Met) variant of the SMC3 gene. The variant has not been recorded in the population databases, and was predicted to be deleterious by several bioinformatic software and evolutionarily conserved based on multiple sequence alignment analysis. Sanger sequencing showed that neither parent has carried the same variant. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was predicted to be likely pathogenic (PS2+PM2_Supporting+PP3). CONCLUSION: The fetus was diagnosed with Cornelia de Lange syndrome, for which the novel heterozygous c.3298G>A variant of the SMC3 gene may be accountable.

Chondroitin Sulfate Proteoglycan 4 Provides New Treatment Approach to Preventing Peritoneal Dissemination in Ovarian Cancer.

Most epithelial ovarian cancer (EOC) patients are diagnosed with peritoneal dissemination. Cellular interactions are an important aspect of EOC cells when they detach from the primary site of the ovary. However, the mechanism remains underexplored. Our study aimed to reveal the role of chondroitin sulfate proteoglycan 4 (CSPG4) in EOC with a major focus on cell-cell interactions. We examined the expression of CSPG4 in clinical samples and cell lines of EOC. The proliferation, migration, and invasion abilities of the CSPG4 knockdown cells were assessed. We also assessed the role of CSPG4 in spheroid formation and peritoneal metastasis in an in vivo model using sh-CSPG4 EOC cell lines. Of the clinical samples, 23 (44.2%) samples expressed CSPG4. CSPG4 was associated with a worse prognosis in patients with advanced EOC. Among the EOC cell lines, aggressive cell lines, including ES2, expressed CSPG4. When CSPG4 was knocked down using siRNA or shRNA, the cell proliferation, migration, and invasion abilities were significantly decreased compared to the control cells. Proteomic analyses showed changes in the expression of proteins related to the cell movement pathways. Spheroid formation was significantly inhibited when CSPG4 was inhibited. The number of nodules and the tumor burden of the omentum were significantly decreased in the sh-CSPG4 mouse models. In the peritoneal wash fluid from mice injected with sh-CSPG4 EOC cells, significantly fewer spheroids were present. Reduced CSPG4 expression was observed in lymphoid enhancer-binding factor 1-inhibited cells. CSPG4 is associated with aggressive features of EOC and poor prognosis. CSPG4 could be a new treatment target for blocking peritoneal metastasis by inhibiting spheroid formation.

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

Hindbrain boundaries as niches of neural progenitor and stem cells regulated by the extracellular matrix proteoglycan chondroitin sulphate.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

Chondroitin Sulfate Proteoglycans Revisited: Its Mechanism of Generation and Action for Spinal Cord Injury.

Reactive astrocytes (RAs) produce chondroitin sulfate proteoglycans (CSPGs) in large quantities after spinal cord injury (SCI) and inhibit axon regeneration through the Rho-associated protein kinase (ROCK) pathway. However, the mechanism of producing CSPGs by RAs and their roles in other aspects are often overlooked. In recent years, novel generation mechanisms and functions of CSPGs have gradually emerged. Extracellular traps (ETs), a new recently discovered phenomenon in SCI, can promote secondary injury. ETs are released by neutrophils and microglia, which activate astrocytes to produce CSPGs after SCI. CSPGs inhibit axon regeneration and play an important role in regulating inflammation as well as cell migration and differentiation; some of these regulations are beneficial. The current review summarized the process of ET-activated RAs to generate CSPGs at the cellular signaling pathway level. Moreover, the roles of CSPGs in inhibiting axon regeneration, regulating inflammation, and regulating cell migration and differentiation were discussed. Finally, based on the above process, novel potential therapeutic targets were proposed to eliminate the adverse effects of CSPGs.

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in hepatocellular carcinoma.

