Integrin expression at the bone/biomaterial interface

The aim of this study was to visualize integrin expression by cells in interface tissue in relation to their ligands. Tissue samples were obtained from 25 patients undergoing revision of aseptically loose total joint replacements. Serial sections were immunolabeled for the integrins alpha (2)beta (1) alpha (v)beta (3), alpha (4)beta (1) alpha (L)beta (2) (CD11a), alpha (M)beta (2) (CD11b), and alpha (x)beta (2) (CD11c), and the ligands fibronectin, laminin, vitronectin, intercellular adhesion molecule-1, and vascular adhesion molecule-1. Most cells were found to express alpha (2)beta (1) most macrophages and giant cells expressed CD11b, and the majority of CD11a was found on perivascular T lymphocytes. From the small amount of alpha (4)beta (1) and vascular adhesion molecule-1 expression in the interface tissue and the combination of CD11a, CD11b, and intercellular adhesion molecule-1 expression, it would seem that macrophages use beta (2) integrins to transmigrate. (C) 2001 John Wiley & Sons, Inc.

The Deubiquitinating Enzyme USP17 is Essential for GTPase Subcellular localization and Cell Motility

Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease.

Sequence analysis of the 3' hypoxia-responsive element of the human erythropoietin gene in patients with erythrocytosis

Erythrocytosis arises from a variety of pathogenic mechanisms. We sequenced a 256-bp region 3' to the erythropoietin (Epo) gene which included a 24- to 50-bp minimal hypoxia-responsive element spanning HIF-1- and HNF-4-binding sites in 12 patients with erythrocytosis and 4 normal subjects. Four polymorphisms were found, none of which affected the HIF-1-binding site, although one polymorphism was present in the HNF-4 consensus region. The data indicate that none of these polymorphisms cause erythrocytosis.

Rod photoreceptor loss in Rho(-/-) mice reduces retinal hypoxia and hypoxia-regulated gene expression

PURPOSE. This study was conducted to evaluate whether regions of the retinal neuropile become hypoxic during periods of high oxygen consumption and whether depletion of the outer retina reduces hypoxia and related changes in gene expression.

METHODS. Retinas from rhodopsin knockout (Rho(-/-)) mice were evaluated along with those of wild-type (WT) control animals. Retinas were also examined at the end of 12-hour dark or light periods, and a separate group was treated with L-cis-diltiazem at the beginning of a 12-hour dark period. Hypoxia was assessed by deposition of hypoxyprobe (HP) and HP-protein adducts were localized by immunohistochemistry and quantified using ELISA. Also, hypoxia-regulated gene expression and transcriptional activity were assessed alongside vascular density.

RESULTS. Hypoxia was observed in the inner nuclear and ganglion cell layers in WT retina and was significantly reduced in Rho (-/-) mice (P < 0.05). Retinal hypoxia was significantly increased during dark adaptation in WT mice (P < 0.05), whereas no change was observed in Rho(-/-) or with L-cis-diltiazem-treated WT mice. Hypoxia-inducible factor (HIF)-1 alpha DNA-binding and VEGF mRNA expression in Rho(-/-) retina was significantly reduced in unison with outer retinal depletion (P < 0.05). Retina from the Rho(-/-) mice displayed an extensive intraretinal vascular network after 6 months, although there was evidence that capillary density was depleted in comparison with that in WT retinas.

CONCLUSIONS. Relative hypoxia occurs in the inner retina especially during dark adaptation. Photoreceptor loss reduces retinal oxygen usage and hypoxia which corresponds with attenuation of the retinal microvasculature. These studies suggest that in normal physiological conditions and diurnal cycles the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle reductions in perfusion.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

A narrow-band ultraviolet B course improves vitamin D balance and alters cutaneous CYP27A1 and CYP27B1 mRNA expression levels in haemodialysis patients supplemented with oral vitamin D

Background: Chronic kidney disease (CKD) patients on dialysis are prone to vitamin D insufficiency despite oral vitamin D supplementation. Here, we studied whether narrow-band ultraviolet B (NB-UVB) exposures improve vitamin D balance.

