Versican Staining Research Articles

Usefulness of a Nanostructured Fibrin-Agarose Bone Substitute in a Model of Severely Critical Mandible Bone Defect.

Critical defects of the mandibular bone are very difficult to manage with currently available materials and technology. In the present work, we generated acellular and cellular substitutes for human bone by tissue engineering using nanostructured fibrin-agarose biomaterials, with and without adipose-tissue-derived mesenchymal stem cells differentiated to the osteogenic lineage using inductive media. Then, these substitutes were evaluated in an immunodeficient animal model of severely critical mandibular bone damage in order to assess the potential of the bioartificial tissues to enable bone regeneration. The results showed that the use of a cellular bone substitute was associated with a morpho-functional improvement of maxillofacial structures as compared to negative controls. Analysis of the defect site showed that none of the study groups fully succeeded in generating dense bone tissue at the regeneration area. However, the use of a cellular substitute was able to improve the density of the regenerated tissue (as determined via CT radiodensity) and form isolated islands of bone and cartilage. Histologically, the regenerated bone islands were comparable to control bone for alizarin red and versican staining, and superior to control bone for toluidine blue and osteocalcin in animals grafted with the cellular substitute. Although these results are preliminary, cellular fibrin-agarose bone substitutes show preliminary signs of usefulness in this animal model of severely critical mandibular bone defect.

Abstract 1904: Versican production is driven by both epithelial and stromal cells in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to resistance to chemotherapy and immunotherapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma. We investigated the role of two major stromal cell types, pancreatic stellate cells (PSCs) and macrophages, in the production of the immunosuppressive proteoglycan versican (VCAN) as a precursor to the identification of novel mechanisms that may enhance therapeutic response. Methods: B6 KPC mice were utilized as a murine model of PDAC. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissue across 131 patients. Immunohistochemistry (IHC) was used to determine levels of VCAN and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were differentiated from BALB/c mouse femurs and polarized to M1 (antitumor) or M2 (protumor) status. Additionally, PDAC organotypic spheroids were derived from both human and murine tissues whereas PSCs were derived solely from human tissue. Results: IHC analysis of KPC tumors revealed elevated levels of stromal VCAN compared to normal pancreatic tissue (p<0.001, n=20). VCAN accumulation was increased even in the earliest stages of acinar-to-ductal metaplasia in KPC mice. Areas of intense stromal VCAN staining trended toward reduced CD8+ T cell infiltration (4.9 vs 7.3 cells/high power field (hpf), p=0.3). IHC analysis of human PDAC revealed elevated VCAN accumulation across all stages compared to the normal adjacent tissue (n=231, p<0.001). Areas of high VCAN accumulation demonstrated reduced CD8+ T cells compared to areas of low VCAN (0.6 vs 2.9 cells/hpf, p<0.001). To investigate cell types responsible for enhanced VCAN accumulation in the tumor microenvironment, relative expression (RE) of VCAN was compared in vitro to M0 macrophages. Organotypic cancer spheroids demonstrated increased expression of VCAN from KPC mice (RE=49, n=2) and patient-derived PDAC tissue (RE=14, p=0.01, n=3). M1-polarized BMDMs had increased expression of VCAN (RE=24) compared to M2 BMDMs (RE=8) (p<0.001, n=3). Interestingly, M1 BMDMs cultured in PDAC-conditioned media had reduced RE of M1 markers: TNFα (395 vs 37, p=0.03) and iNOS (24723 vs 4813, p=0.003), and increased M2 markers: Arg-1 (127 versus 1049, p=0.02) and YM-1 (0.5 vs 3, p=0.02). PDAC-conditioned media also reduced VCAN expression of M1 BMDMs (24 vs 9, p=0.02). PSCs derived from human PDAC also demonstrated enhanced RE of VCAN compared to negative controls (RE=68, p=0.017) with no significant change in the presence of PDAC conditioned media (p=0.4). Conclusions: The accumulation of VCAN is common in PDAC and correlates with CD8+ T cell exclusion. Epithelial and stromal components are responsible for VCAN production. VCAN deserves further investigation as a target for therapeutic interventions for PDAC. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Chelsie K. Sievers, Connor J. Maloney, Rosabella T. Pitera, Susan N. Payne, Mitchell G. Depke, Cheri A. Pasch, Linda Clipson, Jillian K. Johnson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican production is driven by both epithelial and stromal cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1904.

