Versican Expression Research Articles

Stress-deprivation induces an up-regulation of versican and connexin-43 mRNA and protein synthesis and increased ADAMTS-1 production in tendon cells in situ

ABSTRACT Purpose: The proper function of the tenocyte network depends on cell-matrix as well as intercellular communication that is mechanosensitive. Building on the concept that the etiopathogenic stimulus for tendon degeneration is the catabolic response of tendon cells to mechanobiologic under-stimulation, we studied the pericellular matrix rich in versican and its predominant proteolytic enzyme ADAMTS-1, as well as Connexin-43 (Cx43), a major gap junction forming protein in tendons, in stress-deprived rat tail tendon fascicles (RTTfs). Materials and Methods: RTTfs were stress-deprived for up to 7 days under tissue culture conditions. RT-qPCR was used to measure mRNA expression of versican, ADAMTS-1, and Cx43. Protein synthesis was determined using Western blotting and immunohistochemistry. Results: Stress-deprivation (SD) caused a statistically significant up-regulation of versican, ADAMTS-1, and Cx43 mRNA expression that was persistent over the 7-day test period. Western blot analysis and immunohistochemical assessment of protein synthesis revealed a marked increase of the respective proteins with SD. Inhibition of proteolytic enzyme activity with ilomastat prevented the increased versican degradation and Cx43 synthesis in 3 days stress-deprived tendons when compared with non-treated, stress-deprived tendons. Conclusion: In the absence of mechanobiological signaling the immediate pericellular matrix is modulated as tendon cells up-regulate their production of ADAMTS-1, and versican with subsequent proteoglycan degradation potentially leading to cell signaling cues increasing Cx43 gap junctional protein. The results also provide further support for the hypothesis that the cellular changes associated with tendinopathy are a result of decreased mechanobiological signaling and a loss of homeostatic cytoskeletal tension.

Interferon Regulatory Factor 9 Promotes Lung Cancer Progression via Regulation of Versican.

Simple SummaryLung cancer is the leading cause of cancer-related deaths worldwide, accounting for more than 1.6 million deaths per year. The tumor microenvironment (TME) has been shown to play a crucial role in tumor progression and metastasis, and transcription factors link TME signaling to oncogenesis. Type I interferons (IFNs) are strong immune modulators that possess antiproliferative and proapoptotic properties. In this study, we investigated the role of the transcription factor interferon regulatory factor 9 (IRF9) in the IFN pathway in lung cancer. We performed in vitro and in vivo experiments to reveal the oncogenic properties of IRF9, which was highly upregulated in lung adenocarcinoma. For the first time, we showed that IRF9 binds to the promoter of the known oncogene versican, regulates its expression, and thereby promotes oncogenic activity. Transcription factors can serve as links between tumor microenvironment signaling and oncogenesis. Interferon regulatory factor 9 (IRF9) is recruited and expressed upon interferon stimulation and is dependent on cofactors that exert in tumor-suppressing or oncogenic functions via the JAK-STAT pathway. IRF9 is frequently overexpressed in human lung cancer and is associated with decreased patient survival; however, the underlying mechanisms remain to be elucidated. Here, we used stably transduced lung adenocarcinoma cell lines (A549 and A427) to overexpress or knockdown IRF9. Overexpression led to increased oncogenic behavior in vitro, including enhanced proliferation and migration, whereas knockdown reduced these effects. These findings were confirmed in vivo using lung tumor xenografts in nude mice, and effects on both tumor growth and tumor mass were observed. Using RNA sequencing, we identified versican (VCAN) as a novel downstream target of IRF9. Indeed, IRF9 and VCAN expression levels were found to be correlated. We showed for the first time that IRF9 binds at a newly identified response element in the promoter region of VCAN to regulate its transcription. Using an siRNA approach, VCAN was found to enable the oncogenic properties (proliferation and migration) of IRF9 transduced cells, perhaps with CDKN1A involvement. The targeted inhibition of IRF9 in lung cancer could therefore be used as a new treatment option without multimodal interference in microenvironment JAK-STAT signaling.

Loss of versican and production of hyaluronan in lung epithelial cells are associated with airway inflammation during RSV infection

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however, whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production, we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) coculture model. RSV infection of BEC/HLF cocultures led to decreased hyaluronidase expression by HLFs, increased accumulation of HA, and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however, BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan−/−] demonstrated that RSV infection led to increased HA accumulation compared with control mice, which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan−/− mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared with SPC-Cre(−) RSV-infected littermates. Taken together, these data demonstrate that altered extracellular matrix accumulation of HA occurs following RSV infection and may contribute to airway inflammation. In addition, loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican’s role in inflammatory regulation is complex and dependent on the microenvironment.

