Irradiation Attenuates Systemic Lupus Erythematosus-Like Morbidity in NZBWF1 Mice: Focusing on CD180-Negative Cells.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies that can induce systemic inflammation. Ultraviolet-A and X-ray irradiation have been reported to have therapeutic effects in patients with SLE. We previously demonstrated that CD180-negative cells, these are radiosensitive, contribute to the development of SLE-like morbidity in NZBWF1 mice. In this study, the effects of irradiation on SLE-like morbidity manifestations in NZBWF1 mice and on CD180-negative cells were investigated. Whole-body irradiation, excluding the head, attenuated SLE-like morbidity in vivo, as indicated by the prevention of the renal lesion development, inhibition of anti-dsDNA antibody production, reduction of urinary protein levels, and prolongation of the lifespan. Irradiation also reduced the proportion of CD180-negative cells in the spleen. Although other immune cells or molecules may be triggered because of the whole-body irradiation treatment, previous research, and the current results suggest a strong relationship between the radiation-induced decrease in CD180-negative cells and the amelioration of SLE-like morbidities. Clinical trials assessing CD180-negative cells as a therapeutic target for SLE have been hampered by the lack of validated cell markers; nonetheless, the present findings suggest that radiotherapy may be a new therapeutic strategy for managing SLE symptoms.

Estrogen Receptor-ß Gene Cytosine-Adenine (ESR2-CA) Repeat Polymorphism in Postmenopausal Colon Cancer.

The pathobiological role of estrogen is controversial in colorectal cancer. Cytosine-adenine (CA) repeat in the estrogen receptor (ER)-ß gene (ESR2-CA) is a microsatellite, as well as representative of ESR2 polymorphism. Though its function is unknown, we previously showed that a shorter allele (germline) increased the risk of colon cancer in older women, whereas it decreased it in younger postmenopausal women. ESR2-CA and ER-ß expressions were examined in cancerous (Ca) and non-cancerous (NonCa) tissue pairs from 114 postmenopausal women, and comparisons were made considering tissue types, age/locus, and the mismatch repair protein (MMR) status. ESR2-CA repeats <22/≥22 were designated as 'S'/'L', respectively, resulting in genotypes SS/nSS (=SL&LL). In NonCa, the rate of the SS genotype and ER-ß expression level were significantly higher in right-sided cases of women ≥70 (≥70Rt) than in those in the others. A decreased ER-ß expression in Ca compared with NonCa was observed in proficient-MMR, but not in deficient-MMR. In NonCa, but not in Ca, ER-ß expression was significantly higher in SS than in nSS. ≥70Rt cases were characterized by NonCa with a high rate of SS genotype or high ER-ß expression. The germline ESR2-CA genotype and resulting ER-ß expression were considered to affect the clinical characteristics (age/locus/MMR status) of colon cancer, supporting our previous findings.

Estrogen concentration and estrogen receptor-ß expression in postmenopausal colon cancer considering patient/tumor background.

PURPOSE: A large number of studies have suggested an inhibitory role of estrogens against colorectal cancer (CRC), but persistent controversy exists. CRC characteristics are affected by sex, age, and tumor locus, suggesting the need for a systematic study considering these factors. The purpose of this study was to verify the difference in the pathobiological role of estrogens in CRC according to patient/tumor backgrounds. METHODS: Surgical specimens from 116 postmenopausal women (≥ 70 years/o, n = 74; < 70 years/o, n = 42) were studied. Estrogen receptor-ß (ER-ß), the main ER in the colorectal epithelium, was immunohistochemically examined. The concentrations of estradiol (E2) and estrone (E1) were examined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These factors were compared according to the tissue type (cancerous or non-cancerous), patients' age, tumor backgrounds (locus, histology, pathological stage, status of mismatch repair protein = MMR), and clinical outcome. RESULTS: ER-ß-positivity, higher E2 concentration, deficient-MMR, and medullary/mucinous histology (Med/Muc) were closely related to right-sided tumors in women who were aged ≥ 70 years /o (R-Ca ≥ 70) and also closely related to each other. ER-ß reduction compared with non-cancerous counterparts was observed only in left-sided tumors of patients < 70 years /o (L-Ca < 70), non-Med/Muc, or proficient-MMR tumors. CONCLUSION: The present results suggest that estrogens do not suppress, but rather promote, R-Ca ≥ 70, Med/Muc, or deficient-MMR tumors, whereas estrogens suppress L-Ca < 70, non-Med/Muc, or proficient-MMR tumors, confirming the difference in pathobiological role of estrogens in postmenopausal colon cancer according to the patients' age and tumor background. This may at least partly explain the controversy regarding the association between estrogens and CRC.

