Matrix Remodeling Factors Research Articles

Biomarkers for modeling of cancer-specifc tumorassociated macrophages ex vivo

Introduction. Tumor-associated macrophages (TAMs) are essential innate immune cells in the tumor microenvironment. TAMs can stimulate cancer cell proliferation and primary tumor growth, angiogenesis, lymphangiogenesis, cancer cell invasiveness in vessels and metastatic niche formation as well as support chemotherapy resistance. TAMs are phenotypically diverse both in various cancer localizations and in intratumoral heterogeneous compartments. Tumor-specific modeling of TAMs is necessary to understand the fundamental mechanism of pro- and anti-tumor activity, to test their interaction with existing therapies, and to develop TAM- targeted immunotherapy. Aim of study: To investigate cancer-specific transcriptomic features of ex vivo human TAM models. Material and Methods. Here we compared transcriptomic profiles of TAMs for breast, colorectal, ovarian, lung, and prostate cancers ex vivo. Human monocytes were isolated from buffy coats, and then stimulated by the tumor cell conditioned medium ex vivo. Using real-time PCR, we quantified the expression of key TAM biomarkers including inflammatory cytokines, scavenger-receptors, angiogenesis-regulating genes, and matrix remodeling factors. Results. PCR analysis allowed revealing cancer-specific expression profiles of modeled TAMs. By comparing the existing knowledge about TAM phenotypes in human tumors in vivo with the collected data, we discuss the advantages and limitation of ex vivo TAM models derived from human blood monocytes. Conclusion. Monocytes-derived macrophages stimulated with cancer cell-conditioned medium can, to a certain extent, allow modeling of cancer-specific programming of TAMs. Our model system is valuable to examine agents reprogramming key TAM pro-tumoral activities, and for the reproducible analysis of mechanistic events that program tolerogenic status of TAMs towards cancer cells.

Characterization of Angiogenic, Matrix Remodeling, and Antimicrobial Factors in Preterm and Full-Term Human Umbilical Cords.

Little is known about morphogenetic changes in the umbilical cord during the maturation process. Extracellular matrix remodeling, angiogenesis, progenitor activity, and immunomodulation are represented by specific markers; therefore, the aim of this study was to determine the expression of matrix metalloproteinase-2 (MMP2), tissue inhibitor of metalloproteinases-2 (TIMP2), CD34, vascular endothelial growth factor (VEGF), and human β-defensin 2 (HBD2) in preterm and full-term human umbilical cord tissue. Samples of umbilical cord tissue were obtained from 17 patients and divided into two groups: very preterm and moderate preterm birth umbilical cords; late preterm birth and full-term birth umbilical cords. Routine histology examination was conducted. Marker-positive cells were detected using the immunohistochemistry method. The number of positive structures was counted semi-quantitatively using microscopy. Statistical analysis was carried out using the SPSS Statistics 29 program. Extraembryonic mesenchyme cells are the most active cell producers, expressing MMP2, TIMP2, VEGF, and HBD2 at notable levels in preterm and full-term umbilical cord tissue. Statistically significant differences in the expression of CD34, MMP2, and TIMP2 between the two patient groups were found. The expression of VEGF was similar in both patient groups, with the highest number of VEGF-positive cells seen in the extraembryonic mesenchyme. The expression of HBD2 was the highest in the extraembryonic mesenchyme and the amniotic epithelium, where mostly moderate numbers of HBD2-positive cells were detected. Extracellular matrix remodeling in preterm and term umbilical cords is strongly regulated, and tissue factors MMP2 and TIMP2 take part in this process. The expression of VEGF is not affected by the umbilical cord's age; however, individual patient factors can affect the production of VEGF. Numerous CD34-positive cells in the endothelium of the umbilical arteries suggest a significant role of progenitor cells in very preterm and moderate preterm birth umbilical cords. Antimicrobial activity provided by HBD2 is essential and constant in preterm and full-term umbilical cords.

Resistance training-induced changes in muscle proteolysis and extracellular matrix remodeling biomarkers in the untrained and trained states.