Age-Related Changes in Female Scalp Dermal Sheath and Dermal Fibroblasts: How the Hair Follicle Environment Impacts Hair Aging

In women, aging leads to reduced hair density and thinner fibers and can result in female-pattern hair loss. However, the impact of the aging dermal environment on female scalp hair follicles remains unclear. In this study, we document in situ changes in 22 women (aged 19-81 years) and primary cultures of dermal fibroblast and dermal sheath cells. In situ, the papillary reticular boundary was indistinguishable in the young scalp but prominent in the scalp of those aged >40 years, accompanied by reduced podoplanin (PDPN) expression, increased versican expression, and changes in collagen organization. Hair follicles were shorter, not reaching the adipose layer. Hyaluronic acid synthase 2 was highly expressed, whereas matrix metalloproteinase 1 was elevated in the dermal papilla and dermal sheath in situ. Primary dermal fibroblast cultures confirmed that matrix metalloproteinase 1 mRNA, MMP1, increased with aging, whereas in dermal sheath cells, hyaluronic acid synthase 2, HAS2, and PDPN increased and α-smooth muscle actin αSMA mRNA decreased. Both exhibited increased cartilage oligomeric protein, COMP mRNA expression. Proteomics revealed an increase in dermal sheath proteins in the dermal fibroblast secretome with aging. In summary, aging female scalp shows striking structural and biological changes in the hair follicle environment that may impact hair growth.

MiRNA microarray profiling in patients with androgenic alopecia and the effects of miR-133b on hair growth

ObjectiveAndrogenetic alopecia (AGA), a common alopecia, is often accompanied by abnormal expression of multiple miRNAs. This study aims to investigate abnormally expressed miRNAs in patients with AGA and their specific molecular mechanism. MethodsmiRNA microarray profiling and qRT-PCR validation were used to screen and verify abnormally expressed miRNAs in patients with AGA. Human hair follicles (HFs) were treated with different concentrations of dihydrotestosterone (DHT, 10−5, 10−6, 10−7 and 10−8 mol/L) for 10 days. The effects of DHT on HF growth, proliferation, and miRNA expression in cultured HFs were investigated using immunofluorescence staining and qRT-PCR. Moreover, human dermal papilla cells (HDPCs) were treated/transfected with a Wnt/β-catenin pathway activator and/or miR-133b mimic, and then the CCK-8 assay was used to evaluate HDPC proliferation. qRT-PCR and Western blotting were used to measure the expression of Versican, ALP and β-catenin ResultsmiRNA microarray profiling identified 43 miRNAs that were significantly differentially expressed in AGA patients, and qRT-PCR verified that 8 miRNAs were significantly differentially expressed. The expression of miR-133b was abnormally high in AGA patients. DHT (10−5 mol/L) inhibited human HF growth and upregulated miR-133b expression, and DHT (10−7 mol/L) induced human HF growth and downregulated miR-133b expression. HDPC proliferation was inhibited, and the expression of β-catenin was downregulated in the miR-133b mimic-transfected group compared with the control group (P < 0.05). Wnt/β-catenin pathway activator treatment significantly promoted HDPC proliferation and upregulated the expression of β-catenin (P < 0.05). In addition, the proliferation of HDPCs was not significantly different between the group cotreated with a Wnt/β-catenin pathway activator and miR-133b mimic, and the control group (P > 0.05), but the expression of Versican and ALP was suppressed in the cotreatment group (P < 0.05) ConclusionOur data indicated that patients with androgenic alopecia have specific miRNA expression profiles and that the abnormal expression of miR-133b may inactivate the Wnt/β-catenin pathway and ultimately regulate hair growth.

The Role of CCL20-CCR6 Axis in Ovarian Cancer Metastasis.

BackgroundChemokine networks play a key and complex role in tumor progression. CCL20 and its unique receptor CCR6 have been reported to mediate malignant biological activities in various cancers, but their role in ovarian cancer metastasis remains unclear.PurposeOur study aims to explore the effect of CCL20-CCR6 axis on ovarian cancer metastasis and its potential mechanism.MethodsThe transwell assay was used to detect the cell migration and invasion after CCL20 treatment. The CCK-8 assay was used to detect the cell viability after CCL20 treatment and CCR6 depletion. The mRNA and protein expression were assayed through qRT-PCR and Western blotting. The siRNAs and CRISPR-Cas9 system were adopted to suppress CCR6 expression. Intraperitoneal xenograft mouse model was constructed to test the pro-metastasis effect of CCL20-CCR6 axis in vivo. The differentially expressed genes induced by CCL20 were identified through RNA-sequencing, and immunohistochemistry staining was used to detect their protein expression in tumor tissues.ResultsOur results revealed that CCL20 treatment selectively promoted the migration and invasion of CCR6high ovarian cancer cells, but had no effect on CCR6low cells. Blockade of CCR6 expression effectively reversed the cell migration and invasion induced by CCL20 stimulation. Animal experiment proved that CCL20-CCR6 axis mediated ovarian cancer metastasis in vivo. The differentially expressed genes after CCL20 stimulation were associated with metastasis, and CCL20 induced an increased expression of CDH2 and VCAN and decreased CDH1 expression in cancer cells. Moreover, CCL20 stimulated the expression of N-cadherin and versican in tumor tissues and inhibited the expression of E-cadherin, while CCR6 knockout successfully blocked the expression changes.ConclusionOur findings revealed that CCL20-CCR6 axis promotes ovarian cancer metastasis both in vivo and in vitro, probably through increasing cancer cell adhesion and epithelial–mesenchymal transition. Blockade of CCL20-CCR6 axis might become a novel anti-tumor therapeutic target for ovarian cancer.