The Role for miR-146b-5p in the Attenuation of Dermal Fibrosis and Angiogenesis by Targeting PDGFRα in Skin Wounds.

As a candidate microRNA antifibrotic effector in skin wounds, miR-146b-5p was upregulated by basic FGF, and PDGFRα was identified as a direct target of miR-146b-5p in fibroblasts. The treatment of fibroblasts with a miR-146b-5p mimic markedly downregulated the expression of PDGFRα and collagen type I. miR-146b-5p mimic transfection in wounds markedly attenuated cutaneous fibrosis, whereas a miR-146b-5p inhibitor strongly promoted fibrosis, with increases in PDGFRα and collagen I levels. These results indicate the positive effects of miR-146b-5p for the suppression of fibrosis, possibly through the inhibition of PDGFRα. The miR-146b-5p inhibitor markedly increased CD34+ vessel numbers and CD34 expression in wounds. We found miR-146b-5p+ cells in close contact with S100+ adipocytes. Moreover, we discovered the specific colocalization of the exosome marker CD81 and miR-146b-5p in the adipose tissue cells of mimic-transfected wounds, with miR-146b-5p signals being detected in the FSP1+ fibroblastic cells of adipose tissues. Therefore, fibroblastic cells of adipose tissues, which may specifically pick up and contain miR-146b-5p by exosome after transfection, may play an important role in the suppression of fibrosis. In this process, the inhibition of PDGFRα in adipose tissue cells by miR-146b-5p may lead to the loss of their PDGFRα-induced profibrotic activities, thereby suppressing fibrosis.

Angiogenesis in mammary Paget disease: histopathological analyses of blood vessel density and angiogenic factors.

BACKGROUND: We examined the vascularity of mammary Paget disease histologically to confirm the increased blood flow observed previously by clinical imaging. The relationships among blood vessel density (BVD), histopathological parameters of blood flow in the nipple, and the expression of angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) were examined. METHODS: We calculated the average CD34-positive BVD and podoplanin (D2-40)-positive lymphatic vessel density (LVD) and the proportion of proliferating of endothelial cells in 14 Paget disease, 3 dermatitis biopsy, and 14 age-matched control cases. As a parameter related to blood flow in the nipple, the total CD34-positive blood vessel lumen area relative to the entire nipple area was measured in each Paget disease and control case using an automated image analysis system. Immunohistochemical expression of bFGF and VEGFA in Paget cells was also examined. RESULTS: The average BVD and LVD were significantly higher in the Paget disease cases than in the dermatitis (p = 0.003) and control (p < 0.001) cases. The proportion of proliferating endothelial cells was also increased in the Paget disease cases. The ratio of the CD34-positive blood vessel lumen area to nipple area was also significantly higher in the Paget disease than control cases (p = 0.003). The average BVD was correlated with the average LVD (r = 0.734, p < 0.001) and ratio of the blood vessel lumen area to nipple area (r = 0.692, p < 0.001). Immunohistochemical expression of bFGF was strong in all Paget disease cases, with a significantly higher expression score in the Paget disease than dermatitis (p = 0.003) and control (p < 0.001) cases. The bFGF, but not VEGFA, expression score, was strongly correlated with the average BVD (r = 0.818, p < 0.001) and ratio of the blood vessel lumen area to nipple area (r = 0.503, p = 0.006). CONCLUSION: These results provide direct histopathological evidence of a marked increase in nipple blood flow in Paget disease detected by clinical imaging. bFGF is considered to play a pivotal role in angiogenesis in mammary Paget disease.

Impact of older age on clinicopathological and prognostic features of colon cancer in postmenopausal women.

