**Expressional Alterations of Versican, Hyaluronan and Microfibril Associated Proteins in the Cancer Microenvironment**

Hiroko Kuwabara, Masahiko Yoneda and Zenzo Isogai

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55067

### **1. Introduction**

152 Carcinogenesis

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[106] Kedersha NL, Gupta M, Li W, Miller I, Anderson P (1999) RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian

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The extracellular matrix (ECM) is composed of fibronectin, collagen, elastic fibers and microfibrils with many associated adaptor proteins and proteoglycans. The microenvironments of cancer tissues differ from those of normal tissues, and intimately communicate with the cancer cell surface. For example, in breast cancer stroma compared with a normal breast lobule, 2,338 genes are upregulated and 1,234 genes are downregulated in ductal carcinoma *in situ*, and a further 76 genes are upregulated and 229 genes are downregulated in invasive carcinoma [1]. Histologically, smooth muscle actin-positive fibroblasts appear in the cancer stroma. Cancer cells and their microenvironments seem to influence each other during early carcinogenesis, and the microenvironments appear to play important roles in cancer development. The tumor microenvironment is unique in containing higher amounts of versican and hyaluronan, and lower amounts of latent transforming growth factor β-binding proteins. These stromal changes in hyaluronanbinding proteins and microfibril-associated molecules may facilitate cancer spreading, and targeting of specific components of the cancer stroma could lead to effective anticancer therapies. In this chapter, we review the dynamics of ECM proteins such as versican, hyaluronan, fibrillin-1 and latent transforming growth factor β-binding proteins in cancer tissues. In addition, hyaluronan-binding proteins are discussed.

### **2. Versican**

Versican is a type of ECM proteoglycan that was initially identified in cultured human fibroblasts, followed by isolation of its chicken homolog, PG-M, from chondrogenic condensation areas of developing limb buds [2]. Versican is a macromolecule composed of a

© 2013 Kuwabara et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

specific core protein and covalently linked glycosaminoglycan chains named chondroitin sulfate (CS), which are linear, negatively charged polysaccharides comprising repeating disaccharides of acetylated hexosamines [2,3]. It is produced by cancer, lymphoma and leukemia cells, as well as activated peritumoral fibroblasts. Imunohistochemical staining with an anti-versican antibody (clone; 2B1 [4]) showed that versican is accumulated in the stroma of cancers such as melanoma, breast cancer and ovarian cancer (Figure 1). Versican regulates cell adhesion, proliferation, migration, survival, differentiation and angiogenesis, and its expression is increased with higher tumor grade and worse outcome [3].

Expressional Alterations of Versican,

Hyaluronan and Microfibril AssociatedProteins in the Cancer Microenvironment 155

cccagtgtggaggtggtctac-3' and 5'-ctcaaatcactcattcgacgtt-3'; V2, 5' tcagagaaaataagacaggacctgatc-3', and 5'-catacgtaggaagtttcagtaggataaca-3'; and V3, 5' ccctccccctgatagcagat-3' and 5'-ggcacgggttcattttgc-3'. Real-time quantitative RT-PCR was performed using a Thermal Cycler Dice Real Time System (Takara Biochemicals, Shiga, Japan). Each PCR mixture contained 12.5 μl of Master Mix (SYBR Premix ExTaqTMII; Takara Biochemicals), 0.01 mol/l forward and reverse primers, and 100 ng of first-strand cDNA in deionized water. After heating each sample to 95℃ for 10 s, 45 cycles of 95℃ for 5 s and 60 ℃ for 30 s were performed. The fluorescence was measured as a function of temperature. All samples were run in triplicate, and PCR amplifications of the GAPDH and target genes were run in parallel for each sample. The results were analyzed by the Thermal Cycler Dice Real Time System TP800 software, version 2.00. To obtain estimates of the mRNA expression levels of the target genes by real-time RT-PCR, the GAPDH mRNA expression level was given a value of 1.0 and used to normalize the other mRNA expression levels. The cancer and normal tissues expressed all four versican isoforms, with the V1 isoform showing the most abundant expression in both tissues (0.18±0.14 in cancer tissues and 0.032±0.028 in normal tissues) [7]. In the cancer tissues, the mRNA levels of V0 and V1 were increased by about 12 fold and 6-fold, respectively, compared with the normal tissues. The mRNA levels of V2 and V3 were very low in the normal tissues, and did not change in the cancer tissues. The versican V1-transfected Swarm rat chondrosarcoma cells show enhanced cell motility and migration, and produce tumors with more spindle-shaped cells and more myxomatous stroma in SD rats [8]. These findings suggest that versican V1 enhances the invasive capacity and leads to the formation of a cell-associated matrix in the cancer tissues. V2 has the opposite functions to V1, by inhibiting cell proliferation and lacking any association with apoptotic resistance, whereas

V3 expression has a dual role in tumor growth and metastasis [9,10].

**Figure 2.** Schematic representation of human versican isoforms and domains.

**Figure 1.**  Versican is expressed in cancer stroma (x200).

Versican can be expressed as one of four splice variants (Figure 2) [5]. The amino- and carboxy-terminal globular domains (G1 and G3, respectively) are present in all isoforms, which differ in whether they contain two alternatively spliced CS-attachment regions (CS-α and CS-β). Specifically, versican V1 contains only the CS-β region, V2 contains only the CS-α region, V0 contains both CS regions and V3 contains neither of the CS regions. Each versican isoform has different functional roles. The V1 isoform enhances cell proliferation and protects NIH3T3 mouse fibroblasts against apoptosis [6]. We investigated the alterations in versican isoforms in breast cancer tissues in comparison with matched normal tissues using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) [7]. Total RNA was isolated using an SV total RNA isolation system (Promega, Madison, WI). First-strand cDNA was synthesized from the total RNA using a SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA), and amplified using specific primers for versican V0, V1, V2 and V3. The housekeeping gene glyceraldhyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control. The designed primers were as follows: V0, 5'-gcacaaaatttcaccctgacatt-3' and 5'-cttctttagattctgaatctattggatgac-3'; V1, 5'-

