Small Leucine-rich Proteoglycan Research Articles

Abstract PO4-28-09: Upregulation of the immune checkpoint protein B7-H3 is associated with an immune suppressive microenvironment in progression from in situ to invasive lobular breast cancer

Abstract Invasive lobular breast cancer (ILC) is an understudied subtype of breast cancer characterized by late recurrence, metastasis to serosal surfaces including the peritoneum, and poor long-term outcomes. Lobular carcinoma in situ (LCIS) is a non-obligate precursor to ILC but is associated with a 30% increased risk of developing ILC. Therefore, understanding the underlying changes that occur in the invasive lobular cells as well as their tumor microenvironment (TME) during the transition from LCIS to ILC is critical for the development of novel therapeutic targets that could be used in early disease. To address this, we used combined spatial whole genome transcriptomics and a 97-protein proteomics assay to examine the spatial molecular profiles of coexistent LCIS and ILC. PanCK-positive tumor cells and panCK-negative stromal cells were segmented and analyzed separately for both RNA and protein. Despite the close spatial proximity of LCIS and ILC, there were notable differences in gene and protein expression in the tumor cells as well as the tumor microenvironment (TME). RNA profiling revealed significant upregulation of genes encoding essential components of the extracellular matrix, including type I, 3 and 5 collagens, small leucine-rich proteoglycans byglycan (BGN) and lumican (LUM), periostin (POSTN) and secreted protein acidic and cysteine rich (SPARC). Metalloprotease genes MMP2 and MMP11 were also upregulated. Genes highly expressed in the LCIS tumor compartment were KRT5, KRT14 KRT17 and MYLK which are highly expressed in myoepithelial cells at the periphery of the LCIS. One of the most upregulated proteins in ILC cells was B7 homology 3 protein (B7-H3) or Cluster of Differentiation 276 (CD276). B7-H3 is a transmembrane immunoregulatory protein belonging to the B7 family (that also includes PD-L1 and PD-L2). This immune checkpoint protein is overexpressed in many tumors but is barely expressed in normal cells. B7-H3 has been associated with several aspects of cancer progression, such as evasion of tumor immune surveillance and metastasis, and is strongly linked to poor prognosis in cancer. Compared to the LCIS TME, B7-H3 was also the most upregulated protein in the ILC TME along with the tumor-promoting M1 macrophage marker CD68 and the regulatory T-cell marker FOXP3. The lymphocyte marker CD45 was downregulated as well as T-cell proteins CD3, CD40 and granzyme B. Moreover, CD27, a memory B-cell marker was most highly expressed in the TME surrounding LCIS compared to ILC. Overall, these molecular profiles support a transition to a toward a much more immunosuppressive environment for invasive tumor cells compared to LCIS. Based on these data, our working hypothesis is that upregulation of B7-H3 on lobular tumor cells promotes invasion allowing interaction with laminins, integrins and other ECM proteins in the basement membrane and the interstitial membrane as LCIS acquire invasive properties of ILC. Furthermore, upregulation of B7-H3 on CD3+ HLA-DR+ expressing antigen presenting cells also promotes a suppressive immune microenvironment by inhibiting T-cell proliferation and downregulating cytokine production. In sum, these strongly concurrent spatial transcriptome and protein data suggest that B7-H3 and the immune suppressive network of ILC may be a promising target for early stage lobular breast cancer. B7-H3 Inhibitors could be explored in the neoadjuvant setting to evaluate the efficacy of immunotherapy in primary ILC. Citation Format: Lynda Bennett, Sunati Sahoo, Cheryl Lewis, Indu Raman, Candace Frerich, Guanchun Chen, Min Xu, Suzanne Conzen. Upregulation of the immune checkpoint protein B7-H3 is associated with an immune suppressive microenvironment in progression from in situ to invasive lobular breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-09.

Decorin suppresses tumor lymphangiogenesis: A mechanism to curtail cancer progression

The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a prosurvival program and to sustain a proangiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we identified that decorin down-regulated a cluster of tumor-associated genes involved in lymphatic vessel (LV) development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of LVs, were markedly suppressed at both the mRNA and protein levels, and this suppression correlated with a significant reduction in tumor LVs. We further identified that soluble decorin, but not its homologous proteoglycan biglycan, inhibited LV sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with vascular endothelial growth factor receptor 3 (VEGFR3), the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we identified that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a biological factor with antilymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.