Abstract 3146: Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer

Abstract Background: Infiltration of CD8+ T cells has shown important prognostic implications in colorectal cancers (CRC). Our group has recently correlated proteolysis of an extracellular matrix proteoglycan, versican (VCAN), with infiltration of CRCs by CD8+ T cells. Cleavage by ADAMTS proteases generates a bioactive fragment of versican, versikine (VKINE). VKINE stimulates Batf3-driven dendritic cell activation and maturation, which are critical for immunotherapy efficacy. Methods: CT26 murine CRC cells were transfected to constitutively express VKINE. BALB/c mice between 50 and 70 days of age were orthotopically injected with either 105 empty vector (CT26-EV) or VKINE-expressing (CT26-VKINE) cells. Tumor growth was tracked weekly with colonoscopy and growth rate assessed using percent lumen occlusion. Mice were euthanized and dissected once moribund. Tissues were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemical (IHC) staining was performed using standard procedures. Additionally, FFPE samples from human CRC liver metastases (n=9) were obtained under the University of Wisconsin Translational Science Biocore Institutional Review Board-approved protocol and IHC performed for VCAN, VKINE and CD8. The number of CD8+ tumor-infiltrating lymphocytes (TILs) per high-powered field (HPF) within the malignant epithelium was calculated using four separate 400x magnification fields of view. Results: CT26-EV and CT26-VKINE injected mice both had a ~70% tumor initiation rate and these tumors averaged ~50% lumen occlusion by 14 days post injection. Survival data for each group were compared using a log-rank test. A trend towards improved survival was observed in those mice injected with CT26-VKINE (average survival of 27 days for CT26-VKINE compared to 22 days for CT26-EV, p=0.41, n=13). The average number of CD8+ TILs for each tumor were compared. For mice with CT26-VKINE, the average number of CD8+ TILs per HPF was 7.6, compared to 4.7 for mice with CT26-EV (p=0.39). Human CRC liver metastases were analyzed for VCAN, VKINE, and CD8+ T cells. Several lesions had little to no VCAN staining, whereas others had intense pockets or peripheral staining. VKINE staining was more abundant in metastatic CRC lesions than in primary lesions. CD8+ T cells were excluded in the setting of increased VCAN staining and infiltration was noted specifically in areas of VCAN proteolysis (low VCAN and high VKINE staining). These areas of proteolysis were more often present at the tumor margin and correlated with ADAMTS1 expression. Conclusions: Immunotherapies offer a unique treatment option for cancer patients, but their use is limited in CRC. VCAN proteolysis and the generation of bioactive VKINE has potential to influence the ability of CD8+ T cells to infiltrate the tumor in both primary and metastatic CRC, indicating the potential to utilize VCAN and VKINE as immune biomarkers or therapeutic targets. Citation Format: Philip B. Emmerich, Susan N. Payne, Connor J. Maloney, Rosabella T. Pitera, Hanna Rainiero, Gioia Sha, Athanasios T. Papadas, Adam C. Pagenkopf, Michael F. Bassetti, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3146.

Abstract 5013: A 3D culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer

Abstract We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated within type-I collagen in 3D. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent malignant properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained the parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), cetuximab strongly inhibited growth of CC, whereas SC was resistant to growth inhibition and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, to cetuximab in SC restored cetuximab sensitivity. To characterize genome-wide divergence between CC and SC, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most upregulated genes in CC was the tumor suppressor 15-PGDH/HPGD and the most upregulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a human CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a new mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic sensitivities and disease-relevant genes for CRC. Citation Format: Bhuminder Singh, Cunxi Li, Ramona Graves-Deal, Haiting Ma, Alina Starchenko, William H. Fry, Yuanyuan Lu, Yang Wang, Galina Bogatcheva, Mohseen P. Khan, Ginger L. Milne, Shilin Zhao, Gregory D. Ayers, Nenggan Li, Mary K. Washington, Timothy J. Yeatman, Oliver G. McDonald, Qi Liu, Robert J. Coffey. A 3D culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5013. doi:10.1158/1538-7445.AM2017-5013