cccagtgtggaggtggtctac-3' and 5'-ctcaaatcactcattcgacgtt-3'; V2, 5' tcagagaaaataagacaggacctgatc-3', and 5'-catacgtaggaagtttcagtaggataaca-3'; and V3, 5' ccctccccctgatagcagat-3' and 5'-ggcacgggttcattttgc-3'. Real-time quantitative RT-PCR was performed using a Thermal Cycler Dice Real Time System (Takara Biochemicals, Shiga, Japan). Each PCR mixture contained 12.5 μl of Master Mix (SYBR Premix ExTaqTMII; Takara Biochemicals), 0.01 mol/l forward and reverse primers, and 100 ng of first-strand cDNA in deionized water. After heating each sample to 95℃ for 10 s, 45 cycles of 95℃ for 5 s and 60 ℃ for 30 s were performed. The fluorescence was measured as a function of temperature. All samples were run in triplicate, and PCR amplifications of the GAPDH and target genes were run in parallel for each sample. The results were analyzed by the Thermal Cycler Dice Real Time System TP800 software, version 2.00. To obtain estimates of the mRNA expression levels of the target genes by real-time RT-PCR, the GAPDH mRNA expression level was given a value of 1.0 and used to normalize the other mRNA expression levels. The cancer and normal tissues expressed all four versican isoforms, with the V1 isoform showing the most abundant expression in both tissues (0.18±0.14 in cancer tissues and 0.032±0.028 in normal tissues) [7]. In the cancer tissues, the mRNA levels of V0 and V1 were increased by about 12 fold and 6-fold, respectively, compared with the normal tissues. The mRNA levels of V2 and V3 were very low in the normal tissues, and did not change in the cancer tissues. The versican V1-transfected Swarm rat chondrosarcoma cells show enhanced cell motility and migration, and produce tumors with more spindle-shaped cells and more myxomatous stroma in SD rats [8]. These findings suggest that versican V1 enhances the invasive capacity and leads to the formation of a cell-associated matrix in the cancer tissues. V2 has the opposite functions to V1, by inhibiting cell proliferation and lacking any association with apoptotic resistance, whereas V3 expression has a dual role in tumor growth and metastasis [9,10].

154 Carcinogenesis

specific core protein and covalently linked glycosaminoglycan chains named chondroitin sulfate (CS), which are linear, negatively charged polysaccharides comprising repeating disaccharides of acetylated hexosamines [2,3]. It is produced by cancer, lymphoma and leukemia cells, as well as activated peritumoral fibroblasts. Imunohistochemical staining with an anti-versican antibody (clone; 2B1 [4]) showed that versican is accumulated in the stroma of cancers such as melanoma, breast cancer and ovarian cancer (Figure 1). Versican regulates cell adhesion, proliferation, migration, survival, differentiation and angiogenesis,

and its expression is increased with higher tumor grade and worse outcome [3].

**Figure 1.**  Versican is

Versican can be expressed as one of four splice variants (Figure 2) [5]. The amino- and carboxy-terminal globular domains (G1 and G3, respectively) are present in all isoforms, which differ in whether they contain two alternatively spliced CS-attachment regions (CS-α and CS-β). Specifically, versican V1 contains only the CS-β region, V2 contains only the CS-α region, V0 contains both CS regions and V3 contains neither of the CS regions. Each versican isoform has different functional roles. The V1 isoform enhances cell proliferation and protects NIH3T3 mouse fibroblasts against apoptosis [6]. We investigated the alterations in versican isoforms in breast cancer tissues in comparison with matched normal tissues using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) [7]. Total RNA was isolated using an SV total RNA isolation system (Promega, Madison, WI). First-strand cDNA was synthesized from the total RNA using a SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA), and amplified using specific primers for versican V0, V1, V2 and V3. The housekeeping gene glyceraldhyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control. The designed primers were as follows: V0, 5'-gcacaaaatttcaccctgacatt-3' and 5'-cttctttagattctgaatctattggatgac-3'; V1, 5'-

expressed in cancer stroma (x200).

**Figure 2.** Schematic representation of human versican isoforms and domains.

Globular domains G1 and G3 include a hyaluronan binding portion and epidermis growth like repeats, respectively, and they play a crucial role in the function of versican [2,3]. For these reasons, G1 and G3 in breast cancer and non-cancerous lesions were compared by dot blot analyses [7]. Frozen samples (200 mg) were minced into small pieces using a Tissue Grinder System (Biomedical Polymers, Gardener, MA), homogenized in chilled lysis buffer comprising 4 M guanidine-HCl and 1 % protease inhibitor cocktail (Sigma, St. Louis, MO), and extracted at 4℃ for 48 h. After centrifugation, the supernatants were dialyzed against 50 mM Tris-HCl (pH 8.0) and 0.15 M NaCl at 4℃ overnight using Slide-A-Lyzer MINI Dialysis Units (Pierce, Rockford, IL). The protein contents in the lysates were measured using a Micro BCA Protein Assay Reagent Kit (Pierce), and aliquots containing 30 μg of protein were immobilized on nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK) using a PR648 Slot Blot Manifold dot blot apparatus (GE Healthcare). The membranes were blocked with 10 % non-fat milk, and incubated with the 6084 antibody recognizing G1 [11] or the 2B1 antibody recognizing G3 [4]. In the breast cancer tissues, the staining intensities with the 6084 and 2B1 antibodies were increased by about two-fold and four-fold, respectively, compared with the non-cancerous tissues [7]. Immunohistochemical staining supported these findings.

Expressional Alterations of Versican,

Hyaluronan and Microfibril AssociatedProteins in the Cancer Microenvironment 157

an important role in tissue remodeling during development [16,17]. The high concentrations of HA in embryonic tissues are correlated with their rates of cell migration and proliferation, and HA accumulation induces epithelial-mesenchymal transition, at regions such as the future valve sites of the embryonic heart tube. In addition, HA can bind large amounts of water, and form viscous gels at relatively low concentrations. HA is synthesized by HA synthase (HAS) enzymes at the cytoplasmic face of the plasma membrane, and its secretion takes place by extrusion during polymer elongation [18]. HAS enzymes have three different isoforms, and HAS-2 and HAS-3 are common in cancer tissues [19]. HAS-2 in breast cancer stem-like cells (CSCs) promotes tumor progression in bone by stimulating the interactions of CSCs with macrophages and stromal cells [20]. HA plays a critical role in generating a favorable microenvironment by promoting the interactions of macrophages and CSCs, and

HA synthesis is correlated with the level of HAS mRNA, suggesting that transcriptional regulation is important [21]. In addition to measuring the HAS mRNA levels, several different methods have been utilized for detecting HA. Histologically, the expression of HA was evaluated by staining with biotinylated HA-binding protein (5 μg/ml) prepared from bovine nasal cartilage. Another method used was HA-dependent pericellular coat assays. Cells were cultured in dishes, and the medium was then replaced with phosphate-buffered saline containing 1 x 1016 formalin-fixed horse erythrocytes. After 20 min, the dishes were

HA is rich in cancer stroma, including that of lung, bladder, prostate, colon and breast cancers (Figure 3), and stromal HA accumulation is typical for high-grade and poorly differentiated carcinoma [23]. An HA rich matrix supports tumor growth and spreading by regulating cell proliferation, and migration or by enhancing tumor angiogenesis in the cancer. In breast cancer in transgenic mice, HA overproduction accelerates tumor angiogenesis through stromal reactions, most notably in the presence of versican [24]. On the other hand, the HAS suppressor, 4-methylumbelliferone (MU), suppresses cell adhesion, locomotion, and matrix metalloproteinase (MMP)-9 and N-cadherin expression, and inhibits tumor metastasis [25-27]. HA is composed of high and low molecular weight forms that have been shown to have opposite functions. High- molecular-weight HA suppresses vascularization, and has anti-inflammatory and immunosuppressive functions. Lowmolecular-weight HA, including HA oligosaccharides (less than 50 oligomers (mers)), has an angiogenic function, and induces cytokines and chemokines in inflammation and ECM degradation [28]. HA changes its biological activities depending on its molecular weight, and HA oligosaccharides (12 mers) suppress the growth of breast cancer [29]. MU and HA

HA is present not only in the ECM, but also in the cellular cytoplasm and nucleus. Although the mechanism for HA internalization remains controversial, one possibility is that HA is transported in a retrograde manner through the Golgi, endoplasmic reticulum and cytoplasm. HA accumulation is seen in the cytoplasm of malignant cells, such as colon and gastric cancer cells and lymphoma cells. A high level of HA on tumor cells is a strong indicator of an unfavorable outcome [30]. The intracellular HA-binding proteins include

observed and areas without erythrocytes were considered to be HA [22].

oligosaccharides may be candidates for anticancer therapies.