Resistance training (RT) induces muscle growth at varying rates across RT phases, and evidence suggests that the muscle-molecular responses to training bouts become refined or attenuated in the trained state. This study examined how proteolysis-related biomarkers and extracellular matrix (ECM) remodeling factors respond to a bout of RT in the untrained (UT) and trained (T) state. Participants (19 women and 19 men) underwent 10weeks of RT. Biopsies of vastus lateralis were collected before and after (24h) the first (UT) and last (T) sessions. Vastus lateralis cross-sectional area (CSA) was assessed before and after the experimental period. There were increases in muscle and type II fiber CSAs. In both the UT and T states, calpain activity was upregulated and calpain-1/-2 protein expression was downregulated from Pre to 24h. Calpain-2 was higher in the T state. Proteasome activity and 20S proteasome protein expression were upregulated from Pre to 24h in both the UT and T. However, proteasome activity levels were lower in the T state. The expression of poly-ubiquitinated proteins was unchanged. MMP activity was downregulated, and MMP-9 protein expression was elevated from Pre to 24h in UT and T. Although MMP-14 protein expression was acutely unchanged, this marker was lower in T state. TIMP-1 protein levels were reduced Pre to 24h in UT and T, while TIMP-2 protein levels were unchanged. Our results are the first to show that RT does not attenuate the acute-induced response of proteolysis and ECM remodeling-related biomarkers.

Neuronally differentiated macula densa cells regulate tissue remodeling and regeneration in the kidney.

Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here, we identified neuronal differentiation features of MD cells that sense the local and systemic environment and secrete angiogenic, growth, and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models, and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors, including CCN1, as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ-specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue-regenerative therapeutic strategies.

Cell Survival, Death, and Proliferation in Senescent and Cancer Cells: the Role of (Poly)phenols.

Cellular senescence has long been considered a permanent state of cell cycle arrest occurring in proliferating cells subject to different stressors, used as a cellular defense mechanism from acquiring potentially harmful genetic faults. However, recent studies highlight that senescent cells might also alter the local tissue environment and concur to chronic inflammation and cancer risk by secreting inflammatory and matrix remodeling factors, acquiring a senescence-associated secretory phenotype (SASP). Indeed, during aging and age-related diseases, senescent cells amass in mammalian tissues, likely contributing to the inevitable loss of tissue function as we age. Cellular senescence has thus become one potential target to tackle age-associated diseases as well as cancer development. One important aspect characterizing senescent cells is their telomere length. Telomeres shorten as a consequence of multiple cellular replications, gradually leading to permanent cell cycle arrest, known as replicative senescence. Interestingly, in the large majority of cancer cells, a senescence escape strategy is used and telomere length is maintained by telomerase, thus favoring cancer initiation and tumor survival. There is growing evidence showing how (poly)phenols can impact telomere maintenance through different molecular mechanisms depending on dose and cell phenotypes. Although normally, (poly)phenols maintain telomere length and support telomerase activity, in cancer cells this activity is negatively modulated, thus accelerating telomere attrition and promoting cancer cell death. Some (poly)phenols have also been shown to exert senolytic activity, thus suggesting both antiaging (directly eliminating senescent cells) and anticancer (indirectly, via SASP inhibition) potentials. In this review, we analyze selective (poly)phenol mechanisms in senescent and cancer cells to discriminate between in vitro and in vivo evidence and human applications considering (poly)phenol bioavailability, the influence of the gut microbiota, and their dose-response effects.

The Premetastatic Lymph Node Niche in Gynecologic Cancer

It has been suggested that a primary tumor can "prepare" the draining of lymph nodes to "better accommodate" future metastatic cells, thus implying the presence of a premetastatic lymph node niche. However, this phenomenon remains unclear in gynecological cancers. The aim of this study was to evaluate lymph-node draining in gynecological cancers for premetastatic niche factors, such as myeloid-derived suppressor cells (MDSCs), immunosuppressive macrophages, cytotoxic T cells, immuno-modulatory molecules, and factors of the extracellular matrix. This is a monocentric retrospective study of patients who underwent lymph-node excision during their gynecological-cancer treatment. In all, 63 non-metastatic pelvic or inguinal lymph nodes, 25 non-metastatic para-aortic lymph nodes, 13 metastatic lymph nodes, and 21 non-cancer-associated lymph nodes (normal controls) were compared for the immunohistochemical presence of CD8 cytotoxic T cells, CD163 M2 macrophages, S100A8/A9 MDSCs, PD-L1+ immune cells, and tenascin-C, which is a matrix remodeling factor. PD-L1-positive immune cells were significantly higher in the control group, in comparison to the regional and distant cancer-draining lymph nodes. Tenascin-C was higher in metastatic lymph nodes than in both non-metastatic nodes and control lymph nodes. Vulvar cancer-draining lymph nodes showed higher PD-L1 values than endometrial cancer and cervical cancer-draining lymph nodes. Endometrial cancer-draining nodes had higher CD163 values and lower CD8 values, compared to vulvar cancer-draining nodes. Regarding regional draining nodes in low- and high-grade endometrial tumors, the former showed lower S100A8/A9 and CD163 values. Gynecological cancer-draining lymph nodes are generally immunocompetent, but vulvar cancer draining nodes, as well as high-grade endometrial cancer draining nodes, are more susceptible to harboring premetastatic niche factors.