Fetal alcohol and maternal stress modify the expression of proteins controlling postnatal development of the male rat hippocampus

ABSTRACT Background: Developing brains can partially get over prenatal alcohol exposure-related detrimental conditions by activating some mechanisms involved in survival. Objectives: This study aimed to shed light on the molecular correlates of compensatory mechanisms by examining temporal profiles in the expression of proteins controlling postnatal development in the rat hippocampus prenatally exposed to intubation stress/ethanol. Methods: Male pups were randomly assigned to age subgroups (n = 21/age) which were sacrificed on postnatal day (PD)1, PD10, PD30, and PD60. Ethanol (6 g/kg/day) were intragastrically intubated to the dams throughout 7–21 gestation days. The expression of neurogenesis and angiogenesis markers, extracellular matrix proteins, and growth-promoting ligands were examined by western blot. Results: The most rapid increase in the index of neuronal maturation was noted between PD10-PD30 (p < .05). Prenatal stress caused a decrease of neurogenesis markers at birth and an increase of their expressions at PD10 and PD30 to reach control levels (p < .001). The impact of fetal-alcohol was observed as a decrease in the expression of synaptic plasticity protein versican at birth (p < .001), an increase in the synaptic repulsion protein ephrin-B2 at PD10 (p < .001), and a decrease in the maturation of BDNF at PD30 (p < .001) with a decrease in the mature neuron markers at PD30 (p < .001) and PD60 (p = .005) which were compensated with upregulation of angiogenesis and increasing brevican expression, a neuronal maturation protein (p < .001). Conclusion: These data provide in vivo evidence for the potential therapeutic factors related to neurogenesis, angiogenesis, and neurite remodeling which may tolerate the alcohol/stress dependent teratogenicity in the developing hippocampus.

Abstract 403: Versican proteolysis in endometrial cancer

Abstract Background: Endometrial cancer exhibits differential immunogenicity across molecular subtypes. Specifically, mismatch repair (MMR) deficiency in a subset of endometrial cancers increases mutational load, potentially leading to improved detection of tumor neoantigens within this context. The surrounding tumor microenvironment also plays a role in modulating the immune response to tumor neoantigens. The extracellular matrix protein versican (VCAN) has been characterized as an immunosuppressive molecule that is overexpressed in multiple cancer types, while its cleavage product, versikine (Vkine), has immunostimulatory properties. The objective of this study is to examine the relationship between VCAN proteolysis and CD8+ T cell tumor infiltration in endometrial cancer. Methods: An endometrial cancer tissue microarray (TMA) was developed containing tumor cores from 258 patients. TMA slides were stained via immunohistochemistry. VCAN and Vkine stains were scored on a scale of 0 to 3 based on intensity of stromal staining. Tumor-infiltrating lymphocytes (TILs) was quantified as the number of CD8+ T cells touching malignant epithelial cells 400X magnification. MMR proteins were scored as absent or present in each sample by pathology. Samples were classified into three groups based on strength of proteolysis: high proteolysis = VCAN 0 or 1 and Vkine 3, low proteolysis = VCAN 3 and Vkine 0 or 1. All other samples were put into the intermediate proteolysis group. Results: Proteolysis of VCAN correlates with increased CD8+ TILs in endometrial cancer. The CD8 mean in the proteolytic high group was 4-fold higher than that of the proteolytic low group (16.8 vs 3.6; Wilcoxon rank sum test, p=0.059; n=55 and n=8, respectively). High VCAN proteolysis, as well as mismatch repair deficiency, correlate with high CD8+ T cell infiltration of endometrial tumors; 78% of the MMR deficient samples, and 62% of the proteolysis high samples, were above the median CD8 count of 5.3. Furthermore, our results suggest that endometrial cancer recurrence is associated with increased VCAN expression in both type I endometrioid (estrogen-dependent) and type II non-endometrioid (estrogen-independent) cancers. Of the type I endometrioid cancers, 11% of those expressing VCAN recurred whereas 0% of non-VCAN expressing tumors recurred (n=92). Similarly, of the type II non-endometrioid cancers, 47% of VCAN expressing tumors recurred, whereas 20% of the non-VCAN expressing tumors recurred (n=80). Conclusions: VCAN and its proteolysis correlated with CD8+ TILs in endometrial cancer. These data indicate potential for VCAN as both a prognostic and an immune biomarker. Citation Format: Kristen Moriah Bischel, Philip Emmerich, Tonela Qyli, Stephanie McGregor, Mitchell Depke, Nathaniel Verhagen, Dustin Deming, Philip Emmerich, Nathaniel Verhagen, Tonela Qyli, Stephanie McGregor, Mitchell Depke. Versican proteolysis in endometrial cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 403.