Decorin (DCN) Downregulation Activates Breast Stromal Fibroblasts and Promotes Their Pro-Carcinogenic Effects through the IL-6/STAT3/AUF1 Signaling.

Decorin (DCN), a member of the small leucine-rich proteoglycan gene family, is secreted from stromal fibroblasts with non-cell-autonomous anti-breast-cancer effects. Therefore, in the present study, we sought to elucidate the function of decorin in breast stromal fibroblasts (BSFs). We first showed DCN downregulation in active cancer-associated fibroblasts (CAFs) compared to their adjacent tumor counterpart fibroblasts at both the mRNA and protein levels. Interestingly, breast cancer cells and the recombinant IL-6 protein, both known to activate fibroblasts in vitro, downregulated DCN in BSFs. Moreover, specific DCN knockdown in breast fibroblasts modulated the expression/secretion of several CAF biomarkers and cancer-promoting proteins (α-SMA, FAP- α, SDF-1 and IL-6) and enhanced the invasion/proliferation abilities of these cells through activation of the STAT3/AUF1 signaling. Furthermore, DCN-deficient fibroblasts promoted the epithelial-to-mesenchymal transition and stemness processes in BC cells in a paracrine manner, which increased their resistance to cisplatin. These DCN-deficient fibroblasts also enhanced angiogenesis and orthotopic tumor growth in mice in a paracrine manner. On the other hand, ectopic expression of DCN in CAFs suppressed their active features and their paracrine pro-carcinogenic effects. Together, the present findings indicate that endogenous DCN suppresses the pro-carcinogenic and pro-metastatic effects of breast stromal fibroblasts.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (-32.6%) were recorded, as well as less collagen enzymatic crosslinks (-25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (-37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (-29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

Low- and high-grade glioma-associated vascular cells differentially regulate tumor growth.

A key feature distinguishing high-grade glioma (HG) from low-grade glioma (LG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor-associated vasculature from HG is molecularly and functionally distinct from normal brain vasculature and expresses higher levels of pro-tumorigenic factors that promote glioma growth and progression. However, it remains unclear whether vessels from LG also express pro-tumorigenic factors, and to what extent they functionally contribute to glioma growth. Here, we profile the transcriptomes of glioma-associated vascular cells (GVC) from IDH-mutant (mIDH) LG and IDH-wildtype (wIDH) HG and show that they exhibit significant molecular and functional differences. LG-GVC show enrichment of extracellular matrix-related gene sets and sensitivity to anti-angiogenic drugs, whereas HG-GVC display an increase in immune response-related gene sets and anti-angiogenic resistance. Strikingly, conditioned media from LG-GVC inhibits the growth of wIDH glioblastoma cells, whereas HG-GVC promotes growth. In vivo co-transplantation of LG-GVC with tumor cells reduces growth, whereas HG-GVC enhances tumor growth in orthotopic xenografts. We identify ASPORIN (ASPN), a small leucine-rich repeat proteoglycan, highly enriched in LG-GVC as a growth suppressor of wIDH glioblastoma cells in vitro and in vivo. Together, these findings indicate that GVC from LG and HG are molecularly and functionally distinct and differentially regulate tumor growth. Implications: This study demonstrated that vascular cells from IDH-mutant LG and IDH-wildtype HG exhibit distinct molecular signatures and have differential effects on tumor growth via regulation of ASPN-TGFβ1-GPM6A signaling.

Research Progress on Extracellular Matrix Involved in the Development of Preeclampsia.

Preeclampsia (PE) is a serious pregnancy complication, and its primary clinical manifestations are gestational hypertension and proteinuria. Trophoblasts are responsible for the basic functions of the placenta during placental development; recent studies have revealed that placental "shallow implantation" caused by the decreased invasiveness of placental trophoblasts plays a crucial role in PE pathogenesis. The interaction between the cells and the extracellular matrix (ECM) plays a crucial role in trophoblast proliferation, differentiation, and invasion. Abnormal ECM function can result in insufficient migration and invasion of placental trophoblasts, thus participating in PE. This article summarizes the recent studies on the involvement of ECM components, including small leucine-rich proteoglycans, syndecans, glypicans, laminins, fibronectin, collagen, and hyaluronic acid, in the development of PE. ECM plays various roles in PE development, most notably by controlling the activities of trophoblasts. The ECM is structurally stable and can serve as a biological diagnostic marker and therapeutic target for PE.