HAS-2 may promote cancer cell proliferation.

The G1 and G3 domains are thought to be related to the proliferation and migration of cells. For example, the G1 domain stimulates cell migration and proliferation by destabilizing cell adhesion in NIH3T3 cells and astrocytoma cells [12,13]. The G3 domain promotes breast cancer cell proliferation, invasion and bone metastasis and enhances cancer apoptosis, by upregulating the epidermal growth factor receptor (EGFR)-mediating signaling pathway. Activation of phosphorylated extracellular regulated protein kinases (ERKs) is correlated with high levels of G3 expression [14]. The G3 domain contains two epidermal growth factor (EGF)–like motifs, and the activated EGF-EGFR-ERK pathway plays important roles in cell cycle progression and apoptosis.

Versican is cleaved by proteases, which play important roles in tissue remodeling and modulation of cell microenvironments through degradation of the ECM and processing of growth factors and adhesion molecules. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteins, a family of proteases, are composed of the following seven domains: signal peptide, prodomain, metalloproteinase domain, disintegrin domain, thrombospondin type 1 motif, cysteine-rich domain and spacer region. Versican is cleaved by ADAMTS-1 and ADAMTS-4 at Glu441-Ala442, and a 70-kDa neoepitope DPEAAE sequence is generated. ADAMTS-1 in breast cancer cells can promote their growth and metastasis through local accumulation of versican fragments and angiogenesis [15]. This indicates that the concentration of versican is influenced by the cleavage as well as the production, and ADAMTS plays a role in the cancer development.

### **3. Hyaluronan and receptors for hyaluronan-mediated motility**

Hyaluronan (HA) is a nonsulfated glycosaminoglycan of repeating D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units. It is a major constituent of the ECM, and plays an important role in tissue remodeling during development [16,17]. The high concentrations of HA in embryonic tissues are correlated with their rates of cell migration and proliferation, and HA accumulation induces epithelial-mesenchymal transition, at regions such as the future valve sites of the embryonic heart tube. In addition, HA can bind large amounts of water, and form viscous gels at relatively low concentrations. HA is synthesized by HA synthase (HAS) enzymes at the cytoplasmic face of the plasma membrane, and its secretion takes place by extrusion during polymer elongation [18]. HAS enzymes have three different isoforms, and HAS-2 and HAS-3 are common in cancer tissues [19]. HAS-2 in breast cancer stem-like cells (CSCs) promotes tumor progression in bone by stimulating the interactions of CSCs with macrophages and stromal cells [20]. HA plays a critical role in generating a favorable microenvironment by promoting the interactions of macrophages and CSCs, and HAS-2 may promote cancer cell proliferation.

156 Carcinogenesis

supported these findings.

cycle progression and apoptosis.

Globular domains G1 and G3 include a hyaluronan binding portion and epidermis growth like repeats, respectively, and they play a crucial role in the function of versican [2,3]. For these reasons, G1 and G3 in breast cancer and non-cancerous lesions were compared by dot blot analyses [7]. Frozen samples (200 mg) were minced into small pieces using a Tissue Grinder System (Biomedical Polymers, Gardener, MA), homogenized in chilled lysis buffer comprising 4 M guanidine-HCl and 1 % protease inhibitor cocktail (Sigma, St. Louis, MO), and extracted at 4℃ for 48 h. After centrifugation, the supernatants were dialyzed against 50 mM Tris-HCl (pH 8.0) and 0.15 M NaCl at 4℃ overnight using Slide-A-Lyzer MINI Dialysis Units (Pierce, Rockford, IL). The protein contents in the lysates were measured using a Micro BCA Protein Assay Reagent Kit (Pierce), and aliquots containing 30 μg of protein were immobilized on nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK) using a PR648 Slot Blot Manifold dot blot apparatus (GE Healthcare). The membranes were blocked with 10 % non-fat milk, and incubated with the 6084 antibody recognizing G1 [11] or the 2B1 antibody recognizing G3 [4]. In the breast cancer tissues, the staining intensities with the 6084 and 2B1 antibodies were increased by about two-fold and four-fold, respectively, compared with the non-cancerous tissues [7]. Immunohistochemical staining

The G1 and G3 domains are thought to be related to the proliferation and migration of cells. For example, the G1 domain stimulates cell migration and proliferation by destabilizing cell adhesion in NIH3T3 cells and astrocytoma cells [12,13]. The G3 domain promotes breast cancer cell proliferation, invasion and bone metastasis and enhances cancer apoptosis, by upregulating the epidermal growth factor receptor (EGFR)-mediating signaling pathway. Activation of phosphorylated extracellular regulated protein kinases (ERKs) is correlated with high levels of G3 expression [14]. The G3 domain contains two epidermal growth factor (EGF)–like motifs, and the activated EGF-EGFR-ERK pathway plays important roles in cell

Versican is cleaved by proteases, which play important roles in tissue remodeling and modulation of cell microenvironments through degradation of the ECM and processing of growth factors and adhesion molecules. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteins, a family of proteases, are composed of the following seven domains: signal peptide, prodomain, metalloproteinase domain, disintegrin domain, thrombospondin type 1 motif, cysteine-rich domain and spacer region. Versican is cleaved by ADAMTS-1 and ADAMTS-4 at Glu441-Ala442, and a 70-kDa neoepitope DPEAAE sequence is generated. ADAMTS-1 in breast cancer cells can promote their growth and metastasis through local accumulation of versican fragments and angiogenesis [15]. This indicates that the concentration of versican is influenced by the cleavage as well as the

production, and ADAMTS plays a role in the cancer development.

**3. Hyaluronan and receptors for hyaluronan-mediated motility**

Hyaluronan (HA) is a nonsulfated glycosaminoglycan of repeating D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units. It is a major constituent of the ECM, and plays HA synthesis is correlated with the level of HAS mRNA, suggesting that transcriptional regulation is important [21]. In addition to measuring the HAS mRNA levels, several different methods have been utilized for detecting HA. Histologically, the expression of HA was evaluated by staining with biotinylated HA-binding protein (5 μg/ml) prepared from bovine nasal cartilage. Another method used was HA-dependent pericellular coat assays. Cells were cultured in dishes, and the medium was then replaced with phosphate-buffered saline containing 1 x 1016 formalin-fixed horse erythrocytes. After 20 min, the dishes were observed and areas without erythrocytes were considered to be HA [22].