To clarify the clinicopathological features of colorectal cancer in older people, systematic studies considering age, sex, and the tumor locus is needed. We focused on colon cancer in postmenopausal women (<70 years, n = 68 vs. ≥70 years, n = 85), and examined the effect of age on clinicopathological features. Rates of medullary carcinoma /mucinous carcinoma were higher and pathological stages at diagnosis were less advanced in patients ≥70 years compared with <70 years. Matching pathological stages, no significant difference in disease-free interval was observed according to age; however, disease-specific survival (DSS) was poorer in patients ≥70 years than <70 years, being significantly different in stage IV cases. Regarding post-metastasis/recurrence (met/rec) cases, chemotherapy and surgery for metastasis were less frequent in those aged ≥70 years than <70 years. Post-met/rec DSS was poorer in ≥70 years, those with microsatellite instability, and those without surgery for met/rec than in each counterpart; however, post-met/rec chemotherapy exhibited no effect. Multivariate analyses revealed that an older age and no surgery for metastasis were independent predictors of disease-specific death. These findings remained after excluding stage IV cases. Older age was a potent risk factor of rapid disease-specific death after met/rec.

Settlement of Stenotic Site and Enhancement of Risk Factor Load for Atherosclerosis in Left Anterior Descending Coronary Artery by Myocardial Bridge.

OBJECTIVE: The aim of this study was to investigate the influence of a myocardial bridge (MB) on atherosclerosis development in the left anterior descending artery of the normal heart and the importance of traditional risk factors (RFs). An additional objective was to determine the correlation between intimal thickening and luminal narrowing. APPROACH AND RESULTS: The left anterior descending artery from 150 autopsied hearts was treated with formalin perfusion fixation, and each left anterior descending artery was serially cross-sectioned. The intima-media and luminal stenosis ratios were examined using computer-assisted histomorphometry. The luminal stenosis ratio was closely correlated with the intima-media ratio (r=0.792; P<0.001). When an MB was present, the luminal stenosis ratios proximal to the MB in the RF (+) group were significantly greater than those in the RF (-) group (P=0.022 by a multiple comparison test), but there were no differences between the RF (+) and RF (-) groups when an MB was absent. In addition, the site of the greatest stenosis in the MB (+) RF (+) group was 2.5 cm proximal to the MB entrance. Multivariate analyses indicated that age was an independent factor for luminal stenosis ratios ≥50% and 60% (P=0.002 and 0.029, respectively). Furthermore, the presence of an MB plus RFs was an independent factor for a luminal stenosis ratio ≥70% (P=0.037). CONCLUSIONS: An MB enhances left anterior descending artery atherosclerosis development at a site proximal to the MB entrance, particularly in subjects who have some RFs.

Giant Cavernous Hemangioma of the Adrenal Gland in an Elderly Patient.

Cavernous hemangioma is a rare, non-functional, benign adrenal tumor. Adrenal cavernous hemangioma is often diagnosed after surgery with a histologic examination. A 70-year-old man complaining of appetite loss was admitted to our hospital. An incidental large left adrenal mass was found by computed tomography (CT). There were no clinical signs of adrenogenital syndrome, Cushing's syndrome or primary aldosteronism. Total resection was carried out. The pathological diagnosis was cavernous hemangioma. The differentiation of adrenal tumor is necessary in cases of large tumors, and resection is desirable given the risks of hemorrhaging and rupture.

Basic Fibroblast Growth Factor Induces Angiogenic Properties of Fibrocytes to Stimulate Vascular Formation during Wound Healing.