Abstract 4208: In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes

Abstract Preclinical models of Sonic Hedgehog (SHH)-driven tumors support the therapeutic benefit of pharmacological inhibitors, yet clinical outcomes are conflicted due to biphasic tumor responses that include complete remission or faster disease progression. In such studies, the activity of the SHH pathway in the tumor-adjacent stroma supported or restrained tumor progression. Our prior studies show that altered SHH ligand levels can lead to faster tumor growth during pharmacological inhibition suggesting a role for the SHH pathway strength in the biphasic tumor responses of a triple-negative breast cancer (TNBC) model. In this study, the role of SHH pathway strength altered by tumor-fibroblast proximity was examined on the tumor growth, invasion, and transcriptional activity using in vitro and in vivo models. Tumor spheroid models composed of TNBC cell lines and fibroblasts were used to monitor SHH signal strength as a function of the culture modality and SHH ligand levels. In vitro, SHH signal strength was altered by tumor-fibroblast proximity and ligand concentration. Mixed cultures exhibited significantly higher SHH activity and reduced invasion than adjacent cultures. This effect was reversed by pharmacological inhibition of SMO, confirming the involvement of an SHH-dependent mechanism. In a tumor xenograft model, tumor metastasis outcomes were suppressed by pharmacological inhibition of the SHH pathway, but opposite results were observed in tumor xenografts composed of co-injected with fibroblasts indicating that the biphasic tumor response can be driven by the fibroblasts. Spatial transcriptional analysis identified extracellular matrix (ECM) activities in fibroblasts as the top altered bioprocesses in both in vitro and in vivo. This finding was confirmed in cultures, where fibroblast-derived decellularized matrix stimulated tumor invasion of single TNBC spheroids at levels observed in co-cultures. Further analysis of differentially expressed genes (DEGs) identified a small leucine-rich repeat proteoglycan (SLRP) as the primary ECM target altered by variations in SHH signal strength in fibroblasts. Furthermore, we confirmed the role of SLRP cues in governing tumor invasion potency within spheroid cultures embedded in a collagen hydrogel matrix. While high levels of SLRP in the matrix inhibited spheroid invasion, low SLRP matrix promoted spheroid invasion and increased secondary invasion sites. These findings agreed with the Vimentin expression levels observed in TNBC cells and confirmed the potency of SLRP in regulating tumor invasion outcomes. In summary, our study underscores the significance of ECM remodeling activities in fibroblasts and the role of SLRPs in governing tumor invasion, with implications for understanding the impact of altering SHH signaling activity in TNBC. Citation Format: Emanuel A. Carrasquillo-Dones, Heizel M. Rosado-Galindo, Ana M. Reyes-Ramos, Wandaliz Torres-Garcia, Said Cifuentes, Jan P. Rios-Grant, Gabriela Ortiz-Soto, Natalia A. Ramos-Acevedo, Miosotis Acevedo-Esquilin, Monica Colon-Vargas, Israel Almodovar-Rivera, Camilo Mora, Michelle M. Martinez-Montemayor, Maribella Domenech. In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes [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 4208.

Podocan promoting skeletal muscle post-injury regeneration by inhibiting TGF-β signaling pathway.

Podocan, the fifth member of Small Leucine-Rich Proteoglycan (SLRP) family of extracellular matrix components, is poorly known in muscle development. Previous studies have shown that Podocan promotes C2C12 differentiation in mice. In this study, we elucidated the effect of Podocan on skeletal muscle post-injury regeneration and its underlying mechanism. Injection of Podocan protein promoted the process of mice skeletal muscle post-injury regeneration. This effect seemed to be from the acceleration of muscle satellite cell differentiation invivo. Meanwhile, Podocan promoted myogenic differentiation invitro by binding with TGF-β1 to inhibit the activity of the TGF-β signaling pathway. These results indicated that Podocan had the potential roles to enhance skeletal muscle post-injury regeneration. Its mechanism is likely the regulation of the expression of p-Smad2 and p-Smad4 related to the TGF-β signaling pathway by interacting with TGF-β1.