HA is rich in cancer stroma, including that of lung, bladder, prostate, colon and breast cancers (Figure 3), and stromal HA accumulation is typical for high-grade and poorly differentiated carcinoma [23]. An HA rich matrix supports tumor growth and spreading by regulating cell proliferation, and migration or by enhancing tumor angiogenesis in the cancer. In breast cancer in transgenic mice, HA overproduction accelerates tumor angiogenesis through stromal reactions, most notably in the presence of versican [24]. On the other hand, the HAS suppressor, 4-methylumbelliferone (MU), suppresses cell adhesion, locomotion, and matrix metalloproteinase (MMP)-9 and N-cadherin expression, and inhibits tumor metastasis [25-27]. HA is composed of high and low molecular weight forms that have been shown to have opposite functions. High- molecular-weight HA suppresses vascularization, and has anti-inflammatory and immunosuppressive functions. Lowmolecular-weight HA, including HA oligosaccharides (less than 50 oligomers (mers)), has an angiogenic function, and induces cytokines and chemokines in inflammation and ECM degradation [28]. HA changes its biological activities depending on its molecular weight, and HA oligosaccharides (12 mers) suppress the growth of breast cancer [29]. MU and HA oligosaccharides may be candidates for anticancer therapies.

HA is present not only in the ECM, but also in the cellular cytoplasm and nucleus. Although the mechanism for HA internalization remains controversial, one possibility is that HA is transported in a retrograde manner through the Golgi, endoplasmic reticulum and cytoplasm. HA accumulation is seen in the cytoplasm of malignant cells, such as colon and gastric cancer cells and lymphoma cells. A high level of HA on tumor cells is a strong indicator of an unfavorable outcome [30]. The intracellular HA-binding proteins include CDC37 (p50), receptors for HA-mediated motility (RHAMM), P-32 and intracellular HAbinding protein (IHABP)-4 [31]. RHAMM was isolated from culture supernatants of chick embryonic heart fibroblasts [32], and is present in the cell membrane, centrosomes and microtubules. RHAMM is overexpressed in the G2/M phase of cancer cells and in many kinds of cancers [33,34]. These observations suggest that HA and RHAMM play important intracellular roles in mitosis, maintaining the cell shape, and modulating centrosomes and microtubules, which enhance cell proliferation and migration.

Expressional Alterations of Versican,

Hyaluronan and Microfibril AssociatedProteins in the Cancer Microenvironment 159

then evaluated for another GST-RHAMM fusion protein (C-terminal domain) or GST. The transferred proteins from OHK cells were visualized with Coomassie brilliant blue R-250 and the spots corresponding to the proteins bound to RHAMM were excised from the membrane and submitted for sequencing. GRP78 and GRP75 were identified as RHAMMbinding proteins [33]. Double immunostaining revealed that GRP78 and GRP75 colocalized with RHAMM in the interphase of the cell cycle. The RHAMM-Hsp70 complex may stabilize microtubules in the interphase, and the separation of RHAMM and Hsp70 may

HA is cleaved into 10 to 15 disaccharide fragments by hyaluronidases (HYALs). In humans, four HYALs have been identified, namely HYAL1, HYAL2, HYAL3 and PH20 [35]. In lung cancer, wild-type HYAL1 was associated with a poorer prognosis, while the HYAL3-v1 variant was associated with a better prognosis [36]. Increased HYAL1 expression and hyaluronidase activity are associated with an unfavorable prognosis in bladder and

Versican interacts with HA and fibrillin-1 at its N-terminus and C terminus, respectively [2,38]. Fibrillin-1 is a major structural component of connective tissue microfibrils, and has significant roles in the maintenance of microfibrils and elastic fibers. The HA-versicanfibrillin-1 complex and its cleavage products exist in the ciliary nonpigmented epithelium and the vitreous body [39]. Regarding skin tissues, immunoblotting analyses of skin extracts with the 6084 antibody and biotin-conjugated HA revealed that versican was a major HAbinding component in the dermis [11]. MMP-12 (macrophage metalloelastase) degraded versican and abrogated its HA-binding ability. Immunohistochemical analyses revealed that the elastic materials in solar elastosis lesions were negative for 6084 antibody, but positive for 2B1 antibody, indicating loss of the HA-binding regions in the aggregated elastic fibers. In solar elastosis, which is seen in photoaged skin of elderly people, abrogation of the HAbinding ability of versican by MMP-12 is observed. On the other hand, assembly of HAversican aggregates in the pericellular sheath of cancer cells promotes their motility [40]. Similar locations of HA, versican and fibrillin-1 were seen in the ECM of breast cancer

Latent transforming growth factor β binding protein (LTBPs) are large extracellular glycoproteins that are structurally related to fibrillins, and are major regulators of transforming growth factor-β (TGF-β) bioavailability and action [41]. TGF-β is a potent growth inhibitor in the early stage of carcinogenesis, controlling cellular growth and inducing apoptosis. In the advanced stage of carcinogenesis, TGF-β acts as an oncogenic factor and induces invasion-associated epithelial-mesenchymal transition. TGF-β is thought to enhance tumor growth and invasion through regulation of immune functions and angiogenesis, as well as the production of stromal components [42]. TGF-β is secreted as a

**4. HA-versican-fibrillin-1 complex and HA-verscian complex**

tissues [7], but their exact functions in cancer were not elucidated.

**5. Latent transforming growth factor β binding proteins**

play a role in cell division.

prostatic cancers [37].

**Figure 3.** Histochemistry with biotinylated hyaluronan binding protein in human invasive breast cancer. Hyaluronan is expressed in cancer stroma (x200).

CDC37 and RHAMM form complexes with heat shock protein (Hsp) 90 and Hsp70, respectively [31,33]. The CDC37-Hsp90-HA complex stabilizes protein kinases such as Cdk4, p60Src kinase, casein kinase II, MPS1 kinase and Raf-1, and is important for trafficking of kinases from the cytoplasm to the nucleus. On the other hand, the complex of Hsp70 (GRP78 and GRP75) and RHAMM was confirmed by coimmunoprecipitation and glutathione-Stransferase (GST)-RHAMM fusion protein-binding assays [33]. For the GST-RHAMM fusion protein-binding assays, a membrane with transferred proteins from OHK cells (a malignant lymphoma cell line) after two-dimensional gel electrophoresis was used. The membrane was incubated with 0.05 μg/ml of GST-RHAMM fusion protein containing the central domain at room temperature for 1 h. After washing with PBS/Tween-20 at room temperature for 1 h, the localization of the RHAMM fusion protein binding was visualized using a peroxidase-conjugated anti-GST antibody (Amersham, Uppsala, Sweden) and an enhanced chemiluminescence system (Amersham). The membrane was then stripped by incubation in 0.05 M sodium phosphate (pH 6.5), 10 mM SDS and 0.1 M β-mercaptoethanol at 50℃ for 30 min, followed by a wash with PBS/Tween-20. The stripped membrane was then evaluated for another GST-RHAMM fusion protein (C-terminal domain) or GST. The transferred proteins from OHK cells were visualized with Coomassie brilliant blue R-250 and the spots corresponding to the proteins bound to RHAMM were excised from the membrane and submitted for sequencing. GRP78 and GRP75 were identified as RHAMMbinding proteins [33]. Double immunostaining revealed that GRP78 and GRP75 colocalized with RHAMM in the interphase of the cell cycle. The RHAMM-Hsp70 complex may stabilize microtubules in the interphase, and the separation of RHAMM and Hsp70 may play a role in cell division.