271 Ribosome heterogeneity by rRNA methylation in skin cell senescence

The accumulation of senescent cells is associated with several age-related pathologies and represents a primary driver of skin aging. Senescence cells cause tissue damage by secreting a mix of pro-inflammatory cytokines and extracellular matrix remodeling factors. This study aims to elucidate how ribosomes and protein translation change in cellular senescence. Particularly we focus on alterations of the methylation patterns of ribosomal RNA. We exposed human dermal fibroblasts to doxorubicin to induce cellular senescence and tested if specific methylations of ribosomal RNA contribute to the senescent phenotype.

LB1026 Role of skin cellular senescence in chronic wound healing

Wound healing is an essential physiological process to restore normal skin structure and function after injury. Cellular senescence, an irreversible cell cycle state in response to damaging stimuli, has emerged as a paradigm shift to disrupt the traditional concept of wound healing beyond the described inflammatory, proliferative, and remodeling phases. We hypothesized that skin senescent cells, particularly fibroblasts, play an inhibitory role in the wound healing process and contribute to delayed healing. We generated an acute wound versus chronic wound mouse model to compare cellular senescence expression and their senescence-associated secretory phenotype (SASP) including growth factors, cytokines, and extracellular matrix remodeling factors. To validate our model, we first examined wound closure, degree of inflammation, epidermal hyperplasia, and degree of dermal fibrosis among others. Then, we assessed SASP profile in normal skin, acute wound, and chronic wound. Our data demonstrate Mmp3 (matrix metalloproteinase 3), Mmp9, Mcp (membrane cofactor protein), TGF-β (transforming growth factor β) which are key SASP factors involving in delayed would healing, significantly increased in chronic wounds. RNA in situ hybridization indicated increased p16ink4a (Cdkn2a) in chronic wounds, which is an intrinsic mediator in transition to cellular senescence. Furthermore, senescence-associated beta galactosidase staining showed enhanced senescent cells in chronic wounds. Taken together, cellular senescence may play a inhibitory role in chronic wound healing, and senescent cells clearance through systematic or topical senolytic treatment, may present a novel therapeutic option in the treatment of chronic wound disorders, including diabetic ulcers.

Cellular senescence and the tumour microenvironment.

The senescence‐associated secretory phenotype (SASP), where senescent cells produce a variety of secreted proteins including inflammatory cytokines, chemokines, matrix remodelling factors, growth factors and so on, plays pivotal but varying roles in the tumour microenvironment. The effects of SASP on the surrounding microenvironment depend on the cell type and process of cellular senescence induction, which is often associated with innate immunity. Via SASP‐mediated paracrine effects, senescent cells can remodel the surrounding tissues by modulating the character of adjacent cells, such as stromal, immune cells, as well as cancer cells. The SASP is associated with both tumour‐suppressive and tumour‐promoting effects, as observed in senescence surveillance effects (tumour‐suppressive) and suppression of anti‐tumour immunity in most senescent cancer‐associated fibroblasts and senescent T cells (tumour‐promoting). In this review, we discuss the features and roles of senescent cells in tumour microenvironment with emphasis on their context‐dependency that determines whether they promote or suppress cancer development. Potential usage of recently developed drugs that suppress the SASP (senomorphics) or selectively kill senescence cells (senolytics) in cancer therapy are also discussed.