Digital spatial profiling of segmental outflow regions in trabecular meshwork reveals a role for ADAM15.

In this study we used a spatial transcriptomics approach to identify genes specifically associated with either high or low outflow regions in the trabecular meshwork (TM) that could potentially affect aqueous humor outflow in vivo. High and low outflow regions were identified and isolated from organ cultured human anterior segments perfused with fluorescently-labeled 200 nm FluoSpheres. The NanoString GeoMx Digital Spatial Profiler (DSP) platform was then used to identified genes in the paraffin embedded tissue sections from within those regions. These transcriptome analyses revealed that 16 genes were statistically upregulated in high outflow regions and 57 genes were statistically downregulated in high outflow regions when compared to low outflow regions. Gene ontology enrichment analysis indicated that the top three biological categories of these differentially expressed genes were ECM/cell adhesion, signal transduction, and transcription. The ECM/cell adhesion genes that showed the largest differential expression (Log2FC ±1.5) were ADAM15, BGN, LDB3, and CRKL. ADAM15, which is a metalloproteinase that can bind integrins, was upregulated in high outflow regions, while the proteoglycan BGN and two genes associated with integrin signaling (LDB3, and CRKL) were downregulated. Immunolabeling studies supported the differential expression of ADAM15 and showed that it was specifically upregulated in high outflow regions along the inner wall of Schlemm's canal and in the juxtacanalicular (JCT) region of the TM. In addition to these genes, the studies showed that genes for decorin, a small leucine-rich proteoglycan, and the α8 integrin subunit were enriched in high outflow regions. These studies identify several novel genes that could be involved in segmental outflow, thus demonstrating that digital spatial profiling could be a useful approach for understanding segmental flow through the TM. Furthermore, this study suggests that changes in the expression of genes involved in regulating the activity and/or organization of the ECM and integrins in the TM are likely to be key players in segmental outflow.

Relationship between elevated serum decorin levels and acne vulgaris in women with PCOS.

The primary aim of this study was to investigate the serum levels of decorin, a small leucine-rich proteoglycan, in women diagnosed with polycystic ovary syndrome (PCOS) and concurrently presenting with moderate to severe acne vulgaris. Sixty patients with acne vulgaris symptoms, subsequently diagnosed with PCOS according to the revised Rotterdam criteria, were enrolled in the study. Acne severity was assessed using the acne global severity scale (AGSS), with patients fitting AGSS category 4 (moderate) and 5 (severe) grouped into two separate cohorts of 30 individuals each. The moderate acne group comprised patients with few inflammatory lesions and a minor nodule alongside predominantly non-inflammatory lesions, whereas the severe group contained patients with multiple nodules and a mix of non-inflammatory and inflammatory lesions. A control group of twenty healthy women without acne vulgaris or PCOS was also established. The study measured serum testosterone, follicle-stimulating hormone (FSH), Luteinizing Hormone (LH), and insulin levels, and calculated insulin resistance via the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) formula. Quantitative sandwich enzyme immunoassay was employed to determine decorin levels from venous blood samples. While age, body mass index (BMI), serum FSH, LH, testosterone, and HOMA-IR values demonstrated similarity between the moderate and severe acne cohorts, comparisons between the control and both acne groups (AGSS-4 and AGSS-5) revealed significantly elevated values in the latter, excluding age, BMI, and FSH. Importantly, the serum decorin levels in both acne groups were substantially higher than in controls. A significant positive correlation was observed between serum decorin levels and both HOMA-IR (r=7.88, p<0.01) and testosterone (r=0.813, p<0.01). This study suggests that elevated circulating decorin levels play a pivotal role in the manifestation of acne vulgaris in women with PCOS.

