Mucosal Remodeling and Alteration of Stromal Microenvironment in Ulcerative Colitis as Related to Colorectal Tumorigenesis

shortening in mucosal epithelia is an important factor in tumorigenesis. The telomere shortening in colonic

decreased number (/cm), height (μm) and angle (degree) of crypts and increased fused crypts (/100 crypts), metaplastic Paneth cells (/100 crypts) and thickening of muscularis mucosa (μm) quantitatively, and found most of items correlated significantly with the duration of illness in UC (Fig. 2). Fig. 1. A three-dimensional, reconstructed figure of rectal mucosa in UC (Control and UC cases for 8 and 18 years) (Mitsuhashi et al., Pathol Int, 2005) However, there were no significant differences of increased or decreased correlation lines of each marker between the two groups, UC, inactive, without neoplasia and with neoplasia, or between the two groups, UC, active without neoplasia and with neoplasia. For immunohistochemical markers, Ki-67 (for cellular proliferative activity), p53, p21 and ssDNA (for apoptosis) labeling indices (LI) (%) were significantly correlated with the duration of illness (Fig. 3). The period-dependent increase of epithelial p53 and p21 LI is clearly shown. Furthermore, epithelial p53 and p21 LI were significantly higher in the nonneoplastic rectal mucosa of long-standing UC patients with colorectal neoplasia compared with those without neoplasia (Mitsuhashi et al., 2005). Epithelial p53 and p21 overexpression means acceleration of G1 check point due to inflammatory oxidative stress, indicating accumulation of DNA damage in line with pathway for tumorigenesis . Canonical discriminative analysis using duration of UC illness, number of crypts, angles of crypts and thickness of muscularis mucosa gave no clear difference between UC with neoplasia and without neoplasia ( Fig. 4  With this method, mutated crypts increased correlatively with the UC duration. Furthermore, clusters of crypts, positive with mild PAS staining, indicating regenerated crypts covered by a single tissue stem cell after erosion, also increased correlatively with the duration of the UC illness ( Fig. 6). Moreover, non-neoplastic mucosal crypts in cases of sporadic colorectal carcinoma also showed significant increase of mutated crypts with mild PAS staining, although angles of their correlation lines were extremely low compared with those in UC cases. Elongated lines of the correlation between two factors showed 0 crypts at 0 years old, indicating gradual appearance of mutated crypts after birth. Thus, mutated crypts appear extremely higher in UC patients than non-UC patients (Fig. 6).
These results indicate the base of a chronic inflammation-carcinoma sequence (Okayasu et al., 2002;.

Shortening of telomere length of colonic epithelial cells in UC
In addition to chromosomal alterations due to chronic inflammation-driven DNA damage through the generation and effects of reactive oxygen species, telomere shortening in mucosal epithelia is an important factor in tumorigenesis. The telomere shortening in colonic  Canonical discriminative analysis with number and angle of crypts and thickness of muscularis mucosa (MM) among UC active or inactive and with or without neoplasia epithelia is reported in long-standing UC, particularly in patients with colorectal cancers, suggesting an unstable state of chromosomes in which cells can easily mutate (Risques et al., 2008;. In addition to UC, telomere shortening is detected in esophageal Barrett's mucosa-Barrett's adenocarcinoma sequence by our group (Shiraishi et al., 2009). This phenomenon suggests an accelerated aging in inflamed lesions (Risques et al., 2008;.

Alteration of subepithelial (pericryptal) myofibroblasts and interstitial fibrosis in UC
We have clearly shown that subepithelial myofibroblasts forming crypt niches have various phenotypic expressions of -smooth muscle actin (SMA), NCAM, PGP9.5, HSP47 and cytoglobin (Cygb) by immunohistochemistry ( Fig. 7) or immunofluorescence (Fig. 8) and immunoelectron microscope (Fig.9). Therefore, we first identified these subepithelial myofibroblasts as colonic stellate cells (also known as perisinusoidal cells or Ito cells, fat storing cells in perisinusoidal spaces of the liver). Subepithelial myofibroblasts are localized between mucosal epithelia and capillaries, similar to hepatic stellate cells between liver cells and sinusoids. Further, subepithelial myofibroblasts occasionally have small lipid droplets indicating vitamin A storage. Subepithelial myofibroblasts are localized more at the crypt base than they are at the crypt surface, similar to the dense localization of hepatic stellate cells at the periportal area in hepatic lobules. These features indicate that subepithelial myofibroblasts correspond to colonic stellate cells .  It is known that subepithelial myofibroblasts secrete pericryptin, a specific extracellular matrix protein, which recruits activated fibroblasts and forms collagen fibrils, supporting the growth of epithelial components after mechanical stress in tissue repair processes (Shimazaki et al., 2008). Cytoglobin (Cygb), a novel member of the globin family is also expressed in splanchnic fibroblasts-like cells, including hepatic stellate cells and colonic subepithelial myofibroblasts. A recent study demonstrated that Cygb served as a defensive mechanism against oxidative stress under hypoxic conditions in a kidney ischemia-reperfusion experimental system (Nishi et al., 2011). Thus, subepithelial myofibroblasts at the crypt base play important roles to protect and support crypt stem cells and their differentiation and maturation as stem cell niches. Furthermore, we reported the decrease of subepithelial myofibroblasts, along with the inversely correlated increase of interstitial myofibroblasts and fibrosis, in relation to the duration of UC illness   (Table 1). Loss of subepithelial myofibroblasts means dysregulation of colonic crypt stem cell protection and differentiation. Interstitial fibrosis with the increase of stromal myofibroblasts in colonic mucosa, may also accelerate erroneous interactions between epithelial and stromal cells. These alterations might be major components of a microenvironment conductive to tumorigenesis (Fig. 11).