HA is cleaved into 10 to 15 disaccharide fragments by hyaluronidases (HYALs). In humans, four HYALs have been identified, namely HYAL1, HYAL2, HYAL3 and PH20 [35]. In lung cancer, wild-type HYAL1 was associated with a poorer prognosis, while the HYAL3-v1 variant was associated with a better prognosis [36]. Increased HYAL1 expression and hyaluronidase activity are associated with an unfavorable prognosis in bladder and prostatic cancers [37].

### **4. HA-versican-fibrillin-1 complex and HA-verscian complex**

158 Carcinogenesis

CDC37 (p50), receptors for HA-mediated motility (RHAMM), P-32 and intracellular HAbinding protein (IHABP)-4 [31]. RHAMM was isolated from culture supernatants of chick embryonic heart fibroblasts [32], and is present in the cell membrane, centrosomes and microtubules. RHAMM is overexpressed in the G2/M phase of cancer cells and in many kinds of cancers [33,34]. These observations suggest that HA and RHAMM play important intracellular roles in mitosis, maintaining the cell shape, and modulating centrosomes and

**Figure 3.** Histochemistry with biotinylated hyaluronan binding protein in human invasive breast

CDC37 and RHAMM form complexes with heat shock protein (Hsp) 90 and Hsp70, respectively [31,33]. The CDC37-Hsp90-HA complex stabilizes protein kinases such as Cdk4, p60Src kinase, casein kinase II, MPS1 kinase and Raf-1, and is important for trafficking of kinases from the cytoplasm to the nucleus. On the other hand, the complex of Hsp70 (GRP78 and GRP75) and RHAMM was confirmed by coimmunoprecipitation and glutathione-Stransferase (GST)-RHAMM fusion protein-binding assays [33]. For the GST-RHAMM fusion protein-binding assays, a membrane with transferred proteins from OHK cells (a malignant lymphoma cell line) after two-dimensional gel electrophoresis was used. The membrane was incubated with 0.05 μg/ml of GST-RHAMM fusion protein containing the central domain at room temperature for 1 h. After washing with PBS/Tween-20 at room temperature for 1 h, the localization of the RHAMM fusion protein binding was visualized using a peroxidase-conjugated anti-GST antibody (Amersham, Uppsala, Sweden) and an enhanced chemiluminescence system (Amersham). The membrane was then stripped by incubation in 0.05 M sodium phosphate (pH 6.5), 10 mM SDS and 0.1 M β-mercaptoethanol at 50℃ for 30 min, followed by a wash with PBS/Tween-20. The stripped membrane was

microtubules, which enhance cell proliferation and migration.

cancer. Hyaluronan is expressed in cancer stroma (x200).

Versican interacts with HA and fibrillin-1 at its N-terminus and C terminus, respectively [2,38]. Fibrillin-1 is a major structural component of connective tissue microfibrils, and has significant roles in the maintenance of microfibrils and elastic fibers. The HA-versicanfibrillin-1 complex and its cleavage products exist in the ciliary nonpigmented epithelium and the vitreous body [39]. Regarding skin tissues, immunoblotting analyses of skin extracts with the 6084 antibody and biotin-conjugated HA revealed that versican was a major HAbinding component in the dermis [11]. MMP-12 (macrophage metalloelastase) degraded versican and abrogated its HA-binding ability. Immunohistochemical analyses revealed that the elastic materials in solar elastosis lesions were negative for 6084 antibody, but positive for 2B1 antibody, indicating loss of the HA-binding regions in the aggregated elastic fibers. In solar elastosis, which is seen in photoaged skin of elderly people, abrogation of the HAbinding ability of versican by MMP-12 is observed. On the other hand, assembly of HAversican aggregates in the pericellular sheath of cancer cells promotes their motility [40]. Similar locations of HA, versican and fibrillin-1 were seen in the ECM of breast cancer tissues [7], but their exact functions in cancer were not elucidated.

### **5. Latent transforming growth factor β binding proteins**

Latent transforming growth factor β binding protein (LTBPs) are large extracellular glycoproteins that are structurally related to fibrillins, and are major regulators of transforming growth factor-β (TGF-β) bioavailability and action [41]. TGF-β is a potent growth inhibitor in the early stage of carcinogenesis, controlling cellular growth and inducing apoptosis. In the advanced stage of carcinogenesis, TGF-β acts as an oncogenic factor and induces invasion-associated epithelial-mesenchymal transition. TGF-β is thought to enhance tumor growth and invasion through regulation of immune functions and angiogenesis, as well as the production of stromal components [42]. TGF-β is secreted as a latent complex containing one of the LTBPs. Latent TGF-β consists of the mature growth factor and the TGF-β propeptide, also known as the latency-associated peptide (LAP). The LTBPs and TGF-β propeptides have four and three isoforms, respectively. LTBP-1 and LTBP-3 bind all three TGF-β LAP isoforms with high affinity, while LTBP-4 shows a weak binding capacity only for the TGF-β1 LAP isoform. LTBP-1, LTBP-3 and LTBP-4 regulate TGF-β action at multiple levels [41]; (a) they ensure correct folding and efficient secretion; (b) they direct temporal and spatial deposition in the extracellular space; and (c) they regulate activation. These findings show that LTBPs have multiple roles with regard to TGFβ action, and the instability of the TGF-β associations with LTBPs yields inflammation and tumors. It has been reported that LTBP-1 and LTBP-3 expression is downregulated in hepatoma and mesothelioma, respectively, compared with normal tissues [43,44]. In breast cancer tissues, LTBP-4 was decreased (Figure 4) [7]. Downregulation of LTBP-3 expression in malignant mesothelioma contributes to increased TGF-β signaling activity [44], and upregulated TGF-β signaling following LTBP reduction seems to induce carcinoma. TGF-β signaling plays a key role in carcinogenesis, and the LTBP downregulation appears to be critical to cancer progression.

Expressional Alterations of Versican,

Hyaluronan and Microfibril AssociatedProteins in the Cancer Microenvironment 161

future research goals is to elucidate the function of HA-versican-microfibril complex in the

**Figure 5.** A diagram of the breast cancer tissue. Cancer stroma is rich in hyaluronan and versican, while

*Department of Nursing and Health, School of Nursing and Health, Aichi Prefectural University,*

[1] Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC (2009) Gene expression profiling of the tumor microenvironment during breast cancer progression. Breast Cancer Res.

*Department of Advanced Medicine, National Center for Geriatrics and Gerontology, Japan*

cancer stroma.

normal tissues have LTBP-4.

*Department of Pathology, Osaka Medical College, Japan*

**Author details**

Hiroko Kuwabara

Masahiko Yoneda

*Japan*

Zenzo Isogai

**7. References**

11: R7.