Extracellular Matrix Orchestration of Tissue Remodeling in the Chronically Inflamed Mouse Colon

Background & AimsChronic inflammatory illnesses are debilitating and recurrent conditions associated with significant co-morbidities, including an increased risk of developing cancer. Extensive tissue remodelling is a hallmark of such illnesses, and is both a consequence and a mediator of disease progression. Despite previous characterisation of epithelial and stromal remodelling during Inflammatory Bowel Disease, a complete understanding of its impact on disease progression is still lacking. MethodsA comprehensive proteomic pipeline using data-independent acquisition was applied to decellularized colon samples from the Muc2KO mouse model of colitis, for an in-depth characterisation of extracellular matrix remodelling. Unique proteomic profiles of the matrisomal landscape were extracted from pre- and pathological colitis. Integration of proteomics and transcriptomics datasets extracted from the same murine model produced network maps describing the orchestrating role of matrisomal proteins in tissue remodelling during the progression of colitis. ResultsThe in-depth proteomic workflow used here allowed the addition of 34 proteins to the known colon matrisomal signature. Protein signatures of pre- and pathological colitic states were extracted, differentiating the two states by expression of small leucine rich proteoglycans. We outlined the role of this class and other matrisomal proteins in tissue remodelling during colitis, as well as the potential for co-ordinated regulation of cell types by matrisomal ligands. ConclusionOur work highlights a central role for matrisomal proteins in tissue remodelling during colitis and defines orchestrating nodes that can be exploited in selection of therapeutic targets.

Bone Biomarker in Feedlot Cattle from Two Different Production Systems.

The rapid growth rate in feedlot cattle is likely to promote joint abnormalities like osteochondrosis dissecans (OCD) and osteoarthritis (OA) with subsequent lameness. We have identified a small leucine-rich repeat proteoglycan (SLRP) biomarker at cleavage site 262GLGHNQIRM (BGN262) arising from the fragmentation of biglycan (BGN) in subchondral bone associated with OA. With a validated custom-made ELISA, BGN262 has been quantified in serum from cattle. The concentration of BGN262 in serum from cattle raised in the conventional and all-natural production systems increased at harvest compared to the starting period. The limitation of the study is the small sample size. However, the promising results encourage a further evaluation of BGN262 and its potential as a biomarker for subchondral bone pathology in cattle.

Aged tendons lack adaptive response to acute compressive injury.

Rotator cuff tendinopathy has a multifactorial etiology, with both aging and external compression found to influence disease progression. However, tendon's response to these factors is still poorly understood and in vivo animal models make it difficult to decouple these effects. Therefore, we developed an explant culture model that allows us to directly apply compression to tendons and then observe their biological responses. Using this model, we applied a single acute compressive injury to C57BL/6J flexor digitorum longus tendon explants and observed changes in viability, metabolic activity, matrix composition, matrix biosynthesis, matrix structure, gene expression, and mechanical properties. We hypothesized that a single acute compressive load would result in an injury response in tendon and that this effect would be amplified in aged tendons. We found that young tendons had increased matrix turnover with a decrease in small leucine-rich proteoglycans, increase in compression-resistant proteoglycan aggrecan, increase in collagen synthesis, and an upregulation of collagen-degrading MMP-9. Aged tendons lacked any of these adaptive responses and instead had decreased metabolic activity and collagen synthesis. This implies that aged tendons lack the adaptation mechanisms required to return to homeostasis, and therefore are at greater risk for compression-induced injury. Overall, we present a novel compressive injury model that demonstrates lasting age-dependent changes and has the potential to examine the long-term response of tendon to a variety of compressive loading conditions.

SYNOVIAL FLUID PROTEOMICS AND BIOINFORMATICS HIGHLIGHTS POTENTIAL MECHANISMS OF UMBILICAL CORD-MSC INDUCED RADIOLOGICAL IMPROVEMENT IN AN OVINE MODEL OF OSTEOARTHRITIS