Versican proteolysis as a key regulator of CD8+ T-cell infiltration in colorectal cancer.

e15082 Background: Colorectal cancer (CRC) originates within immunologically complex microenvironments. To date the benefits of immunotherapy have been modest except in neoantigen-laden mismatch repair (MMR)-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes (TILS) in the tumor bed may substantially augment clinical immunotherapy responses. Proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) generates a bioactive fragment, versikine, with putative immunostimulatory activities. Methods: Matched normal and CRC tissue samples were collected from 122 patients with cancers across all stages and locations throughout the colon and rectum. These samples were stained for VCAN, αDPEAAE (neoepitope generated in cleaving VCAN to versikine), and CD8 and scored by a pathologist. Tumors were classified as VCAN proteolysis-predominant (VPP) if their staining for total VCAN staining intensity was < 1+ and staining for VCAN proteolysis (αDPEAAE antibody) was > 2. Conversely, tumors were classified as VCAN proteolysis-weak (VPW) if intact VCAN staining intensity was > 1+ or αDPEAAE intensity was < 2+. IHC for mismatch repair (MMR) proteins was also performed. Results: Overall increased VCAN staining was observed in cancer versus (vs) normal tissue. VPP tumors had a 10 fold greater infiltration of CD8+ T-cells vs VPW cancers (p < 0.001). The correlation between VCAN proteolysis and CD8+ T-cell infiltration was maintained in both cancers with proficient (p) MMR and deficient (d) MMR. In both pMMR and dMMR, the VPP tumors had the greatest degree of CD8+ T-cell infiltration (Wilcoxon rank sum tests: pMMR p = 0.006; dMMR p = 0.03). Among the VPP tumors there was a greater degree of CD8+ T cell infiltration in the dMMR cancers vs pMMR cancers (35 versus 14.8 TILs per high power filed, p = 0.04). Nuclear CTNNB1, a marker for activation of WNT signaling, negatively correlated with CD8+ T cell infiltration( p = 0.014). In addition, VCAN accumulation correlated with the presence of nuclear CTNNB1 (p < 0.001) Conclusions: This is the first description indicating that VCAN proteolysis may shape CRC immune contexture and provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker.

Lumican and versican protein expression are associated with colorectal adenoma-to-carcinoma progression.

BackgroundOne prominent event associated with colorectal adenoma-to-carcinoma progression is genomic instability. Approximately 85% of colorectal cancer cases exhibit chromosomal instability characterized by accumulation of chromosome copy number aberrations (CNAs). Adenomas with gain of chromosome 8q, 13q, and/or 20q are at high risk of progression to cancer. Tumor progression is also associated with expansion of the extracellular matrix (ECM) and the activation of non-malignant cells within the tumor stroma. The glycoproteins versican and lumican are overexpressed at the mRNA level in colon carcinomas compared to adenomas, and are associated with the formation of tumor stroma.PurposeThe aim of this study was to characterize versican and lumican protein expression in tumor progression and investigate their association with CNAs commonly associated with adenoma-to-carcinoma progression.MethodsTissue microarrays were constructed with colon adenomas and carcinomas that were characterized for MSI-status and DNA copy number gains of chromosomes 8q, 13q and 20q. Sections were immunohistochemically stained for lumican and versican. Protein expression levels were evaluated using digitized slides, and scores were finally dichotomized into a positive or negative score per sample.ResultsLumican and versican expression were both observed in neoplastic cells and in the tumor stroma of colon adenomas and carcinomas. Lumican expression was more frequently present in epithelial cells of carcinomas than adenomas (49% versus 18%; P = 0.0001) and in high-risk adenomas and carcinomas combined compared to low-risk adenomas (43% versus 16%; P = 0.005). Versican staining in the tumor stroma was more often present in high-risk adenomas combined with carcinomas compared to low-risk adenomas (57% versus 36%; P = 0.03) and was associated with the presence of gain of 13q (71% versus 44%; P = 0.04).ConclusionEpithelial lumican and stromal versican protein expression are increased during colorectal adenoma-to-carcinoma progression.

Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

Versican accumulates in vascular lesions in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a lethal condition for which there is no effective curative pharmacotherapy. PAH is characterized by vasoconstriction, wall thickening of pulmonary arteries, and increased vascular resistance. Versican is a chondroitin sulfate proteoglycan in the vascular extracellular matrix that accumulates following vascular injury and promotes smooth-muscle cell proliferation in systemic arteries. Here, we investigated whether versican may play a similar role in PAH. Paraffin-embedded lung sections from patients who underwent lung transplantation to treat PAH were used for immunohistochemistry. The etiologies of PAH in the subjects involved in this study were idiopathic PAH, scleroderma, and congenital heart disease (atrial septal defect) with left-to-right shunt. Independent of the underlying etiology, increased versican immunostaining was observed in areas of medial thickening, in neointima, and in plexiform lesions. Western blot of lung tissue lysates confirmed accumulation of versican in patients with PAH. Double staining for versican and CD45 showed only occasional colocalization in neointima of high-grade lesions and plexiform lesions. In vitro, metabolic labeling with [(35)S]sulfate showed that human pulmonary artery smooth-muscle cells (hPASMCs) produce mainly chondroitin sulfate glycosaminoglycans. In addition, hypoxia, but not cyclic stretch, was demonstrated to increase both versican messenger RNA expression and protein synthesis by hPASMCs. Versican accumulates in vascular lesions of PAH, and the amount of versican correlates more with lesion severity than with underlying etiology or inflammation. Hypoxia is a possible regulator of versican accumulation, which may promote proliferation of pulmonary smooth-muscle cells and vascular remodeling in PAH.

Subepithelial Accumulation of Versican in a Cockroach Antigen-Induced Murine Model of Allergic Asthma.

The extracellular matrix (ECM) is an important contributor to the asthmatic phenotype. Recent studies investigating airway inflammation have demonstrated an association between hyaluronan (HA) accumulation and inflammatory cell infiltration of the airways. The ECM proteoglycan versican interacts with HA and is important in the recruitment and activation of leukocytes during inflammation. We investigated the role of versican in the pathogenesis of asthmatic airway inflammation. Using cockroach antigen (CRA)-sensitized murine models of allergic asthma, we demonstrate increased subepithelial versican in the airways of CRA-treated mice that parallels subepithelial increases in HA and leukocyte infiltration. During the acute phase, CRA-treated mice displayed increased gene expression of the four major versican isoforms, as well as increased expression of HA synthases. Furthermore, in a murine model that examines both acute and chronic CRA exposure, versican staining peaked 8 days following CRA challenge and preceded subepithelial leukocyte infiltration. We also assessed versican and HA expression in differentiated primary human airway epithelial cells from asthmatic and healthy children. Increases in the expression of versican isoforms and HA synthases in these epithelial cells were similar to those of the murine model. These data indicate an important role for versican in the establishment of airway inflammation in asthma.

Reprint of: A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

Molecular composition of extracellular matrix in the vestibular nuclei of the rat

Previous studies have demonstrated that the molecular and structural composition of the extracellular matrix (ECM) shows regional differences in the central nervous system. By using histochemical and immunohistochemical methods, we provide here a detailed map of the distribution of ECM molecules in the vestibular nuclear complex (VNC) of the rat. We have observed common characteristics of the ECM staining pattern in the VNC and a number of differences among the individual vestibular nuclei and their subdivisions. The perineuronal net (PNN), which is the pericellular condensation of ECM, showed the most intense staining for hyaluronan, aggrecan, brevican and tenascin-R in the superior, lateral and medial vestibular nuclei, whereas the HAPLN1 link protein and the neurocan exhibited moderate staining intensity. The rostral part of the descending vestibular nucleus (DVN) presented a similar staining pattern in the PNN, with the exception of brevican, which was negative. The caudal part of the DVN had the weakest staining for all ECM molecules in the PNN. Throughout the VNC, versican staining in the PNN, when present, was distinctive due to its punctuate appearance. The neuropil also exhibited heterogeneity among the individual vestibular nuclei in ECM staining pattern and intensity. We find that the heterogeneous distribution of ECM molecules is associated in many cases with the variable cytoarchitecture and hodological organization of the vestibular nuclei, and propose that differences in the ECM composition may be related to specific neuronal functions associated with gaze and posture control and vestibular compensation.