BACKGROUND: As a small G protein of Ras family, Ras-like-without-CAAX-1 (RIT1) plays a critical role in various tumors. Our previous study has demonstrated the involvement of RIT1 in promoting malignant progression of hepatocellular carcinoma (HCC). However, its underlying mechanism remains unclear. METHODS: Gene set enrichment analysis (GSEA) was conducted in the TCGA LIHC cohort to investigate the underlying biological mechanism of RIT1. Live cell imaging, immunofluorescence (IF) and flow cytometry assays were used to verify biological function of RIT1 in HCC mitosis. Subcutaneous xenografting of human HCC cells in BALB/c nude mice was utilized to assess tumor proliferation in vivo. RNA-seq, co-immunoprecipitation (Co-IP), mass spectrometry analyses, western blot and IF assays were employed to elucidate the mechanisms by which RIT1 regulates mitosis and promotes proliferation in HCC. RESULTS: Our findings demonstrate that RIT1 plays a crucial role in regulating mitosis in HCC. Knockdown of RIT1 disrupts cell division, leading to G2/M phase arrest, mitotic catastrophe, and apoptosis in HCC cells. SMC3 is found to interact with RIT1 and knockdown of SMC3 attenuates the proliferative effects mediated by RIT1 both in vitro and in vivo. Mechanistically, RIT1 protects and maintains SMC3 acetylation by binding to SMC3 and PDS5 during mitosis, thereby promoting rapid cell division and proliferation in HCC. Notably, we have observed an upregulation of SMC3 expression in HCC tissues, which is associated with poor patient survival and promotion of HCC cell proliferation. Furthermore, there is a significant positive correlation between the expression levels of RIT1, SMC3, and PDS5. Importantly, HCC patients with high expression of both RIT1 and SMC3 exhibit worse prognosis compared to those with high RIT1 but low SMC3 expression. CONCLUSIONS: Our findings underscore the crucial role of RIT1 in regulating mitosis in HCC and further demonstrate its potential as a promising therapeutic target for HCC treatment.

Low oral dose of 4-methylumbelliferone reduces glial scar but is insufficient to induce functional recovery after spinal cord injury.

Spinal cord injury (SCI) induces the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the glial scar and inhibits neuroregeneration. Under normal physiological condition, CSPGs interact with hyaluronan (HA) and other extracellular matrix on the neuronal surface forming a macromolecular structure called perineuronal nets (PNNs) which regulate neuroplasticity. 4-methylumbelliferone (4-MU) is a known inhibitor for HA synthesis but has not been tested in SCI. We first tested the effect of 4-MU in HA reduction in uninjured rats. After 8 weeks of 4-MU administration at a dose of 1.2 g/kg/day, we have not only observed a reduction of HA in the uninjured spinal cords but also a down-regulation of CS glycosaminoglycans (CS-GAGs). In order to assess the effect of 4-MU in chronic SCI, six weeks after Th8 spinal contusion injury, rats were fed with 4-MU or placebo for 8 weeks in combination with daily treadmill rehabilitation for 16 weeks to promote neuroplasticity. 4-MU treatment reduced the HA synthesis by astrocytes around the lesion site and increased sprouting of 5-hydroxytryptamine fibres into ventral horns. However, the current dose was not sufficient to suppress CS-GAG up-regulation induced by SCI. Further adjustment on the dosage will be required to benefit functional recovery after SCI.

Involvement of small leucine-rich proteoglycans and telocytes in thin and thick human endometrium: immunohistochemical and ultrastructural examination.

Thin endometrium, defined as an endometrial thickness of less than 7 mm during the late follicular phase, is a common cause of frequent cancelation of embryo transfers or recurrent implantation failure during assisted reproductive treatment. Small proteoglycans regulate intracellular signaling cascades by bridging other matrix molecules and tissue elements, affecting cell proliferation, adhesion, migration, and cytokine concentration. The aim of the study is to investigate the role of small leucine-rich proteoglycans in the pathogenesis of thin and thick human endometrium and their differences from normal endometrium in terms of fine structure properties. Normal, thin, and thick endometrial samples were collected, and small leucine-rich proteoglycans (SLRPs), decorin, lumican, biglycan, and fibromodulin immunoreactivities were comparatively analyzed immunohistochemically. The data were compared statistically. Moreover, ultrastructural differences among the groups were evaluated by transmission electron microscopy. The immunoreactivities of decorin, lumican, and biglycan were higher in the thin endometrial glandular epithelium and stroma compared to the normal and thick endometrium (p < .001). Fibromodulin immunoreactivity was also higher in the thin endometrial glandular epithelium than in the normal and thick endometrium (p < .001). However, there was no statistical difference in the stroma among the groups. Ultrastructural features were not profoundly different among cases. Telocytes, however, were not seen in the thin endometrium in contrast to normal and thin endometrial tissues. These findings suggest a possible role of changes in proteoglycan levels in the pathogenesis of thin endometrium.

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment.

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in humans and other mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a contributing factor to regeneration failure in mammals. We demonstrate that the SLRPs chondroadherin, fibromodulin, lumican, and prolargin are enriched in rodent and human but not zebrafish CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer mechano-structural properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as inhibitory ECM factors that impair axon regeneration by modifying tissue mechanics and structure, and identifies their enrichment as a feature of human brain and spinal cord lesions. These findings imply that SLRPs may be targets for therapeutic strategies to promote CNS regeneration.

Combining CSPG4-CAR and CD20-CCR for treatment of metastatic melanoma.