The role of fibrocytes in wound angiogenesis remains unclear. We therefore demonstrated the specific changes in fibrocyte accumulation for angiogesis in basic fibroblast growth factor (bFGF)-treated wounds. bFGF-treated wounds exhibited marked formation of arterioles and inhibition of podoplanin+ lymph vessels that were lacking in vascular endothelial growth factor-A-treated wounds. Real-time PCR in bFGF-treated wounds manifested enhanced expression of CD34, CD31, and bFGF mRNA and reduced expression of podoplanin and collagen type I, III, and IV mRNA. Double immunofluorescence staining focusing on fibrocyte detection in bFGF-treated wounds showed increased formation of capillary-like structures composed of CD34+/procollagen I+ fibrocytes, with a lack of capillary-like structures formed by CD45+/procollagen I+ or CD11b+/procollagen I+ fibrocytes. However, vascular endothelial growth factor-A-treated wounds lacked capillary-like structures composed of CD34+/procollagen I+ fibrocytes, with increased numbers of CD34+/fetal liver kinase-1+ endothelial progenitor cells. Furthermore, fibroblast growth factor receptor 1 siRNA injection into wounds, followed by bFGF, inhibited the formation of capillary-like structures composed of CD34+/procollagen I+ fibrocytes, together with inhibited mRNA expression of CD34 and CD31 and enhanced mRNA expression of collagen type I, indicating the requirements of bFGF/fibroblast growth factor receptor 1 system for capillary structure formation. This study highlights the angiogenic properties of CD34+/procollagen I+ fibrocytes specifically induced by bFGF, providing new insight into the active contribution of fibrocytes for vascular formation during wound healing.

Antifibrotic response of cardiac fibroblasts in hypertensive hearts through enhanced TIMP-1 expression by basic fibroblast growth factor.

BACKGROUND: Cardiac fibroblasts (CFs) play a pivotal role in the development of myocardial fibrosis. We previously demonstrated that direct injection of basic fibroblast growth factor (bFGF) into the hypertensive Dahl salt-sensitive (DS) rat heart prevented systolic dysfunction and left ventricular dilation effectively. However, the precise role played by bFGF in fibrotic response of CFs remains unclear. We suggested potential effects of bFGF on the fibrotic response of CFs in vitro. METHODS AND RESULTS: Histopathologic assessment of cardiac fibrosis demonstrated a marked decline in the extent of perivascular and interstitial fibrosis in bFGF-injected hypertensive DS rat hearts. CFs harvested from the hearts of noninjected DS rats demonstrated a significantly increased messenger RNA (mRNA) expression of matrix metalloproteinase (MMP)-2, MMP-9, and both collagen I and III. In contrast, bFGF treatment in the CFs induced a marked increase in tissue inhibitor of MMP (TIMP)-1 expression and a marked decline in MMP-9 activation. bFGF also induced a decline in α-smooth muscle actin and collagen I and III mRNA expression in the CFs accompanied by inhibited differentiation of CFs into myofibroblasts. Small interfering RNA targeting FGF receptor 1 confirmed a specific interference of the mRNA expression changes elicited by bFGF. In vivo examination confirmed many TIMP-1-positive CFs in perivascular spaces of bFGF-injected hearts. CONCLUSIONS: Up-regulated TIMP-1 expression and down-regulated MMP-9 activation by bFGF in CFs could prevent excessive ECM degradation and collagen deposition in perivascular spaces effectively, leading to prevention of cardiac fibrosis during hypertensive heart failure. SUMMARY: Cardiac fibroblasts (CFs) play a pivotal role in myocardial fibrosis. The precise role of CFs in fibrotic response played by growth factors remains unclear. Our results indicates that basic fibroblast growth factor could up-regulate TIMP-1 expression and down-regulate MMP-9 activation in CFs in perivascular spaces, leading to inhibited progression of cardiac fibrosis during hypertensive heart failure.

Association of variance in anatomical elements of myocardial bridge with coronary atherosclerosis.

OBJECTIVES: The myocardial bridge (MB) is an anatomical structure consisting of myocardium covering a part of the left anterior descending coronary artery (LAD). The extent and spatial distribution of atherosclerosis in the LAD with an MB is influenced by the anatomical properties of the MB. In this study, the relationship between the overall anatomical framework of the MB including the periarterial adipose tissue as well as fibrosis of the MB itself and coronary atherosclerosis was histomorphometrically examined. METHODS: Full-length LADs with an MB from 180 autopsied hearts were cross-sectioned at 5-mm intervals. Together with measurements of MB - length, thickness, and location, proportional decrease of the atherosclerosis ratio of LAD segments beneath MB for that of LAD segments proximal to MB was defined as the atherosclerosis suppression ratio. The area ratio of adipose tissue in the periarterial area beneath MB and area ratio of fibrosis in the MB muscle were also measured. RESULTS: The atherosclerosis suppression ratio was significantly proportional to MB length and thickness. Periarterial adipose tissue beneath MB was detected in all cases (100%), and fibrosis within MB muscle for 136 cases (75.6%). The amount of adipose tissue beneath MB and MB fibrosis did not statistically affect the atherosclerosis suppression ratio. Multivariate analysis revealed MB length and thickness were the independent factors affecting the atherosclerosis suppression ratio. CONCLUSIONS: The anatomical properties of an MB, especially of its length and thickness, play decisive roles as regulators of atherosclerosis in the LAD regardless of the amount of adipose tissue around LAD and MB fibrosis.