Transcriptome and unique cytokine microenvironment of Castleman disease

Castleman disease (CD) represents a group of rare, heterogeneous and poorly understood disorders that share characteristic histopathological features. Unicentric CD (UCD) typically involves a single enlarged lymph node whereas multicentric CD (MCD) involves multiple lymph node stations. To understand the cellular basis of CD, we undertook a multi-platform analysis using targeted RNA sequencing, RNA in-situ hybridization (ISH), and adaptive immune receptor rearrangements (AIRR) profiling of archived tissue from 26 UCD, 14 MCD, and 31 non-CD reactive controls. UCD showed differential expression and upregulation of follicular dendritic cell markers (CXCL13, clusterin), angiogenesis factors (LPL, DLL4), extracellular matrix remodeling factors (TGFβ, SKIL, LOXL1, IL-1β, ADAM33, CLEC4A), complement components (C3, CR2) and germinal center activation markers (ZDHHC2 and BLK) compared to controls. MCD showed upregulation of IL-6 (IL-6ST, OSMR and LIFR), IL-2, plasma cell differentiation (XBP1), FDC marker (CXCL13, clusterin), fibroblastic reticular cell cytokine (CCL21), angiogenesis factor (VEGF), and mTORC1 pathway genes compared to UCD and controls. ISH studies demonstrated that VEGF was increased in the follicular dendritic cell-predominant atretic follicles and the interfollicular macrophages of MCD compared to UCD and controls. IL-6 expression was higher along interfollicular vasculature-associated cells of MCD. Immune repertoire analysis revealed oligoclonal expansions of T-cell populations in MCD cases (2/6) and UCD cases (1/9) that are consistent with antigen-driven T cell activation. The findings highlight the unique genes, pathways and cell types involved in UCD and MCD. We identify potential novel targets in CD that may be harnessed for therapeutics.

Pan-Cancer Analysis Reveals a Distinct Neutrophil Extracellular Trap-Associated Regulatory Pattern.

BackgroundNeutrophils form extracellular net-like structures called neutrophil extracellular traps (NETs). Emerging evidence has shown that cancer can induce NET formation; however, it is not fully understood how NETs influence cancer biology, and no consensus has been reached on their pro- or antitumor effects. A comprehensive analysis of the global NET-associated gene regulatory network is currently unavailable and is urgently needed.MethodsWe systematically explored and discussed NET enrichment, NET-associated gene regulatory patterns, and the prognostic implications of NETs in approximately 8,000 patients across 22 major human cancer types. We identified NET-associated regulatory gene sets that we then screened for NET-associated regulatory patterns that might affect patient survival. We functionally annotated the NET-associated regulatory patterns to compare the biological differences between NET-related survival subgroups.ResultsA gene set variation analysis (GSVA) based on 23 major component genes was used to calculate a metric called the NET score. We found that the NET score was closely associated with many important cancer hallmarks, particularly inflammatory responses and epithelial-to-mesenchymal transition (EMT)-induced metastasis. Higher NET scores were related to poor immunotherapy response. Survival analysis revealed that NETs had diverse prognostic impacts among various cancer types. The NET-associated regulatory patterns linked to shorter or longer cancer patient survival were distinct from each other. Functional analysis revealed that more of the NET-associated regulatory genes linked to poor cancer survival were associated with extracellular matrix (ECM) remodeling and pan-cancerous risk factors. SPP1 was found to be highly expressed and correlated with NET formation in cancers with poor survival. We also found that the co-upregulation of NET formation and SPP1 expression was closely linked to increased EMT and poor survival, that SPP1 influenced NET-induced malignant capacity, and that SPP1 overproduction induced a robust formation of metastatic-promoting NETs.ConclusionNETs were common across cancers but displayed a diverse regulatory pattern and outcome readouts in different cancer types. SPP1 is potentially the key to NET-related poor outcomes.

Silencing Survivin: a Key Therapeutic Strategy for Cardiac Hypertrophy.

Cardiac hypertrophy, in its aspects of localized thickening of the interventricular septum and concentric increase of the left ventricle, constitutes a risk factor of heart failure. Myocardial hypertrophy, in the presence of different degree of myocardial fibrosis, is paralleled by significant molecular, cellular, and histological changes inducing alteration of cardiac extracellular matrix composition as well as sarcomeres and cytoskeleton remodeling. Previous studies indicate osteopontin (OPN) and more recently survivin (SURV) overexpression as the hallmarks of heart failure although SURV function in the heart is not completely clarified. In this study, we investigated the involvement of SURV in intracellular signaling of hypertrophic cardiomyocytes and the impact of its transcriptional silencing, laying the foundation for novel target gene therapy in cardiac hypertrophy. Oligonucleotide-based molecules, like theranostic optical nanosensors (molecular beacons) and siRNAs, targeting SURV and OPN mRNAs, were developed. Their diagnostic and therapeutic potential was evaluated in vitro in hypertrophic FGF23-induced human cardiomyocytes and in vivo in transverse aortic constriction hypertrophic mouse model. Engineered erythrocyte was used as shuttle to selectively target and transfer siRNA molecules into unhealthy cardiac cells in vivo. The results highlight how the SURV knockdown could negatively influence the expression of genes involved in myocardial fibrosis in vitro and restores structural, functional, and morphometric features in vivo. Together, these data suggested that SURV is a key factor in inducing cardiomyocytes hypertrophy, and its shutdown is crucial in slowing disease progression as well as reversing cardiac hypertrophy. In the perspective, targeted delivery of siRNAs through engineered erythrocytes can represent a promising therapeutic strategy to treat cardiac hypertrophy. Theranostic SURV molecular beacon (MB-SURV), transfected into FGF23-induced hypertrophic human cardiomyocytes, significantly dampened SURV overexpression. SURV down-regulation determines the tuning down of MMP9, TIMP1 and TIMP4 extracellular matrix remodeling factors while induces the overexpression of the cardioprotective MCAD factor, which counterbalance the absence of pro-survival and anti-apoptotic SURV activity to protect cardiomyocytes from death. In transverse aortic constriction (TAC) mouse model, the SURV silencing restores the LV mass levels to values not different from the sham group and counteracts the progressive decline of EF, maintaining its values always higher with respect to TAC group. These data demonstrate the central role of SURV in the cardiac reverse remodeling and its therapeutic potential to reverse cardiac hypertrophy.