**Figure 4.**  LTBP-4 expression is absent (x100).

### **6. Conclusion**

Versican, HA and LTBP distributions in the cancer ECM differ from those of normal tissues (Figure 5), and the ECM alteration facilitates cancer cell proliferation, migration and angiogenesis. In addition, these proteins can interact with each other, and exert a crucial role in the cancer progression. For these reasons, a novel cancer therapy that targets an individual ECM protein and the complex of these ECM proteins is proposed. One of our future research goals is to elucidate the function of HA-versican-microfibril complex in the cancer stroma.

**Figure 5.** A diagram of the breast cancer tissue. Cancer stroma is rich in hyaluronan and versican, while normal tissues have LTBP-4.

### **Author details**

160 Carcinogenesis

critical to cancer progression.

absent (x100).

**6. Conclusion**

latent complex containing one of the LTBPs. Latent TGF-β consists of the mature growth factor and the TGF-β propeptide, also known as the latency-associated peptide (LAP). The LTBPs and TGF-β propeptides have four and three isoforms, respectively. LTBP-1 and LTBP-3 bind all three TGF-β LAP isoforms with high affinity, while LTBP-4 shows a weak binding capacity only for the TGF-β1 LAP isoform. LTBP-1, LTBP-3 and LTBP-4 regulate TGF-β action at multiple levels [41]; (a) they ensure correct folding and efficient secretion; (b) they direct temporal and spatial deposition in the extracellular space; and (c) they regulate activation. These findings show that LTBPs have multiple roles with regard to TGFβ action, and the instability of the TGF-β associations with LTBPs yields inflammation and tumors. It has been reported that LTBP-1 and LTBP-3 expression is downregulated in hepatoma and mesothelioma, respectively, compared with normal tissues [43,44]. In breast cancer tissues, LTBP-4 was decreased (Figure 4) [7]. Downregulation of LTBP-3 expression in malignant mesothelioma contributes to increased TGF-β signaling activity [44], and upregulated TGF-β signaling following LTBP reduction seems to induce carcinoma. TGF-β signaling plays a key role in carcinogenesis, and the LTBP downregulation appears to be

**Figure 4.**  LTBP-4 expression is

Versican, HA and LTBP distributions in the cancer ECM differ from those of normal tissues (Figure 5), and the ECM alteration facilitates cancer cell proliferation, migration and angiogenesis. In addition, these proteins can interact with each other, and exert a crucial role in the cancer progression. For these reasons, a novel cancer therapy that targets an individual ECM protein and the complex of these ECM proteins is proposed. One of our Hiroko Kuwabara *Department of Pathology, Osaka Medical College, Japan*

Masahiko Yoneda

*Department of Nursing and Health, School of Nursing and Health, Aichi Prefectural University, Japan*

Zenzo Isogai *Department of Advanced Medicine, National Center for Geriatrics and Gerontology, Japan*

### **7. References**

[1] Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC (2009) Gene expression profiling of the tumor microenvironment during breast cancer progression. Breast Cancer Res. 11: R7.

[2] Kimata K, Oike Y, Tani K, Shinomura T, Yamagata M, Uritani M, Suzuki S (1986) A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo. J. Biol. Chem. 261: 13517-13525.

Expressional Alterations of Versican,

Hyaluronan and Microfibril AssociatedProteins in the Cancer Microenvironment 163

[17] Fraser JR, Laurent TC, Laurent UB (1997) Hyaluronan: its nature, distribution, functions

[18] Weigel PH, Hascall VC, Tammi M (1997) Hyaluronan synthases. J. Biol. Chem. 272:

[19] Kuwabara H, Yoneda M, Nagai M, Nishio H, Tasaka T, Suzuki K, Mori H (2003) High levels of hyaluronan production by a malignant lymphoma cell line with primary

[21] Tammi RH, Passi AG, Rilla K, Karousou E, Vigetti D, Makkonen K, Tammi MI (2011) Transcriptional and post-transcriptional regulation of hyaluronan synthesis. FEBS. J.

[22] Kuwabara H, Yoneda M, Nagai M, Hayasaki H, Mori H (2004) A new polyclonal antibody that recognizes a human receptor for hyaluronan mediated motility. Cancer

[23] Tammi RH, Kultti A, Kosma VM, Pirinen R, Auvinen P, Tammi MI (2008) Hyaluronan in human tumors: Pathobiological and prognostic messages from cell-associated and

[24] Koyama H, Hibi T, Isogai Z, Yoneda M, Fujimori M, Amano J, Kawakubo M, Kannagi R, Kimata K, Taniguchi S, Itano N (2007) Hyperproduction of hyaluronan in neuinduced mammary tumor accelerates angiogenesis through stromal cell recruitment.

[25] Yoshihara S, Kon A, Kudo D, Nakazawa H, Kakizaki I, Sasaki M, Endo M, Takagaki K (2005) A hyaluronan synthase suppressor, 4-methylumbelliferone, inhibits liver

[26] Kuwabara H, Yoneda M, Hayasaki H, Nakamura T, Shibayama Y (2011) A hyaluronan synthase suppressor, 4-methylumbelliferone, inhibits the tumor invasion associated

[27] Nakamura R, Kuwabara H, Yoneda M, Yoshihara S, Ishikawa T, Miura T, Nozaka H, Nanashima N, Sato T, Nakamura T (2007) Suppression of matrix metalloproteinase-9 by

[28] Noble PW, McKee CM, Cowman M, Shin HS (1996) Hyaluronan fragments activate an NF-κB/I-κBα autoregulatory loop in murine macrophages. J. Exp. Med. 183: 2373-2378. [29] Misra S, Ghatak S, Zoltan-Jones A, Toole BP (2003) Regulation of multidrug resistance

[30] Huang L, Grammatikakis N, Yoneda M, Banerjee SD, Toole BP (2000) Molecular characterization of a novel intracellular hyaluronan-binding protein. J. Biol. Chem. 275:

[31] Yoneda M (2001) Key molecules to an understanding of intracellular hyaluronan

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[17] Fraser JR, Laurent TC, Laurent UB (1997) Hyaluronan: its nature, distribution, functions and turnover. J. Intern. Med. 242: 27-33.

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3902-3908.

270: 958-965.

[2] Kimata K, Oike Y, Tani K, Shinomura T, Yamagata M, Uritani M, Suzuki S (1986) A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in

[3] Wight TN (2002) Versican: a versatile extracellular matrix proteoglycan in cell biology.

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[5] Ito K, Shinomura T, Zako M, Ujita M, Kimata K (1995) Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing. J. Biol. Chem.