Epithelial and stromal genomic instability in UC and its relation to tumorigenesis
We have analyzed genomic instability, microsatellite instability (MSI) and loss of heterozygosity (LOH) in stromal cells, as well as epithelial cells, in order to see the role of stromal cell alterations in UC-associated tumorigenesis, using a combination of lasercaptured microdissection and the Gene-scan approach (Fig. 12). Stromal genomic instability kept the same incidences in dysplasia and invasive carcinoma lesions as those of regenerative mucosa. The frequency of LOH or MSI for 5 tumor suppressor gene (TSG) markers, D9S161 (close to p16 INK4A ), D7S486 (close to ST-7), D13S268 (close to Rb), D18S474 (close to Smad 4 and DCC) and D3S1300 (close to the FHIT [fragile histidine triad]) was almost constantly found to be increased in stromal components of all lesion types (regenerative mucosa, dysplasia and carcinoma). In contrast, the epithelial cells showed a step-up increase of MSI in tumor progression and a constant increase of LOH for TSG markers. In epithelium, LOH for 4 chromosome17 markers, D17S796 (17p13 close to the p53 gene, TP53 (17p13, the p53 gene locus), D17S786 (17p13, close to the p53 gene) and D17S579 (p17q21, close to BRCA1) increased along with histological progression. In stroma, LOH was relatively low, but there was a constant incidence in all types of lesions. When data were combined for TSG and chromosome 17 markers, the tendency was prominent that stromal cells showed a constantly increased incidence of both MSI and LOH in all types of lesions, including regenerative mucosa, dysplasia and carcinoma, compared with the step-up increase in epithelium along with histological progression (Matsumoto et al., 2003a;2003b;Yagishita et al., 2008). On the other hand, genomic instability for NCI-recommended standard microsatellite markers for colorectal cancers, BAT25, BAT26, D2S123, D3S346 and D17S250 (Boland et al., 1998) was not remarkable, indicating that chromosome 17 and tumor suppressor gene markers are more sensitive in UC-associated mucosal lesions (Matsumoto et al., 2003a). Table 1. Summary of stromal cell alterations in the rectal mucosa in UC (Okayasu et al., Pathol Int, 2009) www.intechopen.com   (Yagishita et al., Scand J Gastroenterol, 2008) Genomic instability in sporadic colorectal tumorigenesis, including de novo carcinogenesis and the adenoma-carcinoma sequence, was prominent in epithelium compared with a low incidence in stroma (Ishiguro et al., 2006;Ogawa et al., 2006). We also demonstrated that genomic instability was accelerated in stromal cells in Barrett's mucosa and Barrett's adenocarcinoma sequence in the esophagus similarly to UC tumorigenesis (Shiraishi et al., 2006). Contribution of genomic instability in stromal cells to carcinogenesis is variable in the tumorigenesis of various organs. It has been demonstrated that stromal genomic instability due to hormonal dysfunction precedes in tumorigenesis of the breast (Moinfar et al., 2000;Shekhar et al., 2001). Thus, enhanced genomic instability in stromal cells is important in the chronic inflammation-carcinoma sequence. inflammation, such as TNF-, IL-8, IL-6, toll-like receptors, cell cycle G1-check point and NF-kB (Yoshida et al., , 2004Ohkusa et al., 2009). Compared to de novo cancer development and the adenoma-carcinoma sequence, stochastic pathways to tumor development can be proposed in a chronic inflammation-carcinoma sequence. In a mountain climbing analogy, it is obvious that various climbing pathways connecting with that various climbing pathways connecting with each other showing stochasticity at the mountain-base are reduced to several paths and then even fewer paths as one approaches closer to the top of the mountain. The analogy in the body is that stochastic (probabilistic) pathways to tumor development over time gain commonalty through chronic inflammatory stimulation (Kobayashi and Inoue, 2008;Inoue and Kobayashi, 2011).

Conclusions
Not only chromosomal instability, telomere shortening and genomic instability of epithelial cells due to chronic inflammation-driven DNA damage, we also stressed the important role of mucosal remodeling, including morphological alterations and genomic instability of stromal cells, suggesting insufficient crosstalk between epithelial and stromal cells, as it relates to UC-associated tumorigenesis.