The Gene Expression of MMP-2 and TIMP-1 in Non-pathological Paratumoral and Autopsy Lung Tissues

Background: Finding a sample of healthy tissue is a critical challenge in research studies. Non-pathological Tissue adjacent to the tumor (NAT) specimens is usually used as the control in several studies. However, little is known about the similarity of NAT to healthy tissues. Here, we compared the expression of Matrix Metalloproteinase 2 (MMP-2) and its inhibitor, Tissue Inhibitors of MMP (TIMP)-1 as extracellular matrix remodeling factors in NAT and autopsy lung tissue. Materials and Methods: RNA of 7 NAT and 6 Formalin-Fixed Paraffin-Embedded (FFPE) lung autopsies from healthy people as the control group was extracted, and cDNA was synthesized. The gene expression levels of MMP-2 and TIMP-1 were evaluated by real-time PCR. Results: There were no significant differences in the expression of MMP-2, TIMP-1, or their ratio between the two groups. Conclusion: The results showed that NAT could be used as healthy controls in lung tissue studies for MMP-2 and TIMP-1.

Dysregulated Expression of Arterial MicroRNAs and Their Target Gene Networks in Temporal Arteries of Treatment-Naïve Patients with Giant Cell Arteritis.

In this study, we explored expression of microRNA (miR), miR-target genes and matrix remodelling molecules in temporal artery biopsies (TABs) from treatment-naïve patients with giant cell arteritis (GCA, n = 41) and integrated these analyses with clinical, laboratory, ultrasound and histological manifestations of GCA. NonGCA patients (n = 4) served as controls. GCA TABs exhibited deregulated expression of several miRs (miR-21-5p, -145-5p, -146a-5p, -146b-5p, -155-5p, 424-3p, -424-5p, -503-5p), putative miR-target genes (YAP1, PELI1, FGF2, VEGFA, KLF4) and matrix remodelling factors (MMP2, MMP9, TIMP1, TIPM2) with key roles in Toll-like receptor signaling, mechanotransduction and extracellular matrix biology. MiR-424-3p, -503-5p, KLF4, PELI1 and YAP1 were identified as new deregulated molecular factors in GCA TABs. Quantities of miR-146a-5p, YAP1, PELI1, FGF2, TIMP2 and MMP9 were particularly high in histologically positive GCA TABs with occluded temporal artery lumen. MiR-424-5p expression in TABs and the presence of facial or carotid arteritis on ultrasound were associated with vision disturbances in GCA patients. Correlative analysis of miR-mRNA quantities demonstrated a highly interrelated expression network of deregulated miRs and mRNAs in temporal arteries and identified KLF4 as a candidate target gene of deregulated miR-21-5p, -146a-5p and -155-5p network in GCA TABs. Meanwhile, arterial miR and mRNA expression did not correlate with constitutive symptoms and signs of GCA, elevated markers of systemic inflammation nor sonographic characteristics of GCA. Our study provides new insights into GCA pathophysiology and uncovers new candidate biomarkers of vision impairment in GCA.