[6] Sheng W, Wang G, Wang Y, Liang J, Wen J, Zheng PS, Wu Y, Lee V, Slingerland J, Dumont D, Yang BB (2005) The roles of versican V1 and V2 isoforms in cell

[7] Takahashi Y, Kuwabara H, Yoneda M, Isogai Z, Tanigawa N, Shibayama Y (2012) Versican G1 and G3 domains are upregulated and latent transforming growth factor-β binding protein-4 is downregulated in breast cancer stroma. Breast Cancer. 19: 46-53. [8] Wasa J, Nishida Y, Shinomura T, Isogai Z, Futamura N, Urakawa H, Arai E, Kozawa E, Tsukushi S, Ishiguro N (2012) Versican V1 isoform regulates cell-associated matrix formation and cell behavior differentially from aggrecan in Swarm rat chondrosarcoma

[9] Serra M, Miquel L, Domenzain C, Docampo MJ, Fabra A, Wight TN, Bassols A (2005) V3 versican isoform expression alters the phenotype of melanoma cells and their

[10] Miquel-Serra L, Serra M, Hernandez D, Domenzain C, Docampo MJ, Rabanal RM, de Torres I, Wight TN, Fabra A, Bassols A (2006) V3 versican isoform expression has a dual

role in human melanoma tumor growth and metastasis. Lab. Invest. 86: 889-901. [11] Hasegawa K, Yoneda M, Kuwabara H, Miyaishi O, Itano N, Ohno A, Zako M, Isogai Z (2007) Versican, a major hyaluronan-binding component in the dermis, loses its

hyaluronan-binding ability in solar elastosis. J. Invest. Dermatol. 127: 1657-1663. [12] Yang BL, Zhang Y, Cao L, Yang BB (1999) Cell adhesion and proliferation mediated

[13] Ang LC, Zhang Y, Cao L, Yang BL, Young B, Kiani C, Lee V, Allan K, Yang BB (1999) Versican enhances locomotion of astrocytoma cells and reduces cell adhesion through

[14] Du WW, Yang BB, Shatseva TA, Yang BL, Deng Z, Shan SW, Lee DY, Seth A, Yee AJ (2010) Versican G3 promotes mouse mammary tumor cell growth, migration, and

[15] Ricciardelli C, Frewin KM, Tan IA, Williams ED, Opeskin K, Pritchard MA, Ingman WV, Russell DL (2011) The ADAMTS1 protease gene is required for mammary tumor

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the limb bud of chick embryo. J. Biol. Chem. 261: 13517-13525.

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[32] Turley EA (1982) Purification of a hyaluronan-binding protein fraction that modifies cell social behavior. Biochem. Biophys. Res. Commun. 108: 1016-1024.

**Chapter 9**

© 2013 Hernández-Caballero, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Molecular Mechanisms of Metastasis:**

During the process of tumorigenesis, some cells separate themselves from the tumor to invade distant tissues. Cellular migration in this process is similar to what occurs during embryonic development and wound healing. Unlike these two healthy processes, which involve the creation of a structure or the healing of tissue, metastasis results in the formation of a cellular mass that, if not eliminated, leads to the death of the organism. The process of metastasis includes the invasion of the basal membrane and nearby tissue by tumoral cells and the intravasation towards the blood vessels or infiltration of the lymphatic vessels. This is followed by the mechanisms of survival of tumor cells in these vessels and their extravasation to different tissues of the organism, where they may be able to proliferate. This pathogenic process requires a precise coordination of various signaling pathways that allows the tumor cells to move across the cell membrane, remodel the matrix, transport themselves by circulation [1] and create the appropriate conditions, at a distance, for

Although exploring this complex process of motility and invasion by tumoral cells is fundamental for deepening the understanding of metastasis [2], much remains a mystery despite the enormous amount of research contributions. Both *in vitro* and *in vivo*, the time required for analyzing the evolution of this pathogenesis is excessive. Today the best way to make functional evaluations of the genetic changes that take place in humans with metastasis is with animal models. Although time consuming, a complete follow up can be carried out of the entire process, from the moment of the appearance of a primary tumor, to the strategies used by cancerous cells to escape from the controls of adhesion, their interaction with endothelial cells during their migration, and the establishment of a secondary tumor through the preparation of a new microenvironment favorable to tumor growth in the affected organ.

and reproduction in any medium, provided the original work is properly cited.

**Epithelial-Mesenchymal Transition,**

**Anoikis and Loss of Adhesion**

Additional information is available at the end of the chapter

establishing themselves in a different organ (Figure 1).

M.E. Hernández-Caballero

http://dx.doi.org/10.5772/55399

**1. Introduction**

**Chapter 9**


**4. The prodrome**

Although there is great variability between patients in how their prodromes manifest, certain symptoms and signs have been frequently described. These include depressed mood, anxiety, irritability and aggressive behavior, suicidal ideation and attempts, and substance use. The most commonly occurring prodromal symptoms, according to retrospective studies of patients with schizophrenia and schizophreniform disorder, are reduced concentration and attention, reduced drive and motivation, depression, sleep disturbance, social withdrawal, suspiciousness, deterioration in role functioning, and irritability [1].

Studying these symptoms, we observe two things. First, many of them are nonspecific occurring frequently in the prodromes of nonpsychotic threshold syndromes. Second, a considerable amount of psychiatric symptoms, disability, self-harming, and other health-damaging behaviors, occur during this prodromal phase, even in the earliest stages [1, 19, 22, 39].

Cognitive, affective, and social disturbances known as "basic symptoms" are also commonly described in the early prodromal phases. This concept of "basic symptoms," developed in the 1960s, has significantly influenced the new area of prodromal research [1].

5–10% of the general population experience attenuated or subthreshold form of psychotic symptoms like transient perceptual symptoms; suspiciousness; reference and bizarre delusional ideas (e.g., the beliefs that others may be thinking badly about or laughing at); nonattendance at school, university, or work; and altered behavior toward family and friends [1, 16].

The difference between these phenomena and clear psychotic symptoms is due to their intensity, frequency, duration, and deleterious effects on the individual functionality of the person.

Neurocognitive deficits in particular impaired attention, spatial and verbal memory, and speeded information processing are also evident in the prodromal phase but at a lower degree of severity comparing to those found in first-degree relatives of patients with schizophrenia or in fully affected patients [1].

Specific cognitive deficits may be related more directly to affected brain structures and candidate genes and so may be more directly predictive of psychosis.


3.4.4. Scattering from Ship Wakes

The possibility of detecting ship wakes was recognized in the 1970s, but early studies focused on first-order scattering, which is critically dependent on radar geometry and frequency and hence not a practical solution. The development of a second-order theory [38–40] has opened the way to a meaningful capability, but here too, careful consideration of the radar geometry and frequency is required to approach optimum sensitivity.

According to this theory, the wake signature depends not only on the vessel, it is also a function of the ambient sea state, so no simple assertion of optimality of one or other configuration can be made. Nevertheless, extensive modelling has shown that bistatic geometries routinely emerge as optimal solutions.

One indication of the possible advantage of bistatic geometry can be seen from Figure 19, showing a measured omni-directional wave spectrum from the open sea, on which is superimposed the corresponding spectrum for a ship wake. As the annotations reveal, the wake components are concentrated at low frequencies (and hence wavenumbers) for which monostatic first-order Bragg scatter at HF is not available, but at a bistatic angle ϕ the resonant wavenumber is decreased by a factor sin(ϕ/2).

**Figure 19.** The omnidirectional spectrum of an ambient sea, and, superimposed (in red), the wake of a merchant ship at normal sailing speed; note that the abscissa is frequency, not wavenumber, connected here by the familiar deep-water dispersion relation. The radar frequencies corresponding to monostatic Bragg scatter are marked by the dashed lines.