Abstract 3294: PD-L1 status and versican proteolysis predict CD8+ T cell infiltration in esophageal cancer

Abstract Background: Esophageal cancer is a highly lethal cancer with a 20% 5-year overall survival despite standard of care treatment.. Pembrolizumab (anti-PD1, Merck) is FDA-approved for PD-L1 positive esophageal cancer, which comprises roughly 40% of all patients. PD-L1 positive (+) esophageal cancers have previously been shown to demonstrate elevated CD8+ T cell infiltration (CD8+ TILs). Versican (VCAN) is an immunosuppressive extracellular matrix proteoglycan which produces an immunostimulatory fragment, versikine (Vkine), upon proteolytic cleavage by ADAMTS proteases. Previously, VCAN proteolysis has been shown to correlate with enhanced CD8+ TILs in colorectal cancer (CRC), indicating potential for use as an immune biomarker. Methods: A human esophageal cancer tissue microarray (TMA) was developed consisting of multiple samples across 106 representative esophageal cancer patients. Immunohistochemistry (IHC) was used to evaluate VCAN and Vkine expression, and a binning system was used to quantify these stains based on stromal intensity (0: 0-10%, 1: 10-25%, 2:25-50%, 3: 50-100%). IHC was also conducted for CD8, and the number of CD8+ tumor-infiltrating cells per 400x field of view was counted. PD-L1 IHC 22C3 pharmDx was performed and quantified per CPS guidelines for GEJ cancers to quantify PD-L1 status. The VCAN proteolytic predominant (VPP) phenotype was defined as both low VCAN (0 or 1) and high Vkine (2 or 3), and VCAN proteolytic weak phenotype (VPW) was defined as either high VCAN (2 or 3) or low Vkine (0 or 1). Results: Overall, 42.7% of all samples were scored as PD-L1+ (CPS≥1). PD-L1+ samples demonstrated greater CD8+ TILs than PD-L1- samples (44.5±71 vs 14±22; p&amp;lt;0.001). The presence of immunosuppressive VCAN negatively correlated with CD8+ TILs (16.8±27 in VCAN+ samples vs 39.4±67 in VCAN- samples; p&amp;lt;0.01). The VPP phenotype was observed in 47.8% of the cancer samples. VPP samples also demonstrated a trend towards greater CD8+ TILs compared to VPW samples (35.9±63 vs 22.7±46; p=0.09); however, this was not statistically significant. Of all samples, 33.9% were both PD-L1+ and VCAN-. These dual PD-L1+ and VCAN- samples demonstrated even greater CD8+ TILs compared to either PD-L1- or VCAN+ samples (49.5±79 vs 18.1±29; p&amp;lt;0.001). Discussion: Our TMA demonstrates enhanced CD8+ TILs in PD-L1+ samples, as previously reported. Additionally, we discovered the presence of VCAN and its proteolysis are independent predictors of enhanced CD8+ TILs in esophageal cancers. These data indicate the potential importance of the tumor matrix in establishing a permissive tumor microenvironment. These data support the potential for combining VCAN testing with PD-L1 testing to further select appropriate patients for immunotherapeutic trials in esophageal cancer. Citation Format: Phillip B. Emmerich, Jacob Witt, Darya Buehler, Mitchell Depke, Nathaniel Verhagen, Linda Clipson, Kristina Matkowskyj, Nataliya Uboha, Andrew Baschnagel, Dustin Deming. PD-L1 status and versican proteolysis predict CD8+ T cell infiltration in esophageal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3294.