MO620ANTIFIBROTIC EFFECTS OF SGLT2 INHIBITION IN THE KIDNEY AND PROXIMAL TUBULAR CELLS*

Abstract Background and Aims Diabetic kidney disease (DKD) is a major cause of chronic kidney disease and end stage renal disease, therefore identification of novel therapeutic strategies that reduce the risk of DKD is a research priority. Recent large clinical trials suggest that improved renal outcomes by sodium-glucose cotransporter 2 inhibitors (SGLT2i) are partly beyond their glucose lowering effects. Enhanced glucose reabsorption in diabetes leads to tubular hypoxia triggering fibrotic response. Hyperglycemia is in strong association with increased protein O-GlcNAcylation, a post-translational modification contributing to renal fibrosis. Considering the proximal tubular involvement in DKD pathogenesis and the key role of SGLT2 in glucose metabolism, here we investigated the effects of SGLT2i on tubular hypoxia and O-GlcNAcylation. Method Diabetes (D) was induced by streptozotocin (65 mg/bwkg, ip.) in adult, male Wistar rats. Following the onset of diabetes rats were treated for six weeks with dapagliflozin (D+DAPA, 1 mg/bwkg/day, po.). Metabolic parameters and renal function were evaluated. Novel urinary biomarkers of extracellular matrix remodeling (Pro-C3, uC3M, tumstatin) and profibrotic growth factors (TGF-β, CTGF, PDGF) were determined. Histological evaluation of glomerular damage (PAS), tubulointerstitial fibrosis (Masson’s trichrome, Picrosirius red) and fibronectin accumulation were performed. The effect of hyperglycemia was tested in human proximal tubular epithelial cells (HK-2) kept under normal glucose (5.5 mM), high glucose (35 mM) or high mannitol (osmotic control, 35 mM) conditions for 24 hours. HG cells were treated with 10 µM DAPA. O-GlcNAc, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) were measured. To test the effect of hypoxia cells were treated with 10 µM DAPA and were placed in a hypoxic chamber (1% O2) for 2 hours. Hypoxic injury was investigated using three different methods (qRT-PCR, Western blot, immunofluorescence analysis). HIF-1α, EPO, VEGFA and profibrotic factors were measured. Results DAPA decreased blood glucose levels (D: 37±2.7 vs. D+DAPA: 18±5.6 mmol/L; p<0.05) and improved renal function (creatinine clearance: D: 3.8±0.4 vs. D+DAPA: 8.9±1.0 mL/min; p<0.01). In parallel, novel urinary biomarkers of extracellular matrix remodeling, profibrotic growth factor expressions and extensive fibrotic tissue accumulation were reduced in the kidney. DAPA minimized hyperglycemia-induced total protein O-GlcNAcylation in HK-2 cells. Hypoxia-induced HIF-1α elevation was suspended by DAPA treatment. Moreover, DAPA treatment prevented HIF-1α translocation to the nucleus, thereby confirming abolished HIF-1α activation. EPO, VEGFA and profibrotic factor levels were also increased in hypoxia and DAPA prevented EPO, TGFB and PDGF elevation. Conclusion These data highlight the role of ameliorated O-GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia and fibrosis, a vicious trio, which seem to be targeted by SGLT2i. All these mechanisms are important parts in the puzzle of the complex system behind the protective effect of SGLT2i. OTKA-FK124491-K135398, 2017-1.3.1-VKE-2017-00006, 2020-4.1.1.-TKP2020-6183169273, 2020-4.1.1.-TKP2020-6183069269

Tumor necrosis factor α, protein gene product 9.5, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 presence in congenital intra-abdominal adhesions in children under one year of age.

IntroductionThe regulatory role of cytokines and extracellular matrix remodeling factors in congenital intra-abdominal adhesions has not yet been defined. The aim of this study was to assess the presence and relative distribution of tumor necrosis factor α (TNF-α), protein gene product 9.5 (PGP 9.5), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in adhesions.Material and methodsTNF-α, PGP 9.5, MMP-2 and TIMP-2 were detected using immunohistochemical methods and their relative distribution was evaluated by means of the semiquantitative counting method. The results were analyzed using non-parametric statistical methods.ResultsA moderate number of TNF-α positive macrophages and fibroblasts was found. A positive correlation was observed between the immunoreactive structures for TNF-α and PGP 9.5. A positive reaction for PGP 9.5 was observed in nerve fibers and shape modified fibroblasts. In control group tissues, positive structures were seen in significantly higher counts for PGP 9.5. Few to moderate numbers of MMP-2 positive macrophages, epithelioid cells, fibroblasts and endotheliocytes were detected. There was no significant difference between the groups. A positive reaction for TIMP-2 was seen in fibroblasts, macrophages and endotheliocytes. In control group tissues, positive structures were found in significantly higher counts for TIMP-2.ConclusionsThe positive correlation between the immunoreactive structures for TNF-α and PGP 9.5 suggests that nerve in-growth into intraabdominal adhesions might be induced by TNF-α and PGP 9.5 could have a role in maintaining inflammation. The down-regulation of PGP 9.5 suggests that pathogenesis of congenital intraabdominal adhesions may be related to hypoxia induced damage. The imbalance between MMP-2 and TIMP-2 may prove tissue fibrosis as a response to congenital peritoneal adhesions.