The prognosis for patients with metastatic melanoma is poor and treatment options are limited. Genetically-engineered T cell therapy targeting chondroitin sulfate proteoglycan 4 (CSPG4), however, represents a promising treatment option, especially as both primary melanoma cells as well as metastases uniformly express CSPG4. Aiming to prevent off-tumor toxicity while maintaining a high cytolytic potential, we combined a chimeric co-stimulatory receptor (CCR) and a CSPG4-directed second-generation chimeric antigen receptor (CAR) with moderate potency. CCRs are artificial receptors similar to CARs, but lacking the CD3ζ activation element. Thus, T cells expressing solely a CCR, do not induce any cytolytic activity upon target cell binding, but are capable of boosting the CAR T cell response when both CAR and CCR engage their target antigens simultaneously. Here we demonstrate that co-expression of a CCR can significantly enhance the anti-tumor response of CSPG4-CAR T cells in vitro as well as in vivo. Importantly, this boosting effect was not dependent on co-expression of both CCR- and CAR-target on the very same tumor cell, but was also achieved upon trans activation. Finally, our data support the idea of using a CCR as a powerful tool to enhance the cytolytic potential of CAR T cells, which might open a novel therapeutic window for the treatment of metastatic melanoma.

Targeting the melanoma-associated antigen CSPG4 with HLA-C*07:01-restricted T-cell receptors.

Intorduction: Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen, is expressed in melanoma but also other tumor entities and constitutes an attractive target for immunotherapeutic approaches. While recent preclinical reports focused on anti-CSPG4 chimeric antigen receptors (CAR), we here explore T-cell receptor (TCR)-based approaches targeting CSPG4. Methods: The TCRs of two CSPG4-reactive T-cell clones (11C/73 and 2C/165) restricted by the highly prevalent HLA-C*07:01 allele were isolated and the respective αßTCR pairs were retrovirally expressed in CRISPR/Cas9-edited TCR-knockout T cells for functional testing. We also combined alpha and beta TCR chains derived from 11C/73 and 2C/165 in a cross-over fashion to assess for hemichain dominance. CSPG4+ melanoma, glioblastoma and lung cancer cell lines were identified and, if negative, retrovirally transduced with HLA-C*07:01. Results: Functional tests confirmed specific recognition of CSPG4+HLA-C*07:01+ target cells by the αßTCR retrieved from the parental T-cell clones and in part also by the cross-over TCR construct 2Cα-11Cß. Despite high surface expression, the 11Cα-2Cß combination, however, was not functional. Discussion: Collectively, 11C/73- and 2C/165-expressing T cells specifically and efficiently recognized CSPG4+HLA-C*07:01+ cancer cells which warrants further preclinical and clinical evaluation of these TCRs.

Molecular cues for immune cells from small leucine-rich repeat proteoglycans in their extracellular matrix-associated and free forms.

In this review we highlight emerging immune regulatory functions of lumican, keratocan, fibromodulin, biglycan and decorin, which are members of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). These SLRPs have been studied extensively as collagen-fibril regulatory structural components of the skin, cornea, bone and cartilage in homeostasis. However, SLRPs released from a remodeling ECM, or synthesized by activated fibroblasts and immune cells contribute to an ECM-free pool in tissues and circulation, that may have a significant, but poorly understood foot print in inflammation and disease. Their molecular interactions and the signaling networks they influence also require investigations. Here we present studies on the leucine-rich repeat (LRR) motifs of SLRP core proteins, their evolutionary and functional relationships with other LRR pathogen recognition receptors, such as the toll-like receptors (TLRs) to bring some molecular clarity in the immune regulatory functions of SLRPs. We discuss molecular interactions of fragments and intact SLRPs, and how some of these interactions are likely modulated by glycosaminoglycan side chains. We integrate findings on molecular interactions of these SLRPs together with what is known about their presence in circulation and lymph nodes (LN), which are important sites of immune cell regulation. Recent bulk and single cell RNA sequencing studies have identified subsets of stromal reticular cells that express these SLRPs within LNs. An understanding of the cellular source, molecular interactions and signaling consequences will lead to a fundamental understanding of how SLRPs modulate immune responses, and to therapeutic tools based on these SLRPs in the future.

Hyperprolactinemia modifies extracellular matrix components associated with collagen fibrillogenesis in harderian glands of non- and pregnant female mice.