Versican expression in canine carcinomas in benign mixed tumours: is there an association with clinical pathological factors, invasion and overall survival?

BackgroundComponents of the extracellular matrix have been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The presence of the proteoglycan versican has been strongly associated with cancer development and progression. However, relationship between versican expression and clinical pathological factors and overall survival has not been previously studied in veterinary medicine. Carcinomas in benign mixed tumours (CBMTs) are one of the most common malignant tumours in female canines and can serve as models for studies of tumour progression. The aim of this study was to evaluate the expression of versican in in situ and invasive carcinomatous areas of canine CBMTs and to evaluate possible associations of versican expression with other classic prognostic factors and overall survival.ResultsClinical staging; histological grade determination; immunohistochemical staining for versican, E-cadherin and Ki-67; and confirmation of invasion areas by staining for p63 and smooth muscle α-actin (α-SMA) were performed on 49 canine cases of CBMT. Tumour invasion was considered when suspicious Haematoxylin-Eosin (HE)-stained areas showed a total loss of α-SMA and p63 immunoreactivity. Versican immunoreactivity was less intense in the areas adjacent to the in situ carcinomatous regions, compared to invasive regions, which showed extensive and strong staining.ConclusionsOur data reveal that in canine CBMTs, versican expression differs significantly between invasive and in situ areas, suggesting a role for this molecule in tumour progression. Although a direct relationship exists between versican and invasiveness, our results indicate that the isolated evaluation of this proteoglycan does not represent an independent prognostic factor in canine CBMTs.

Gene and Protein Expression Alterations in the Myocardium following Mechanical Circulatory Assist in Heart Failure Patients

Purpose: The number of patients suffering from end-stage heart failure (HF) continues to mount alongside an aging demographic. Management of the failing heart involves medical therapy, mechanical circulatory assist (MCA) devices and ultimately heart transplantation. Unloading of the heart by MCA may induce reverse remodeling and normalize cardiac parameters (chamber geometry, heart size, ventricular volume, ejection fraction, fetal gene expression). We undertook the identification of markers within the heart correlating with patient response to MCA, and of changes in gene and protein expression in the myocardium following MCA. Methods: Myocardial tissue cores removed during left ventricular assist device (LVAD) implantation, and explanted hearts obtained at the time of cardiac transplantation are archived for flash frozen, paraffin-fixed formalin embedded (PPFE), RNAlater and OCT preserved samples. Serial sections from PPFE samples of left ventricular (LV) apical tissue cores were prepared with standard histology stains (H&E, Movat's pentachrome and picrosirius red) for quantification of fibrosis and degree of myocardial remodeling. Immunohistochemical staining of markers of myocardial dysfunction and remodeling (brain natriuretic peptide (BNP), galectin-3, versican, matrix metalloproteinases (MMP)-2 and -9, tissue inhibitor of metalloproteinases (TIMP)-1 were performed. RNA extracted from LV apical tissue cores was analyzed on Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Differential staining, gene expression and degree of remodeling were quantitated and correlated with patient response (non-reponders: death, cardiac transplantation; responders: recovery with explantation of device). Comparison of gene and protein signatures from LV core samples at time of MCA initiation with myocardial tissue samples from the LV in matched explanted hearts was also performed. Results and Conclusions: Increased galectin&-3 and versican staining was seen in patients who did not respond to MCA, suggesting that activation of fibrosing pathways may impair the ability of the myocardium to recover despite chronic unloading. Differential gene expression of MMP-2 and -9 was observed in patients who subsequently required cardiac transplantation following MCA. Differential gene and protein expression was also seen in explanted hearts following MCA when compared to initial LV cores removed during MCA device implantation.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

BackgroundRemodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.MethodsUteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR.ResultsIn diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus.ConclusionThese results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.