Methods: 14 haemodialysis patients and 15 healthy subjects receiving oral cholecalciferol 20 µg daily got nine NB-UVB exposures on the entire body. Serum 25-hydroxyvitamin D (25(OH)D) was measured by radioimmunoassay. Cutaneous mRNA expression levels of CYP27A1 and CYP27B1, two enzymes required for hydroxylation of vitamin D into its active metabolite, were also measured.

Results: The baseline serum 25(OH)D concentration was 57.6 ± 18.2 nmol/l in the CKD patients and 74.3 ± 14.8 nmol/l in the healthy subjects. The NB-UVB course increased serum 25(OH)D by 14.0 nmol/l (95% CI 8.7-19.5) and 17.0 nmol/l (CI 13.7-20.2), respectively. At baseline the CKD patients showed significantly increased CYP27B1 levels compared to the healthy subjects.

Conclusions: A short NB-UVB course is an efficient way to improve vitamin D balance in CKD patients on dialysis who are receiving oral vitamin D supplementation. The increased cutaneous CYP27B1 levels in the CKD patients suggest that the loss of renal activity of this enzyme is at least partially compensated for by the skin.

Narrow-band ultraviolet B treatment boosts serum 25-hydroxyvitamin D in patients with psoriasis on oral vitamin D supplementation

A course of treatment with narrow-band ultraviolet B (NB-UVB) improves psoriasis and increases serum 25-hydroxyvitamin D (25(OH)D). In this study 12 patients with psoriasis who were supplemented with oral cholecalciferol, 20 µg daily, were given a course of NB-UVB and their response measured. At baseline, serum 25(OH)D was 74.14 ± 22.9 nmol/l. At the 9th exposure to NB-UVB 25(OH)D had increased by 13.2 nmol/l (95% confidence interval (95% CI) 7.2–18.4) and at the 18th exposure by 49.4 nmol/l (95% CI 35.9–64.6) above baseline. Psoriasis Area Severity Index score improved from 8.7 ± 3.5 to 4.5 ± 2.0 (p < 0.001). At baseline, psoriasis lesions showed low vitamin D metabolizing enzyme (CYP27A1, CYP27B1) and high human β-defensin-2 mRNA expression levels compared with those of the healthy subjects. In conclusion, NB-UVB treatment significantly increases serum 25(OH)D in patients with psoriasis who are taking oral vitamin D supplementation, and the concentrations remain far from the toxicity level. Healing psoriasis lesions show similar mRNA expression of vitamin D metabolizing enzymes, but higher antimicrobial peptide levels than NB-UVB-treated skin in healthy subjects.

Narrow-band ultraviolet B exposure increases serum vitamin D levels in haemodialysis patients

Chronic kidney disease (CKD) patients are especially prone to vitamin D insufficiency. Narrow-band ultraviolet B (NB-UVB) treatment increases serum 25-hydroxyvitamin D [25(OH)D] in dermatological patients, and we studied whether it also improves vitamin D balance in CKD patients on haemodialysis.

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes.

Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells

OBJECTIVE: The efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of hypoxia-inducible-factor 1α (HIF1A). Overexpression of the hypoxia-responsive gene class III β-tubulin (TUBB3) has been associated with docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel.

METHODS: The cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with methylation status at the HIF1A binding site.

RESULTS: Prostate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression.

CONCLUSION: These data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer patients.

Microtubule-targeting-compound PBOX-15 radiosensitizes cancer cells in vitro

BACKGROUND: We proposed to investigate the radiosensitizing properties of PBOX-15, a novel microtubule-disrupting agent, in a panel of cancer cell lines.

RESULTS: PBOX-15 treatment was associated with significant cell kill and increased radiosensitivity in all three cell lines tested. The number of surviving cells in response to the combined treatment was significantly less than PBOX -15 alone in 22Rv1 cells. In these cells, radiosensitisation correlated with induction of G2/M cell cycle arrest by PBOX-15. The compound sustained its activity and increased HIF-1Α expression under hypoxic conditions. PBOX-15 prevented onset of hypoxia-induced radioresistance in hypoxic prostate cells and reduced the surviving fraction of irradiated hypoxic cells to levels similar to those achieved under aerobic conditions.