**25. Visual outcome in endogenous endophthalmitis**

to have fungal isolates with a predominance of Candida albicans. The most common Gram positive organisms are Staphylococcus aureus, group B streptococci, Streptococcus pneumo‐ niae,and Listeria monocytogenes; the most common Gram negative organisms are Klebsiella spp., Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Neisseria meningitidis. [2,

The optimal treatment for endogenous endophthalmitis is controversial. When indicated, these patients may require systemic antibiotics in addition to the PPV. While EVS has provid‐ ed guidelines for the role of early vitrectomy and intra-vitreal antibiotics in post-operative en‐ dophthalmitis, no such study has addressed endogenous endophthalmitis. Data from the EVS may not be applicable to cases of endogenous endophthalmitis because the spectrum of caus‐ ative organisms differs significantly in endogenous endophthalmitis as compared to post-op‐ erative endophthalmitis. Although systemic and intra-vitreal antibiotics may be sufficient in milder forms of infection, PPV has been shown to be helpful in severe cases of endogenous en‐ dophthalmitis. More virulent organisms such as endotoxin-producing Streptococcus and Ba‐ cillus species are commonly involved in endogenous endopthalmitis. [67, 71] In addition, material from vitrectomy may provide a better source for culture. This is particularly true in children because of the variety of pediatric cases and lack of sufficient experience in diagnos‐ ing in this age group. [7] In the adults, early intervention with PPV has been found to be high‐ ly effective, no such data has been proven for cases of pediatric endogenous endophthalmitis. Suggested medical treatment in these patients include topical, sub-conjunctival and intra-vi‐ treal injection of antibiotics having broad coverage with consideration for corticosteroids in cases of severe inflammation. Patients with endogenous endophthalmitis should be evaluat‐ ed for underlying systemic conditions. Systemic anti-microbial therapy is the mainstay of en‐ dogenous endophthalmitis. Intravitreal antibiotic selection is similar to exogenous endophthalmitis including Vancomycin (1.0 mg/0.1 mL) for Gram-positive coverage or in combination with Ceftazidime (2.25 mg/0.1 mL) or Amikacin (400 ug/0.1 mL) for Gram-nega‐ tive coverage.. In general, systemic therapy must be continued for several weeks to ensure eradication of the infection. Generally, a combination of intra-vitreal antibiotics is injected that may include Vancomycin, Cephazolin or Ceftazidime and Amikacin after the tap has been performed. Systemic antibiotics are administered according to the focus of the infection. Infec‐ tious diseases consultation may be sought in cases of endocarditis and early vitrectomy should be planned if indicated. [88] Immediate vitrectomy is performed in eyes with lightperception-only vision at the initial visit. Routine immediate vitrectomy is not necessary in eyes presenting with better than light-perception vision. Aggressive therapy and early vitrec‐ tomy may be considered in endogenous endophthalmitis caused by virulent pathogens such Pseudomonas aeruginosa and in cases of Klebsiella endophthalmitis. [81, 97, 99] Patients with endogenous endophthalmitis who undergo PPV early in the course of endogenous endoph‐

**24. Management of endogenous endophthalmitis**

thalmitis may end up with some useful vision.

3, 69, 91]

198 Common Eye Infections

Endogenous endophthalmitis is generally associated with high mortality and poor visual outcomes, particularly when caused by more virulent species such as Aspergillus. [98] Fun‐ gal endopthalmitis has a poor visual outcome as compared to bacterial endophthalmitis. [100] The visual outcome in cases of treated Streptococcal endophthalmitis is generally poor than some of the Staphylococcal species. Patients with good initial VA typically have good final VA. It is believed that an active therapeutic approach including intra-vitreal antibiotics and vitreo-retinal surgery may save eyes from blindness. In the past, the visual outcome has been poor with most cases leading to blindness in the affected eye. [70, 81] In an experimen‐ tally model of endogenous endophthalmitis, infant rats inoculated by either intra-nasal or intra-peritoneal injection of Haemophilus influenzae type b, suppurative endophthalmitis occurred in 50% of bacteremic animals who survived. [101] This experimental induced en‐ dogenous endophthalmitis ultimately progressed to panophthalmitis followed by organiza‐ tion of the exudate and phthisis bulbi. Recent data for the effectiveness of vitrectomy and intra-vitreal antibiotics to save some vision has been encouraging. [88] Endophthalmitis due to Streptococcal species may result in earlier onset and perhaps worse visual outcome. On the other hand, endophthalmitis which yields no positive results from culture usually have delayed onset of infection and better visual results.


**12. PLP supplementation as immunodulatory and anti-inflammatory agent**

Pyridoxal 5′ phosphate (PLP) is an active component of pyridoxine and is a cofactor vital for several inflammatory disorder processes that contribute to immune disorder. In chronic inflammatory diseases, PLP has a reverse relation with plasma TNF-α and IL-6. Throughout inflammation, PLP use enhances its depletion, which means that COVID-19 can be deficient in patients with high inflammatory response. In patients with type-2 diabetes and cardiovascular disorder, groups at greater risk for relatively poor COVID-19 outcomes were reported with low levels

*Novel Treatment Strategy against COVID-19 through Anti-Inflammatory, Antioxidant… DOI: http://dx.doi.org/10.5772/intechopen.100251*

of PLP [40, 41]. Elevated incidence of coagulopathy between many COVID-19 patients was also observed as a result of immune disregulation. In a recent manuscript, PLP intake was associated to ameliorate COVID-19 symptoms by regulate immune functions, lower pro-inflammatory cytokines, preserve endothelial stability and avoid hypercoagulability [42]. Indeed, it was found 30 years ago that platelet accumulation and blood coagulation defects were reduced by PLP amounts [43]. Vitamin B6 (along with B2 and B9) prediculated IL 10, a potent anti-inflammatory and immune suppressant cytokine which can disable monocytes and prevent T cells as well as cells present with the antigen [44]. Patients with COVID-19 often frequently react to the virus by implementing a pro-inflammatory T cell response and secretion. PLP may help dampen the cytokine storm and inflammatory processes suffered by a few patients with COVID-19.


*2.5. Ensemble Assessment*

The ability of the multi-model averages to estimate observed VLF frequencies was quantified over the temporal range of the extant MTBS fire occurrence record at three non-overlapping time periods. Two of the three time periods correspond to the tuning (1984–2005), training (2006–2015) datasets that were used to bias correct and fit the initial suite of PETs respectively. Additionally, a testing dataset independent of the information used to build the multi-model averages was constructed using 2016 MTBS occurrence data. For each time period, a sample of 100,000 probability time series were drawn from the relevant multi-model average posterior, which were then used to simulate the distribution of VLF counts predicted during that time period. Note that the 2016 fire data used to independently validate the multi-model averages represent an updated version of the MTBS data that was unavailable during the PET training and tuning stages, and that slight differences in the total number of large (>404 hectares) incidents between 1984–2015 were observed in the two versions. Specifically, the original MTBS dataset reported 10,295 large incidents between 1984–2015, while the updated version reported 10,298 large incidents during that same period.