AbstractObjectivesA promising therapy for early osteoarthritis (OA) is the transplantation of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). The synovial fluid (SF) from a pre-clinical ovine model treated with hUC-MSCs has been profiled using proteomics and bioinformatics to elucidate potential mechanisms of therapeutic effect.MethodsFour weeks after a medial meniscus transection surgery, sheep were injected with 107 hUC-MSCs in Phosphate Buffered Saline (PBS) or PBS only (n=7) and sacrificed at 12 weeks. SF was normalised for protein abundance (ProteoMinerTM) and analysed using label-free quantitation proteomics. Bioinformatics analyses (Ingenuity Pathway Analysis (IPA) and STRING) were used to assess differentially regulated functions from the proteomic data. Human orthologues were identified for the ovine proteins using UniProt and DAVID resources and proteins that were ≥±1.3 fold differentially abundant between treatment groups, were included in the bioinformatics analyses.ResultshUC-MSC treated animals demonstrated significantly less joint space narrowing. Nineteen SF proteins were differentially abundant in treated cf. control sheep (FC±2.0; p&lt;0.05). Biglycan (a small leucine-rich proteoglycan of the cartilage extracellular matrix) abundance was increased by 2.1 fold in treated compared to untreated sheep (p=0.024). IPA indicated that lipid synthesis (z-score=1.772; p=0.00267) and immune cell migration pathways (cell movement of mononuclear leukocytes: z-score=1.761; p=0.00259), amongst others, were likely to be activated in the treated sheep. Conversely, tissue damage (z-score=−2; p=0.00019), senescence (z-score=−1.981; p=0.00007) and necrosis (z-score=−1.728; p=0.00829) associated pathways as well as inflammation (z-score=−1.718; p=0.00057) and vascular permeability (z-score=−1.698; p=0.00002) were likely to be inhibited in treated cf. untreated sheep.ConclusionshUC-MSC treatment prevented/delayed OA progression, demonstrated via a reduction in joint space narrowing. SF proteome bioinformatics revealed potential mechanisms of therapeutic action related to immunomodulation and the inhibition of multiple cell death, and tissue damage associated pathways. Further, a potential predicted upregulation in lipid synthesis in treated sheep represents a novel mechanism warranting further investigation. Additional work is required to validate these discovery phase proteomic findings in studies which specifically target and manipulate the proposed mechanisms highlighted.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Abstract 18691: Asporin Attenuates Pulmonary Arterial Smooth Muscle Cell Proliferation and Regulates Pulmonary Hypertension Development

Introduction: Pulmonary arterial hypertension (PAH) is a devastating pulmonary vascular lung disease characterized by increased pressure in the pulmonary arteries (PA) leading to cardiac right ventricular (RV) hypertrophy, RV failure, and ultimately death. Current PAH therapies merely improve symptoms temporarily and patients often need lung and heart transplantation. There is dire need for new insights into PAH pathophysiology to find novel therapeutic approaches. A key mechanism driving PAH is medial hyperplasia resulting from enhanced pulmonary arterial smooth muscle cell (PASMC) proliferation. Methods: We employed an unbiased bioinformatics approach in online-available microarray expression and single-cell sequencing datasets of dissected PA from PAH patients. In vitro experiments were performed in PASMC isolated from a female donor and PAH patient. Human lung tissue sections were obtained from the Pulmonary Hypertension Breakthrough Initiative Biobank. The Sugen/Hypoxia model was used to induce PH in rats. Results: We discovered that a novel gene, small leucine-rich proteoglycan Asporin (Aspn), is highly and significantly upregulated in PAH compared to donor PA and is most highly expressed in PASMC. We validated higher Aspn mRNA expression in human lungs and PASMC from PAH patients compared to controls, supporting our bioinformatics approach. Moreover, Aspn protein expression is elevated in PASMC in lung tissue sections from PAH patients compared to donors, as well as in PAH patient plasma . Functionally, silencing Aspn in PASMC from PAH patients promoted proliferation, whereas recombinant Aspn exerted opposite effects. To find potential downstream mediators of Aspn, STRING database analysis revealed that Aspn binds to the pro-proliferative mediator TGFβ1. Accordingly, TGFβ1 downstream mediator p-SMAD2/3 is enhanced upon Aspn silencing in PASMC. Intratracheal delivery of Aspn siRNA in rats aggravated Sugen/Hypoxia-induced PH, as assessed by RVSP, echocardiography and vascular remodeling. Conclusions: We hypothesize that elevated Aspn expression in PAH is a compensatory mechanism that inhibits PASMC proliferation, leading to decreased PA vascular remodeling, and that Aspn may be a novel therapeutic target against PAH.

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

ABSTRACT 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.

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.