The harderian gland (HG) is a gland located at the base of the nictating membrane and fills the inferomedial aspect of the orbit in rodents. It is under the influence of the hypothalamic-pituitary-gonadal axis and, because of its hormone receptors, it is a target tissue for prolactin (PRL) and sex steroid hormones (estrogen and progesterone). In humans and murine, the anterior surface of the eyes is protected by a tear film synthesized by glands associated with the eye. In order to understand the endocrine changes caused by hyperprolactinemia in the glands responsible for the formation of the tear film, we used an animal model with metoclopramide-induced hyperprolactinemia (HPRL). Given the evidences that HPRL can lead to a process of cell death and tissue fibrosis, the protein expression of small leucine-rich proteoglycans (SLRPs) was analyzed through immunohistochemistry in the HG of the non- and the pregnant female mice with hyperprolactinemia. The SRLPs are related to collagen fibrillogenesis and they participate in pro-apoptotic signals. Our data revealed that high prolactin levels and changes in steroid hormones (estrogen and progesterone) can lead to an alteration in the amount of collagen, and in the structure of type I and III collagen fibers through changes in the amounts of lumican and decorin, which are responsible for collagen fibrillogenesis. This fact can lead to the impaired functioning of the HG by excessive apoptosis in the HG of the non- and the pregnant female mice with HPRL and especially in the HG of pregnancy-associated hyperprolactinemia.

Recovery of Forearm and Fine Digit Function After Chronic Spinal Cord Injury by Simultaneous Blockade of Inhibitory Matrix Chondroitin Sulfate Proteoglycan Production and the Receptor PTPσ.

Spinal cord injuries (SCI), for which there are limited effective treatments, result in enduring paralysis and hypoesthesia, in part because of the inhibitory microenvironment that develops and limits regeneration/sprouting, especially during chronic stages. Recently, we discovered that targeted enzymatic removal of the inhibitory chondroitin sulfate proteoglycan (CSPG) component of the extracellular and perineuronal net (PNN) matrix via Chondroitinase ABC (ChABC) rapidly restored robust respiratory function to the previously paralyzed hemi-diaphragm after remarkably long times post-injury (up to 1.5 years) following a cervical level 2 lateral hemi-transection. Importantly, ChABC treatment at cervical level 4 in this chronic model also elicited improvements in gross upper arm function. In the present study, we focused on arm and hand function, seeking to highlight and optimize crude as well as fine motor control of the forearm and digits at lengthy chronic stages post-injury. However, instead of using ChABC, we utilized a novel and more clinically relevant systemic combinatorial treatment strategy designed to simultaneously reduce and overcome inhibitory CSPGs. Following a 3-month upper cervical spinal hemi-lesion using adult female Sprague Dawley rats, we show that the combined treatment had a profound effect on functional recovery of the chronically paralyzed forelimb and paw, as well as on precision movements of the digits. The regenerative and immune system related events that we describe deepen our basic understanding of the crucial role of CSPG-mediated inhibition via the PTPσ receptor in constraining functional synaptic plasticity at lengthy time points following SCI, hopefully leading to clinically relevant translational benefits.

SMC3 epigenetic silencing regulates Rab27a expression and drives pancreatic cancer progression.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is expected to soon surpass colorectal cancer as a leading cause of cancer mortality in both males and females in the US, only lagging behind lung cancer. The lethality of PDAC is driven by late diagnosis and inefficient therapies. The complex biology of PDAC involves various cellular components, including exosomes that carry molecular information between cells. Thus, recipient cells can be reprogrammed, impacting tumorigenesis. Rab27a is a GTPase responsible for the last step of exosomes biogenesis. Hence, dissecting the mechanisms that regulate the expression of Rab27a and that control exosomes biogenesis can provide fundamental insights into the molecular underpinnings regulating PDAC progression. METHODS: To assess the mechanism that regulates Rab27a expression in PDAC, we used PDAC cell lines. The biological significance of these findings was validated in PDAC genetically engineered mouse models (GEMMs) and human samples. RESULTS: In this work we demonstrate in human PDAC samples and GEMMs that Rab27a expression decreases throughout the development of the disease, and that Rab27a knockout promotes disease progression. What is more, we demonstrate that Rab27a expression is epigenetically regulated in PDAC. Treatment with demethylating agents increases Rab27a expression specifically in human PDAC cell lines. We found that SMC3, a component of the cohesin complex, regulates Rab27a expression in PDAC. SMC3 methylation is present in human PDAC specimens and treatment with demethylating agents increases SMC3 expression in human PDAC cell lines. Most importantly, high levels of SMC3 methylation are associated with a worse prognosis in PDAC. Mechanistically, we identified an enhancer region within the Rab27a gene that recruits SMC3, and modulates Rab27a expression. CONCLUSION: Overall, we dissected a mechanism that regulates Rab27a expression during PDAC progression and impacts disease prognosis.