Abstract 4699: Decline in Arylsulfatase B (ARSB) increases PD-L1 expression in melanoma, hepatic, prostate, and mononuclear cells

Abstract Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) is the enzyme that removes 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate. In previous work, we have shown decline in ARSB with increasing aggressiveness of melanoma cell lines and in association with more aggressive prostate cancers. As ARSB decreases, chondroitin 4-sulfate increases, leading to increased binding of SHP2 (PTP11; non-receptor tyrosine phosphatase) and reduced binding of galectin-3. Subsequent effects include increase in phospho-ERK1/2 and transcriptional effects, including hypermethylation of the DKK3 promoter and reduced expression of DKK (Dickkopf WNT pathway signaling inhibitor) 3, leading to activation of Wnt signaling. Other transcriptional events following silencing of ARSB include: increased expression of CSPG4 and MMP-2 in melanoma cells; increased expression of versican in prostate epithelial cells; increased expression of Wnt9A in colonic epithelial cells; and increased MITF-mediated expression of GPNMB in hepatic cells. Invasiveness of cultured melanoma cells increased when ARSB was silenced by siRNA. To further address the role of ARSB, metastatic melanoma tissue was obtained from the biorepository of the University of Illinois at Chicago. Expression of CSPG4 and MMP2 was increased in the metastatic tissues and SHP2 activity was reduced. When ARSB was silenced by siRNA in cultured normal melanocytes (PCS 200-013, ATCC), Programmed Death-Ligand (PD-L)1 increased from 78 ± 7 pg/mg protein to 128 ± 11 pg/mg protein (ELISA assay; p&amp;lt;0.001). In metastatic melanoma cells (1205 Lu), PD-L1 increased from baseline value of 407 ± 34 pg/mg to 462 ± 26 pg/mg when ARSB was silenced. ARSB activity and expression were reduced in the skin metastases, attributable to increased ARSB promoter methylation. In the skin metastases, PD-L1 increased from 103 ± 2.4 pg/mg protein to 457 ± 49 pg/ml protein. In malignant HepG2 cells, when ARSB was silenced, PD-L1 mRNA expression increased to 1.6 times baseline. In human prostate cell lines, including metastatic PC-3 cells, normal prostate epithelial cells and normal prostate stromal cells, PD-L1 mRNA expression increased to 1.8 times, 2.2 times, and 1.6 times the baseline level when ARSB was silenced. In mononuclear cells of prediabetes participants in a dietary study of carrageenan withdrawal, PD-L1 expression declined by 62% in participants on the no-carrageenan diet over a 12-week period, as ARSB activity increased by 34%. These findings indicate that changes in ARSB contribute to the expression of PD-L1 in human cells and can thereby affect the immune checkpoint response. Further studies are required to determine how decline in ARSB and the resulting increase in N-acetylgalactosamine-4-sulfation lead to increased PD-L1 expression and how treatment with recombinant ARSB might affect the immune responses. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Decline in Arylsulfatase B (ARSB) increases PD-L1 expression in melanoma, hepatic, prostate, and mononuclear cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4699.

Abstract 6204: Prostate cancer exosomes alter extracellular matrix composition in bone marrow and enhance metastasis

Abstract Introduction: Exosomes produced by neoplastic cells can promote the development of a pro-tumorigenic microenvironment via intercellular communication, and in some cases promote metastasis. The role of exosomes in the spread of prostate cancer, however, remains poorly understood. Here, we show that exosomes derived from enzalutamide resistant prostate cancer cells (EnzR exos) mediate intercellular communication with bone marrow-resident cells, alter the expression of extracellular matrix (ECM) proteins, and enhance metastasis in a clinically relevant mouse model of metastatic prostate cancer. Methods: Exosomes were isolated via ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells. Mice were primed with exosomes via three systemic injections of 10 ug exosomal protein. Exosome-mediated changes in bone marrow ECM composition were assessed via RNA sequencing of CD11b+ bone marrow cells and immunohistochemistry of formalin-fixed, decalcified, paraffin-embedded mouse femurs and tibias. For metastasis studies, intracardiac injection of luciferase-expressing enzalutamide resistant CWR-R1 cells into C.B.-17 SCID mice was used to establish metastatic tumor burden, and animals were monitored via bioluminescence imaging. Exosomal RNA sequencing was performed on EnzR exos and clinical specimens, with exosomes from normal prostate epithelial cells (PNT2) used as a control. Results: Mice primed with EnzR exos developed significantly enhanced metastatic tumor burden compared to unprimed controls. EnzR exo priming also led to decreased expression of thrombospondin-1 in the bone marrow compartment, and increased expression of versican, as determined by RNA sequencing and immunohistochemistry. Exosomal RNA sequencing revealed that five miRNAs were significantly enriched in EnzR exos compared to PNT2 exosome controls. One of these, miR-4443, was found to target thrombospondin-1. Prostate cancer patient serum samples were also enriched in miR-4443 over healthy controls, and exosomal miR-4443 correlated with disease progression. Conclusions: These data indicate that exosomes derived from enzalutamide resistant prostate cancer cells can alter ECM composition in the bone marrow compartment in a pro-tumorigenic manner. Evidence for the clinical relevance of these data was observed in both serum samples and bone biopsies from prostate cancer patients, demonstrating abundant serum exosomal miR-4443 and similarly altered bone marrow ECM composition in advanced prostate cancer patients. Exosomal miR-4443 may be a promising diagnostic and prognostic marker, as well as a potential therapeutic target. Citation Format: Stephen E. Henrich, Kaylin McMahon, Michael Plebanek, Fabio Tavora, Andre De Souza, Anthony Mega, Benedito Carneiro, C. Shad Thaxton. Prostate cancer exosomes alter extracellular matrix composition in bone marrow and enhance metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6204.