Histopathologic profiles of coronary atherosclerosis by myocardial bridge underlying myocardial infarction.

OBJECTIVE: Anatomic properties of myocardial bridge (MB) are sometimes responsible for myocardial infarction (MI) through the changes in the atherosclerosis distribution in the left ascending coronary artery (LAD). The purpose of this study was to investigate histopathologic profiles of atherosclerotic lesions resulting from the MB presence in the LAD in the MI cases. METHODS: In 150 consecutive autopsied MI hearts either with MBs [MI(+)MB(+); n = 67] or without MBs [MI(+)MB(-); n = 83] and 100 normal hearts with MBs [MI(-)MB(+)], LADs were consecutively cross-sectioned at 5-mm intervals. The most advanced intimal lesion and unstable plaque-related lesion characteristics (UPLCs) in each section were histopathologically evaluated in conjunction with the anatomic properties of the MB, such as its thickness, length, location, and MB muscle volume burden (MMV: the total volume of MB thickness multiplied by MB length). RESULTS: The MB showed a significantly greater thickness (P = 0.0090), length (P = 0.0300), and MMV (P = 0.0019) in MI(+)MB(+) than in MI(-)MB(+). Mean age of acute MI cases was significantly younger (P = 0.0227) in MI(+)MB(+) than in MI(+)MB(-). Frequency of plaque fissure/rupture in the proximal LAD was significantly higher in acute MI cases of MI(+)MB(+) than in MI(+)MB(-). UPLCs tended to be located proximally in MI(+)MB(+) and frequent 2.0 cm or more proximal to the MB entrance in MI(+)MB(+). CONCLUSION: In MI(+)MB(+), UPLCs tend to be located more proximally, and a plaque in the LAD proximal to the MB is prone to rupture, resulting in MI at younger age.

Pathogenesis of lupus-like nephritis through autoimmune antibody produced by CD180-negative B lymphocytes in NZBWF1 mouse.

Toll-like receptors appear to play an important role in the pathogenesis of lupus-like nephritis in mice. In human and mouse, CD180 is a homologue of TLR4. In SLE patients, the number of CD180-negative B cells in peripheral blood changes in parallel with disease activity. In the present study using NZBWF1 mice, the population of splenic CD180-negative B cells increased with progression of renal lesions and aging. These cells produced both anti-dsDNA and histone antibodies; the peripheral blood levels of anti-dsDNA antibody increased markedly with aging. B cells infiltrating into renal lesions were CD180-negative and produced anti-dsDNA antibody. Considered together, these findings indicate that CD180-negative B cells contribute significantly to development of SLE-like morbidity in NZBWF1 mice by autoantibody production.

Histopathological predictors of regional lymph node metastasis at the invasive front in early colorectal cancer.