METHODS: Clonogenic assays were used to determine sensitivity of a panel of cancer cell lines (22Rv1, A549, U87) to PBOX-15 alone or in combination with a single 2Gy dose fraction. Induction of cell cycle arrest and apoptosis was investigated in 22Rv1 prostate cancer cells. The cytotoxic properties of the compound under hypoxic conditions were correlated with Hypoxia Inducible Factor 1 alpha (HIF-1Α) gene and protein expression levels and its radiosensitisation potential was investigated in hypoxic 22Rv1 using clonogenic assays.

CONCLUSIONS: This preliminary data identifies the potential of PBOX-15 as a novel radiosensitising agent for the management of solid tumours and eradication of hypoxic cells.

The HIF-1alpha C1772T polymorphism may be associated with susceptibility to clinically localised prostate cancer but not with elevated expression of hypoxic biomarkers

We investigated the role of the C1772T polymorphisms in exon 12 of the Hypoxia-inducible factor-1 alpha (HIF-1alpha) gene C1772T genotype in prostate cancer (PCa) and amplification of the hypoxic response. We identified the heterozygous germline CT genotype as an increased risk factor for clinically localised prostate cancer (Odds ratio = 6.2; p < 0.0001). While immunostaining intensity for HIF-1alpha and VEGF was significantly enhanced in 75% of PCa specimens when compared to matched benign specimens (p < 0.0001), the CT genotype did not modulate the kinetics of HIF-1alpha protein expression in hypoxia in vitro, and was not associated with enhanced expression of hypoxic biomarkers. This study provides the first evidence of an increased risk for clinically localised prostate cancer in men carrying the C1772T HIF-1alpha gene polymorphism. Although our results did not suggest an association between expression of hypoxic biomarkers and genotype status, the correlation may merit further investigation.

Hypoxia in prostate cancer:a powerful shield against tumour destruction?

Tumour hypoxia is progressively emerging as a common feature of prostate tumours associated with poor prognosis. While the molecular basis of disease progression is increasingly well documented, the potential role of hypoxia in these processes remains poorly evaluated. By dissecting the impact of hypoxia-inducible factor 1 alpha on molecular responses, this review provides evidence for a powerful protecting role of oxygen deprivation against oxidative stress injury, androgen deprivation, chemotherapeutic and radiation cytotoxicity. We propose hypoxia as a potent tumour-induced shield against destruction and suggest its targeting may need to be routinely addressed in the management of prostate cancer.

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Serum Cytokines and Adipokines in Periodontitis

Objective: To investigate whether there was an association between serum levels of pro-inflammatory cytokines, adipokines and periodontitis in 60-70 year-old men in Northern Ireland. Methods: Test subjects (n=61) with established periodontitis, categorised by loss of periodontal attachment (PAL) and pocketing of at least 5mm, were matched for age, smoking and BMI with controls (n=60) who were periodontitis resistant (no PAL or pocketing over 3mm). Serum levels of IL 1 alpha, TNF alpha, IL 6, adiponectin, resistin, leptin and C-reactive protein (CRP) were measured from serum using a multiplex array (Randox, UK). Mann Whitney analysis was used with the level of significance set at p<0.05. Results: There was considerable inter-individual variability in all the analytes measured. There was a significantly higher level of IL 6 in men with periodontitis (3.25 pg/ml, SD 2.29) than in those with no periodontitis (2.38 pg/ml, SD1.62), p=0.0041 (after Bonferroni correction for multiple testing p=0.029). Serum IL 6 was strongly correlated with CRP: r=0.52 (95% CI 0.38-0.64), p<0.0001. There was a lower serum level of adiponectin in men with periodontitis (3662.1 ng/ml, SD 2383.4) compared with those who were periodontitis resistant (4265.9 ng/ml, SD 2266.0), which just escaped statistical significance at p=0.068. There were no significant differences in the serum levels of any of the other analytes measured in relation to periodontal status. Conclusions: There was evidence in the 60-70 year-old male population investigated that periodontitis was associated with an increased serum level of IL 6.