VUp-Regulation of VCAN Promotes the Proliferation, Invasion and Migration and Serves as a Biomarker in Gastric Cancer.

BackgroundVersican (VCAN), a significant protein of extracellular matrix (ECM), is capable of accumulating in tumor stroma and critically impacts malignant transforming process and tumor progressing process. Promoted VCAN expression was identified in numerous malignant tumors and showed relationships to cancer relapse and ineffective breast, prostate, and many other cancer types of patients. Nevertheless, the molecular capability and prognosis importance exhibited by VCAN are infrequently presented in gastric cancer (GC).MethodsAccording to 5 GC tissues and corresponding general tissues, mRNA expression profiles were taken here. VCAN expression in tissues was confirmed by quantitative reverse transcription polymerase reaction (qRT-PCR). The effect generated by VCAN expression on cell proliferating, invading and migrating processes was assessed in vitro with knockdown and overexpression strategies. Moreover, the relationships between immune response and VCAN expression in GC were assessed with the use of the software online.ResultsThere are 181 genes up-regulated and 530 genes down-regulated in GC. According to pathway study, the mentioned differently expressed mRNAs showed correlations with a number of vital physiological processes, cellular components, molecular functions and critical cancer signal pathways. VCAN was reported to be noticeably promoted in GC tissues and related to individual cancer age, race, and stages. VCAN was up-regulated in 16 GC tissues compared to adjacent non-tumorous tissue specimens via qRT-PCR. GC patients exhibiting higher VCAN expression had less post-progression survival (PPS), first progression (FP) and overall survival (OS). Experimental processes in vitro revealed VCAN knockdown hindered, proliferated, invaded, and migrated levels of GC cells, whereas overexpression of VCAN played the opposite effect. Immune factors may interact with VCAN mRNA in GC, and VCAN was found noticeably linked with regulatory T cells (Tregs).ConclusionAccording to the mentioned results, VCAN critically impacts GC progression. Accordingly, VCAN is likely to be a potentially feasible prognosis marking element and a prominent cancer drug for GC patients.

Expression of Versican and Its Related Molecules in Patient with Multiple Myeloma and Its Clinical Significance

To investigate the expression and clinical significant of VCAN and its related molecules in patients with MM. Ficoll density gradient centrifugation method was used to speared the bone marrow mononuclear cell in 25 cases of MM before and after treatment, the relative mRNA expression of VCAN and their related molecules (FAK, FN, MK, and HAS) in bone marrow was detected by real-time quantitative PCR, and their protein expression was determined by Western bolt. The expression of VCAN, FK and FN in the effective group after treatment was significantly lower than that before treatment (P＜0.05), however, the expression of MK and HAS showed no statistically significantly different before and after treatment (P＜0.05). The expression of VCAN of patients in non remission group was significantly higher than that in control group (P＜0.05). The expression of FAK and FN of patients in no remission group was significant increased as compared with the patients in newly diagnosed group (P＜0.05). The relative expression of VCAN mRNA in the patients at 3rd stage was significantly higher than those at the 1st stage (P＜0.05) and control group but showed no significant difference to the patients at 2nd stage (P＜0.05). The expression of VCAN and its related proteins (FAK, MK, FN) showed positively correlation in bone marrow mononuclear cells of MM patients (P＜0.05). The correlation between VCAN and HAS was not statistically significant (r=0.259，P＞0.05). Survival analysis showed that the relative expression of VCAN mRNA was associated with OS (P=0.049) and PFS (P=0.041) in MM patients. VCAN and its related molecules are highly expressed in MM patients; VCAN may act as potential biomarker in the development of multiple myeloma.

Cerebral cavernous malformations are driven by ADAMTS5 proteolysis of versican.

Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.