Expression and localization of versican during postnatal development of rat temporomandibular joint disc

To analyze the growth-related changes in extracellular matrix components in temporomandibular joint (TMJ) discs, the expression and localization of the core protein of a large chondroitin sulphate proteoglycan, versican, in rat TMJ discs during postnatal development (2-32 weeks) were examined using Western blot analysis, real-time quantitative PCR and immunohistochemistry. Western blot analysis showed that rat TMJ discs predominantly expressed one isoform (V1) and the core protein sharply increased after birth, reached a peak at 8 weeks, and then gradually decreased up to 32 weeks. Real-time quantitative PCR with TaqMan probes indicated that mRNA expression of versican was highest at 2 weeks and gradually decreased with growth. An immunohistochemical study showed that staining for versican was weak and evenly distributed in TMJ discs at 2 weeks. Regional differences in staining for versican became prominent after 8 weeks; staining was intense in the anterior and posterior peripheral attachments, and weak in the central part of the discs. These results demonstrate that growth-related changes and regional differences exist in the expression of versican in the TMJ discs of growing rats, and these probably reflect the changes in the biomechanical environment caused by the development of orofacial functions.

Versican in nonsmall cell lung cancer: relation to hyaluronan, clinicopathologic factors, and prognosis

The aim of the study was to investigate the expression and prognostic role of versican in 212 patients with resected nonsmall cell lung cancer. Tumor samples were stained immunohistochemically, and the versican staining was evaluated both in tumor stroma and cancer cells. The staining results were compared to the clinical data of the patients, the tumor cell proliferation, and the expression of hyaluronan. In the whole material, low and high area percentages of stromal versican staining were observed in 135 and 77 carcinomas, respectively. Tumor cell-associated staining signal for versican was observed in 33 cases. In the whole material, the significant relationship between high stromal staining of versican and that of hyaluronan was noticed (P = .001). The expression of stromal versican was related to tumor type (P = .008) and high stromal staining was inversely correlated with poor tumor differentiation (P = .045), but not with tumor cell proliferation. Among adenocarcinomas, the high stromal staining of versican was associated with tumor recurrence (P = .024), higher tumor stage (P = .022), and lymph node metastases (P = .042). Versican expression was not related to patient outcome in the whole material, but among adenocarcinomas, the high stromal staining was related to poor disease-free survival (P = .0056). However, in Cox multivariate analysis with tumor stage, versican expression did not retain its prognostic significance. The results indicate that increased stromal versican is related to higher tumor recurrence rate and more advanced disease. Despite the important role of versican in nonsmall cell lung cancer, traditional clinicopathologic factors remained most significant in the current study.

Versican expression in pharyngeal squamous cell carcinoma: an immunohistochemical study

Aims: To study the expression of versican, a large proteoglycan involved in repressing adhesion between cells and the extracellular matrix in pharyngeal squamous cell carcinoma (PSCC), and its relation to...

Versican in epithelial ovarian cancer: Relation to hyaluronan, clinicopathologic factors and prognosis

Versican, a proteoglycan previously reported to increase in other malignant tumours, was studied immunohistochemically in 299 primary epithelial ovarian cancers, their 43 metastases and 6 normal ovaries to evaluate its prognostic value and relation to hyaluronan, another extracellular matrix molecule increased in cancer and a binding partner of versican. The stainings were scored according to the area percentage of strong versican signal of total peri- and intratumoural stroma as low (<15%) or high (>or=15%). Epithelial staining of the tumours was scored as positive or negative. Low and high area percentage of strong stromal versican staining were observed in 133 and 166 carcinomas, respectively. A low area percentage of strong stromal versican staining correlated with mucinous histology (p = 0.019) and early International Federation of Gynecologists and Obstetritians (FIGO) stage (p < 0.0005), whereas a high percentage was associated with reduced 5-year survival rate of the patients (44% vs. 32%; p = 0.032). Versican was associated with the cancer cells in 151 tumours and correlated with clear cell histology (p < 0.0005), early FIGO stage (p = 0.049) and increased recurrence-free survival (63% vs. 47%; p = 0.032). However, in Cox's multivariate analyses with the conventional prognostic factors included, neither stromal nor cancer cell-associated versican reached a significant prognostic value. Versican is thus enriched in the malignant stroma surrounding and promoting the growth of ovarian cancer, probably acting with hyaluronan, and associates with unfavourable prognosis but does not constitute an independent indicator of patient survival.