AIMS: In early colorectal cancer (ECC), prediction of lymph node (LN) metastasis is vital for the decision of additional surgical treatment after endoscopic mucosal/submucosal resection. The aim of this study was to determine the relationship between LN metastasis and comprehensive histopathological findings including the cancer microenvironment in ECC. METHODS AND RESULTS: Using 111 ECC cases, including 36 cases with LN metastasis, histopathological observations and immunohistochemistry for lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), von Willebrand factor, matrix metalloproteinase-7 (MMP-7), CXC chemokine ligand-12 (CXCL12) and angiopoietin-like-4 (ANGPTL4) were conducted. Relationships between LN metastasis and growth pattern, status of muscularis mucosae, depth of cancer invasion, overall histopathological type, histopathological type at the invasive front, tumour budding, neutrophil infiltration in cancer cells (NIC), fibrotic cancer-stroma type, Crohn's-like lymphoid reaction, microscopic abscess formation and lymphatic invasion were determined. In addition, the expression of MMP-7, CXCL12 and ANGPTL4 in cancer cells at the invasive front were also considered in the context of LN metastasis. By multivariate analysis, lymphatic invasion, NIC and MMP-7 expression at the invasive front were independent predictors of LN metastasis. CONCLUSIONS: LN metastasis is regulated not only by the characteristics of cancer cells but also by microenvironmental factors of lymphatics and neutrophils, especially at the invasive front.

Malignant adrenal rest tumor of the retroperitoneum producing adrenocortical steroids.

We present a case of a malignant adrenal rest tumor arising from the retroperitoneum with Cushing's syndrome in a 31-year-old female. Her serum cortisol and dehydroepiandrosterone sulfate levels were elevated, while adrenocorticotropic hormone levels were low. Computed tomography scans and magnetic resonance imaging revealed a retroperitoneal tumor with no visible lesions in the adrenal glands and ovaries. From those results and the histopathologic findings following biopsy of an enlarged supraclavicular lymph node, the patient was diagnosed as a malignant adrenal rest tumor of the retroperitoneum. Despite chemotherapy, the patient died of rapid development of multiple metastases. Autopsy revealed a large tumor that extended around the abdominal aorta from the levels of the left kidney to the aortic bifurcation with generalized metastases. Tumor cells were characterized by clear and eosinophilic cytoplasm and atypical nuclei that exhibited frequent and atypical mitoses. Immunohistochemistry regarding steroidogenesis was performed and revealed that the tumor cells were immunopositive for adrenal 4 binding protein/steroidogenic factor-1, cholesterol side-chain cleavage enzyme, 17α-hydroxylase, and 21-hydroxylase. We thus elucidated the adrenocortical steroid production in the tumor cells causing Cushing's syndrome. This case report first demonstrates the steroidogenic capacity in a malignant adrenal rest tumor.

Solitary hepatic lymphangioma: report of a case.

A 52-year-old woman presented with upper abdominal pain. Abdominal ultrasonography showed a 4-cm well-defined mass containing solid and cystic components in segment IV of the liver, and contrast-enhanced T1-weighted magnetic resonance imaging revealed heterogeneous enhancement within the tumor, indicating a solid or fibrous component. There were no cystic lesions in any other organs. A partial hepatectomy was performed, based on a preoperative diagnosis of sclerosing hemangioma and biliary cystadenoma or cystadenocarcinoma. Pathologically, the tumor appeared to be a multilocular and cystic lesion lined by attenuated endothelial- like cells with no atypia. Immunohistochemistry demonstrated the endothelial-like cells to be positive for the lymphatic-specific markers D2-40, LYVE-1, and Prox-1, which proved helpful for confirming the diagnosis as solitary hepatic lymphangioma. This case is presented with details of the pathologic and radiologic findings, because solitary hepatic lymphangioma is an extremely rare tumor and no previous reports have provided details of the immunohistochemical characteristics.

Prognostic value of LYVE-1-positive lymphatic vessel in tongue squamous cell carcinomas.

The density of lymphatic vessels in 52 cases of human tongue squamous cell carcinoma (TSCC) and normal portions was analyzed. TSCC specimens were immunostained with antibodies against lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and podoplanin monoclonal antibody (D2-40). The significance of the LYVE-1-positive vessel density (LVD) was calculated in 6 topographic areas and investigated on the basis of specific clinical and histo-pathological parameters. LYVE-1 positivity was more evident in the muscular area than the submucosal area, while small D2-40-positive lymphatic vessels were not demonstrable in muscular endomysium. The LVD in peri-tumoral submucosal and peri-tumoral muscular areas was lower than in normal counterparts (p<0.01). LVD was higher in the tumor invasion front as compared to tumor-associated stroma (p<0.01). Low LVD in invasion front and peri-tumoral submucosal area was significantly related to regional lymph node metastasis (p<0.05 and p<0.01, respectively). The decrease of LYVE-1-positive lymphatic vessels in the invasion front and peri-tumoral submucosal area would seem to predict cervical lymph node metastasis in TSCC.