Genetic reduction of the extracellular matrix protein versican attenuates inflammatory cell infiltration and improves contractile function in dystrophic mdx diaphragm muscles

There is a persistent, aberrant accumulation of V0/V1 versican in skeletal muscles from patients with Duchenne muscular dystrophy and in diaphragm muscles from mdx mice. Versican is a provisional matrix protein implicated in fibrosis and inflammation in various disease states, yet its role in the pathogenesis of muscular dystrophy is not known. Here, female mdx and male hdf mice (haploinsufficient for the versican allele) were bred. In the resulting F1 mdx-hdf male pups, V0/V1 versican expression in diaphragm muscles was decreased by 50% compared to mdx littermates at 20–26 weeks of age. In mdx-hdf mice, spontaneous physical activity increased by 17% and there was a concomitant decrease in total energy expenditure and whole-body glucose oxidation. Versican reduction improved the ex vivo strength and endurance of diaphragm muscle strips. These changes in diaphragm contractile properties in mdx-hdf mice were associated with decreased monocyte and macrophage infiltration and a reduction in the proportion of fibres expressing the slow type I myosin heavy chain isoform. Given the high metabolic cost of inflammation in dystrophy, an attenuated inflammatory response may contribute to the effects of versican reduction on whole-body metabolism. Altogether, versican reduction ameliorates the dystrophic pathology of mdx-hdf mice as evidenced by improved diaphragm contractile function and increased physical activity.

Human Hair Outer Root Sheath Cells and Platelet-Lysis Exosomes Promote Hair Inductivity of Dermal Papilla Cell.

Hair loss is a prevalent medical problem in both men and women. Maintaining the potential hair inductivity of dermal papilla cells (DPCs) during cell culture is the main factor in hair follicle morphogenesis and regeneration. The present study was conducted to compare the effects of different concentrations of human hair outer root sheath cell (HHORSC) and platelet lysis (PL) exosomes to maintain hair inductivity of the human dermal papilla cells (hDPCs). In this study, hDPCs and HHORSCs were isolated from healthy hair samples. Specific markers of hDPCs (versican, α-SMA) and HHORSCs (K15) were evaluated using flow cytometric and immunocytochemical techniques. The exosomes were isolated from HHORSCs and PL with ultracentrifugation technique. Western blot was used to detect specific markers of HHORSCs and PL exosomes. Particle size and distribution of the exosomes were analyzed by NanoSight dynamic light NanoSight Dynamic Light Scattering. Different methods such as proliferation test (MTS assay), migration test (Transwell assay) were used to evaluate the effects of different concentrations of exosomes (2,550,100µg/ml) derived from HHORSC and PL on hDPCs. Expression of specific genes in the hair follicle inductivity, including ALP, versican and α-SMA were also evaluated using real time-PCR. The flow cytometry of the specific cytoplasmic markers of the hDPCs and HHORSCs showed expression of versican (77%), α-SMA (55.2%) and K15 (73.2%). The result of particle size and distribution of the exosomes were analyzed by NanoSight dynamic light NanoSight Dynamic Light Scattering, which revealed the majority of HHORSC and PL exosomes were 30-150nm. For 100µg/ml of HHORSC exosomes, the expressions of ALP, versican and α-SMA proteins respectively increased by a factor of 2.1, 1.7and 1.3 compared to those in the control group. In summary, we applied HHORSC exosomes as a new method to support hair inductivity of dermal papilla cells and improve the outcome for the treatment of hair loss.

Adipocyte-Derived Versican and Macrophage-Derived Biglycan Control Adipose Tissue Inflammation in Obesity

Obesity is characterized by adipose tissue inflammation. Because proteoglycans regulate inflammation, here we investigate their role in adipose tissue inflammation in obesity. We find that adipose tissue versican and biglycan increase in obesity. Versican is produced mainly by adipocytes and biglycan by adipose tissue macrophages. Both proteoglycans are also present in adipose tissue from obese human subjects undergoing gastric bypass surgery. Deletion of adipocyte-specific versican or macrophage-specific biglycan in mice reduces macrophage accumulation and chemokine and cytokine expression, although only adipocyte-specific versican deletion leads to sustained improvement in glucose tolerance. Macrophage-derived biglycan activates inflammatory genes in adipocytes. Versican expression increases in cultured adipocytes exposed to excess glucose, and adipocyte-conditioned medium stimulates inflammation in resident peritoneal macrophages, in part because of a versican breakdown product, versikine. These findings provide insights into the role of adipocyte- and macrophage-derived proteoglycans in adipose tissue inflammation in obesity.