Structural Insights into Protein-Ligand Interactions of Small Leucine Rich Repeat Proteoglycans with a Large Number of Binding Partners: An Overview

Small leucine rich repeat proteoglycans (SLRPs) exist in the extracellular matrix. SLRPs contain tandem arrays of LRRs flanked by cysteine clusters at the both N- and C-termini. The extreme N- and/or C-termini contain low complexity sequences, glycosaminoglycan (GAG) chain and/or sulfated tyrosine residues in some members of SLRPs. The LRR solenoid structure may be divided into four parts consisting of a concave surface, an ascending surface, a convex surface, and a descending surface. SLRPs share many biological functions including collagen fibrillogenesis, signaling, innate immunity, and inflammation through the binding of various ligands. We undertake a comprehensive literature search of publications in order to make a list of ligands of SLRPs. We describe and discuss the interacting sites of SLRPs to binding partners. The protein-ligand interactions occur on not only the concave surface but also the ascending surface and the N- or C-terminal capping regions. In addition, the extreme N- and/or C-terminal regions with the GAG chains or sulfated tyrosine residues participate in ligand-interactions.

Small Leucine-Rich Proteoglycan PODNL1 Identified as a Potential Tumor Matrix-Mediated Biomarker for Prognosis and Immunotherapy in a Pan-Cancer Setting.

The podocan-like protein 1 (PODNL1), an important member of the small leucine-rich proteoglycans (SLRP) family, is a crucial component of the tumor microenvironment (TME). But its prognostic values and the role in the TME have not been systematically estimated in a pan-cancer setting. Targeting PODNL1, a systematic exploration into the TCGA datasets, reconciling with the analyses of single-cell transcriptomes and immunotherapeutic cohorts in cancers, and validation by tissue microarray-based multiplex immunofluorescence staining was performed. PODNL1 was significantly correlated with the poor prognosis and immunotherapeutic responses in various cancers. In-depth demonstration of molecular mechanisms indicated that PODNL1 expressions were notably positively correlated with cancer-associated fibroblast (CAF) infiltration levels in 33 types of cancers. It also positively correlated with the pan-fibroblast TGF-β response signature score, and the hallmarks including TGF-β, TNF-α, inflammatory response, apical junction, epithelial-mesenchymal transition and hedgehog in pan-cancer. Furthermore, high PODNL1 expressions were positively related with the regulation of tumor-promoting TGF-β signaling through downregulating SMAD2/3:4 heterotrimer regulations transcription and up-regulating the pathway restricted SMAD protein phosphorylation. Single-cell transcriptome analyses and immunofluorescence validations indicated that PODNL1 was predominantly expressed in the cancer cells and CAFs in various cancers. Additionally, the heterogeneity of cancer genotype-phenotype cross-talking was also observed associated with PODNL1. Our systematic study indicates that PODNL1 plays an important role in the complex regulation network of tumor progression, and lays a foundation for further exploration to develop PODNL1 as a valuable matrix-mediated biomarker for cancer immunotherapy and prognosis in a pan-cancer setting.

The Landscape of Small Leucine-Rich Proteoglycan Impact on Cancer Pathogenesis with a Focus on Biglycan and Lumican.

Cancer development is a multifactorial procedure that involves changes in the cell microenvironment and specific modulations in cell functions. A tumor microenvironment contains tumor cells, non-malignant cells, blood vessels, cells of the immune system, stromal cells, and the extracellular matrix (ECM). The small leucine-rich proteoglycans (SLRPs) are a family of nineteen proteoglycans, which are ubiquitously expressed among mammalian tissues and especially abundant in the ECM. SLRPs are divided into five canonical classes (classes I-III, containing fourteen members) and non-canonical classes (classes IV-V, including five members) based on their amino-acid structural sequence, chromosomal organization, and functional properties. Variations in both the protein core structure and glycosylation status lead to SLRP-specific interactions with cell membrane receptors, cytokines, growth factors, and structural ECM molecules. SLRPs have been implicated in the regulation of cancer growth, motility, and invasion, as well as in cancer-associated inflammation and autophagy, highlighting their crucial role in the processes of carcinogenesis. Except for the class I SLRP decorin, to which an anti-tumorigenic role has been attributed, other SLPRs' roles have not been fully clarified. This review will focus on the functions of the class I and II SLRP members biglycan and lumican, which are correlated to various aspects of cancer development.