Extracellular Matrix Remodeling Factors as Markers of Carotid Artery Atherosclerosis.

Materials and Methods 20 patients undergoing routine carotid endarterectomy and 40 healthy volunteers were enrolled in this study. MMPs activity and OPG and FN concentrations were measured in atherosclerotic plaques and nonchanged contiguous tissue after homogenization as well as in plasma from patients and reference group. The activity of MMPs was evaluated by gelatin zymography, and the concentration of OPG and FN was assessed by ELISA. Results OPG concentration and MMP-9 activity showed differences between plaque and nonchanged tissue; OPG was higher in adjacent tissue (P=0.0009), whereas MMP-9 was higher in plaque (proMMP-9 P=0.0003; MMP-9 P < 0.0001). The OPG plasma concentration and both MMPs plasma activity were higher in patients (OPG P < 0.001; proMMP-2 P=0.0292; and proMMP-9 P=0.0374), while FN plasma concentration was lower in patients than in the reference group (P=0.0004). The ROC curves analysis showed the highest AUC for OPG (0.943) with 85.0% sensitivity and 92.1% specificity. Conclusions The atherosclerotic plaque and the contiguous artery wall are biochemically different. OPG shows the highest potential to be a marker of advanced carotid atherosclerosis.

A Novel Potential of SGLT2 Inhibition: Antifibrotic Effect in Diabetes

Background and aimsIdentification of novel therapeutic strategies that reduce the risk of diabetic kidney disease (DKD) is a research priority. Clinical data suggest that improved renal outcomes by sodium‐glucose cotransporter 2 inhibitors (SGLT2i) are partly beyond their glucose lowering effects; however, mechanisms are still elusive. Enhanced glucose reabsorption in diabetes leads to tubular hypoxia playing part in fibrogenesis. Hyperglycemia is in strong association with increased protein O‐GlcNAcylation, a post‐translational modification contributing to renal fibrosis. Considering the proximal tubular involvement in DKD pathogenesis and the key role of SGLT2 in glucose metabolism, here we investigated the effects of SGLT2i on tubular hypoxia and O‐GlcNAcylation.MethodsDiabetes (D) was induced by streptozotocin (65 mg/bwkg, ip.) in adult, male Wistar rats. Following the onset of diabetes rats were treated for six weeks with dapagliflozin (D+DAPA, 1 mg/bwkg/day, po.). Metabolic parameters and renal function were evaluated. Novel urinary biomarkers of extracellular matrix remodeling (Pro‐C3, uC3M, tumstatin) and profibrotic growth factors (TGF‐β, CTGF, PDGF) were determined. Histological evaluation of glomerular damage (PAS) and tubulointerstitial fibrosis (Masson’s trichrome, Picrosirius red, fibronectin) were performed. The effect of hyperglycemia was tested in human proximal tubular epithelial cells (HK‐2) kept under normal glucose (5.5 mM), high glucose (35 mM) or high mannitol (osmotic control, 35 mM) conditions for 24 hours. HG cells were treated with 10 μM DAPA. O‐GlcNAc, O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) were measured. To test the effect of hypoxia cells were treated with 10 μM DAPA and were placed in a hypoxic chamber (1% O2) for 2 hours. HIF‐1α, EPO, VEGFA and profibrotic factors were measured. Immunocytochemistry (ICC) staining for HIF‐1α was performed.ResultsDAPA decreased blood glucose levels (D: 37±2.7 vs. D+DAPA: 18±5.6 mmol/L; p&lt;0.05) and improved renal function (creatinine clearance: D: 3.8±0.4 vs. D+DAPA: 8.9±1.0 mL/min; p&lt;0.01). Subsequently novel fibrosis biomarkers, profibrotic growth factor expressions and renal fibrotic tissue accumulation were reduced. DAPA minimized hyperglycemia‐induced total protein O‐GlcNAcylation in HK‐2 cells. Hypoxia‐induced HIF‐1α elevation was suspended by DAPA treatment. EPO, VEGFA and profibrotic factor levels were also increased in hypoxia and DAPA prevented EPO and CTGF elevation.ConclusionsThese data highlight the role of ameliorated O‐GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia and fibrosis, a vicious trio that could be targeted by SGLT2i. All these mechanisms are important parts in the puzzle of the complex system behind the protective effect of SGLT2i.Support or Funding InformationOTKA‐FK124491, VKE‐2017‐00006, FIKP, UNKP‐19‐3‐III‐SE‐6, EFOP‐VEKOP‐16‐2017‐0009