Segmental differences found in aqueous angiographic-determined high - and low-flow regions of human trabecular meshwork

This work sought to compare aqueous angiographic segmental patterns with bead-based methods which directly visualize segmental trabecular meshwork (TM) tracer trapping. Additionally, segmental protein expression differences between aqueous angiographic-derived low- and high-outflow human TM regions were evaluated. Post-mortem human eyes (One Legacy and San Diego eye banks; n = 15) were perfused with fluorescent tracers (fluorescein [2.5%], indocyanine green [0.4%], and/or fluorescent microspheres). After angiographic imaging (Spectralis HRA+OCT; Heidelberg Engineering), peri-limbal low- and high-angiographic flow regions were marked. Aqueous angiographic segmental outflow patterns were similar to fluorescent microsphere TM trapping segmental patterns. TM was dissected from low- and high-flow areas and processed for immunofluorescence or Western blot and compared. Versican expression was relatively elevated in low-flow regions while MMP3 and collagen VI were relatively elevated in high-flow regions. TGF-β2, thrombospondin-1, TGF-β receptor1, and TGF-β downstream proteins such as α-smooth muscle actin were relatively elevated in low-flow regions. Additionally, fibronectin (FN) levels were unchanged, but the EDA isoform (FN-EDA) that is associated with fibrosis was relatively elevated in low-flow regions. These results show that segmental aqueous angiographic patterns are reflective of underlying TM molecular characteristics and demonstrate increased pro-fibrotic activation in low-flow regions. Thus, we provide evidence that aqueous angiography outflow visualization, the only tracer outflow imaging method available to clinicians, is in part representative of TM biology.

Versican G1 Fragment Establishes a Strongly Stabilized Interaction with Hyaluronan-Rich Expanding Matrix during Oocyte Maturation.

In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle. We investigated versican expression in porcine ovarian follicles by real-time (RT)-PCR and western blotting. The aims of the present work were to determine whether 1) versican was produced and cleaved by porcine OCCs during gonadotropin stimulation; 2) these processes were autonomous or required the participation of mural granulosa cells (MGCs); and 3) versican cleavage was involved in the formation or degradation of expanded cumulus ECM. We demonstrate two cleavage products of G1 domain of versican (V1) accumulated in the HA-rich cumulus ECM. One of them, a G1-DPEAAE N-terminal fragment (VG1) of ~70 kDa, was generated from V1 during organization of HA in in vivo and in vitro expanded porcine OCCs. Second, the V1-cleaved DPEAAE-positive form of ~65 kDa was the only species detected in MGCs. No versican cleavage products were detected in OCCs cultured without follicular fluid. In summary, porcine OCCs are autonomous in producing and cleaving V1; the cleaved fragment of ~70 kDa VG1 is specific for formation of the expanded cumulus HA-rich ECM.

Versican expression level in cumulus cells is associated with human oocyte developmental competence

ABSTRACT To study the relationship between the expression of 10 selected genes in cumulus cells and the corresponding oocyte development competence, and the effect of patient age and body mass index on gene expression of cumulus cells, we collected 354 cumulus cell masses associated with individual oocyte from 48 women. The expression levels of the genes involved in glucose metabolism (PFKP, PKM2, LDHA and GFPT) and expansion (HAS2, VCAN, TNFAIP6, PTGS2, PTX3 and SDC4) in cumulus cells were detected by reverse transcription polymerase chain reaction. These were compared among oocyte maturity, fertilization, embryo morphology and implantation, and analyzed the effect of the subject’s age and body mass index. Cumulus cell PFKP expression from mature oocytes was higher than those from immature oocytes (P = 0.014), and VCAN expression was higher from oocytes that developed into high-quality embryos (P = 0.024). TNFAIP6 expression in cumulus cells from fertilized oocytes was lower than that from unfertilized oocytes (P = 0.044). The levels of VCAN, TNFAIP6, PTX3 and SDC4 were changed significantly as a function of the subject’s age and body mass index. In conclusion, the level of VCAN expression in cumulus cells is positively correlated with the early embryo morphology score, and with further development could perhaps be used to evaluate oocyte developmental competence to complement embryonic morphological assessment. Abbreviations CCs: cumulus cells; GDF9: growth differentiation factor 9; BMP15: bone morphogenetic protein 15; PTGS2: prostaglandin synthase 2; HAS2: hyaluronic acid synthase 2; VCAN: versican; GREM1: gremlin 1; PFKP: phosphofructokinase, platelet; PKM2: pyruvate kinase isozyme type M2; LDHA: lactic dehydrogenase; GFPT: glucosaminefructo-6-phosphate transaminase; TNFAIP6: tumor necrosis factor 6 protein; PTX3: penetrin 3; SDC4: syndecan-4; BMI: body mass index; MD: median values; IQR: interquartile range; FSH: follicle-stimulating hormone; LH: luteinizing hormone; HCG: human chorionic gonadotropin; ICSI: intracytoplasmic sperm injection; GnRH: gonadotropin-releasing hormone; hMG: human menopausal gonadotropin; GV: germinal vesicle; M I: metaphase I; M II: metaphase II; cDNA: complementary DNA; SD: standard deviation.