The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing.

While investigating the mechanisms underlying cell death during wound healing processes, we uncovered the pro-apoptotic effects of basic fibroblast growth factor (bFGF) on granulation tissue fibroblasts following pretreatment with transforming growth factor (TGF)-beta1 in vitro. bFGF induced caspase-3 activation and apoptosis in TGF-beta1-pretreated granulation tissue-derived fibroblasts (GF-1) following bFGF treatment for 48 and 96 h. In contrast, fibroblasts that had been treated in the same manner and that originated from the uninjured dermis did not display apoptosis, indicating that the mechanisms underlying apoptosis events in fibroblasts that originate from normal dermal and wound tissues differ. In this process, we also found that bFGF inhibited Akt phosphorylation at serine 473 and induced a rapid loss of phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in pretreated GF-1 cells, an event that coincided with the dissociation of phosphorylated FAK from the focal adhesions. Therefore, inhibition of survival signals relayed via the disrupted focal adhesion structures and inactivated Akt following bFGF treatment may lead to apoptosis in GF-1 cells pretreated with TGF-beta1. Pretreatment of GF-1 with TGF-beta1 followed by the addition of bFGF resulted in significantly greater inhibition of phosphorylation of Akt and FAK compared to treatment with TGF-beta1 or bFGF alone. The combinatorial treatment also led to proteolysis of FAK and inhibition of FAK and Akt protein expression in GF-1 cells. These findings demonstrated a significant role for the two cytokines in apoptosis of granulation tissue fibroblasts during wound healing. In vivo studies also confirmed a marked decline in phosphorylation and protein expression of Akt and FAK in bFGF-injected skin wounds. These results led to the hypothesis that temporal activation of TGF-beta1 and bFGF at the injury site promotes apoptosis in granulation tissue fibroblasts, an event that is critical for the termination of proliferative granulation tissue formation.

Significance of PGP9.5 expression in cancer-associated fibroblasts for prognosis of colorectal carcinoma.

To assess the expression of a cancer-associated fibroblasts (CAFs) marker as an indicator of prognosis, we raised anti-protein gene product 9.5 (PGP9.5) monoclonal antibody against cultured fibroblasts. PGP9.5 expression in cultured normal fibroblasts was increased by transforming growth factor beta stimulation, indicating the phenotypic alteration to activated fibroblast. We immunohistochemically evaluated PGP9.5 expression with the CAFs of 110 colorectal cancer cases under T3 stage. PGP9.5 immunoreactivity in 30% or more of CAFs was defined as high PGP9.5 expression, and the other cases were considered as having low PGP9.5 expression. Patients with high PGP9.5 expression (42.7%) had significantly shorter survival and a higher incidence of recurrence than the low PGP9.5 expression group (P = .002 and P < .001, respectively). Multivariate analysis indicated PGP9.5 expression as an independent prognostic factor for overall and recurrence-free survival partly as well as lymph node metastasis. These results indicate that PGP9.5 expression in CAFs is a helpful finding to represent the overall biologic behavior of advanced colorectal cancer.

Prognostic significance of CXCL12 expression in patients with colorectal carcinoma.

The present study investigated the protein expression level of CXCL12 in colorectal cancer and aimed to elucidate its association with prognosis. CXCL12 positivity in 50% or more of tumor cells was defined as high expression and that in less than 50% of the tumor cells as low expression. CXCL12+ tumor budding at the invasive front was divided into 2 grades: high with 10 or more budding foci per x200 field of view and low grade with fewer than 10 budding foci. Patients with high expression (72.7%) and high grade CXCL12+ tumor budding (43.0%) had significantly shorter survival than patients with low expression (P = .014) and low grade (P = .003), respectively. Patients with a combination of high expression and high grade had the worst outcome (P < .001). Our study demonstrated that CXCL12 expression in colorectal cancer cells and at sites of budding were significant prognostic factors. Furthermore, together with lymph node metastasis, a combination of both expression patterns was a more powerful independent prognostic factor.