Recurrent pregnancy loss is associated with a pro-senescent decidual response during the peri-implantation window

During the implantation window, the endometrium becomes poised to transition to a pregnant state, a process driven by differentiation of stromal cells into decidual cells (DC). Perturbations in this process, termed decidualization, leads to breakdown of the feto-maternal interface and miscarriage, but the underlying mechanisms are poorly understood. Here, we reconstructed the decidual pathway at single-cell level in vitro and demonstrate that stromal cells first mount an acute stress response before emerging as DC or senescent DC (snDC). In the absence of immune cell-mediated clearance of snDC, secondary senescence transforms DC into progesterone-resistant cells that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent decidual response in peri-implantation endometrium and recurrent pregnancy loss, suggesting that pre-pregnancy screening and intervention may reduce the burden of miscarriage.

Abstract B42: MDSCs accumulation within metastatic liver is modulated by CXCR4/CXCL12 axis after HSCs interaction with C26 in the ICAM-1 regulated inflammatory milieu

Abstract Introduction: Inflammation and immune suppression mediate tumor progression. During liver metastasis, tumor cells interact with and cross the hepatic sinusoidal line formed by liver sinusoidal endothelial cells (LSECs). In this inflammatory milieu, hepatic stellate cells (HSCs) are recruited into the nascent foci initiating the contact with the tumor cells. These cross-talks modulate the tumor microenvironment into a more tolerant one, in part by attracting myeloid derived suppressor cells (MDSCs). However, the mechanisms regulating the initiation of the inflammatory response and the recruitment of HSCs and MDSCs during the development of liver metastasis from colorectal cancer is not fully characterized. We aim to unmask mediators of the inflammatory response triggered after tumor cells interaction with LSECs and in the recruitment of HSC. Besides, the involvement of CXCR4/CXCL12 axis in the cross-talk between tumor cells and the recruited HSCs will be analyzed, with focus on the HSCs-mediated recruitment of MDSCs. Methods: To do so, the involvement of primary LSECs and tumor cells in the levels of inflammatory factors were measured by ELISA. Afterwards, the effect of both tumor-activated LSECs and sICAM-1 activated C26 cells in the recruitment of HSCs and their secretion of promigratory factors was studied. Moreover, the expression and role of CXCR4/CXCL12 axis in amplifying the migratory potential of HSCs and C26 was determined by using CXCL12 and the CXCR4 antagonist AMD3100. Finally, the role of CXCL12-CXCR4 in the hepatic stellate cell and MDSCs recruitment during liver metastasis and disease progression was analyzed in vivo by means of AMD3100 treatment. Results: Tumor-endothelial interplay increased secretion of IL-1β, IL-6, PGE2, and TNF-α, partly abrogated by blocking endothelial ICAM-1. Additionally, sICAM-1 activation of C26 could mimic this inflammatory response. Moreover, sICAM-1 stimulated CT26 derived medium significantly increased not only HSCs migration but also the matrix remodeling factors such as MMP-2 and uPA activity and the angiogenic factor VEGF. Additionally, tumor-derived conditioned medium promoted the expression of CXCR4, while CXCL12 increased the migratory potential of HSCs which was abrogated by blocking CXCL12/CXCR4 interaction through AMD3100. In vivo, the administration of AMD3100 revealed impaired infiltration of ASMA expressing HSCs into the metastatic lesions, along with reduced CD11b+Ly6G+ MDSC counts in the invaded organ. These observations were in line with 2-fold reduced metastatic area in AMD3100 treated mice liver. Conclusions: We can conclude that ICAM-1 mediates the early inflammatory response driving to HSC recruitment and their contribution to matrix remodeling and angiogenesis. These recruited HSC increase their expression of CXCR4 which could amplify their response to CXCL12 and thus trigger a positive feedback recruiting more C26 and HSCs. In vivo, these recruited HSCs might induce the recruitment of MDSCs via CXCR4/CXCL12 axis decreasing the local immune response. Therefore, blocking CXCR4/CXCL12 axis might be a potential therapeutic strategy to increase the antitumor immune response by different mechanisms mediated by both tumor and stromal cells. Citation Format: Aitor Benedicto, Irene Romayor, Beatriz Arteta. MDSCs accumulation within metastatic liver is modulated by CXCR4/CXCL12 axis after HSCs interaction with C26 in the ICAM-1 regulated inflammatory milieu [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B42.