Cell therapy for renal regeneration--time for some joined-up thinking?
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Animals↗
Biomedical subjects
Richard Poulsom
Publications and source records attributed to Richard Poulsom.
Expression of HIF-1alpha, HIF-2alpha (EPAS1), and their target genes in paraganglioma and pheochromocytoma with VHL and SDH mutations.
CONTEXT: Activation of the hypoxia-inducible transcription factors HIF-1 and HIF-2 and a HIF-independent defect in developmental apoptosis have been implicated in the pathogenesis of pheochromocytoma (PCC) associated with VHL, SDHB, and SDHD mutations. OBJECTIVE: Our objective was to compare protein (HIF-1alpha, EPAS1, SDHB, JunB, CCND1, CD34, CLU) and gene (VEGF, BNIP3) expression patterns in VHL and SDHB/D associated tumors. RESULTS: Overexpression of HIF-2 was relatively more common in VHL than SDHB/D PCC (12 of 13 vs. 14 of 20, P = 0.02), whereas nuclear HIF-1 staining was relatively more frequent in SDHB/D PCC (19 of 20 vs. 13 of 16, P = 0.04). In addition, CCND1 and VEGF expression (HIF-2 target genes) was significantly higher in VHL than in SDHB/D PCC. These findings suggest that VHL inactivation leads to preferential HIF-2 activation and CCND1 expression as described previously in VHL-defective renal cell carcinoma cell lines but not in other cell types. These similarities between the downstream consequences of VHL inactivation and HIF dysregulation in renal cell carcinoma and PCC may explain how inactivation of the ubiquitously expressed VHL protein results in susceptibility to specific tumor types. Both VHL and SDHB/D PCC demonstrated reduced CLU and SDHB expression. SDHB PCC are associated with a high risk of malignancy, and expression of (proapototic) BNIP3 was significantly lower in SDHB than VHL PCC. CONCLUSION: Although inactivation of VHL and SDHB/D may disrupt similar HIF-dependent and HIF-independent signaling pathways, their effects on target gene expression are not identical, and this may explain the observed clinical differences in PCC and associated tumors seen with germline VHL and SDHB/D mutations.
Adrenal Gland Neoplasms↗
Alterations in the composition of the supramucosal defense barrier in relation to disease severity of ulcerative colitis.
Mucin glycoproteins and trefoil peptides play an important role in protection and repair of the gastrointestinal epithelium. This study investigates alterations in mucin and trefoil peptide gene expression and product localization in ulcerative colitis (UC). Product localization and message expression of mucin MUC1 to 6 and trefoil peptide TFF1 to 3 genes was analyzed in rectosigmoid tissue from a cohort of patients with active UC and compared with that of normal colorectal mucosa. MUC1 expression was upregulated in severe UC at the site of rupture of crypt abscesses. Reduction in MUC2 expression occurred in UC adjacent to ulceration. No alteration in MUC3 or MUC4 gene expression was detectable in UC compared with normal colorectal mucosa. No ectopic expression of MUC5AC, MUC5B, or MUC6 was identified in UC. Ectopic TFF1 expression was identified in tissues eliciting histological features of severe disease. Decreased TFF3 localization was demonstrated in UC tissues, but no TFF2 expression was detected in any colorectal specimens. Subtle alterations in composition of the supramucosal defense barrier exist in UC and vary in relation to clinical severity of disease. There is upregulation in mucin MUC1 at crypt abscesses and neo-expression of TFF1 trefoil peptide in severe disease.
Adult↗
Up-regulation of endothelial delta-like 4 expression correlates with vessel maturation in bladder cancer.
PURPOSE: Angiogenesis and vascular endothelial growth factor (VEGF) expression are associated with a poor outcome in bladder cancer. To understand more about the mechanisms, we studied the role of delta-like 4 (DLL4), an endothelial-specific ligand of the Notch signaling pathway, in bladder cancer angiogenesis. EXPERIMENTAL DESIGN: The expression of DLL4, CD34, and VEGF were studied in a cohort of 60 bladder tumors and 10 normal samples using quantitative PCR. In situ hybridization was used to study the pattern of DLL4 expression in 22 tumor and 9 normal samples. Serial sections were also stained for CD34 and alpha-smooth muscle actin (alpha-SMA) using conventional immunohistochemistry. RESULTS: The expression of DLL4 was significantly up-regulated in superficial (P < 0.01) and invasive (P < 0.05) bladder cancers. DLL4 expression significantly correlated with CD34 (P < 0.001) and VEGF (P < 0.001) expression. The in situ hybridization studies showed that DLL4 was highly expressed within bladder tumor vasculature. Additionally, DLL4 expression significantly correlated with vessel maturation as judged by periendothelial cell expression of alpha-SMA, 98.7% of DLL4-positive tumor vessels coexpressed alpha-SMA, compared with 64.5% of DLL4-negative tumor vessels (P < 0.001). High DLL4 expression may have prognostic value in superficial and invasive bladder. CONCLUSION: DLL4 expression is associated with vascular differentiation in bladder cancer; thus, targeting DLL4 may be a novel antiangiogenic therapy.
Actins↗
Bone marrow-derived cells contribute to podocyte regeneration and amelioration of renal disease in a mouse model of Alport syndrome.
In a model of autosomally recessive Alport syndrome, mice that lack the alpha3 chain of collagen IV (Col4alpha3(-/-)) develop progressive glomerular damage leading to renal failure. The proposed mechanism is that podocytes fail to synthesize normal glomerular basement membrane, so the collagen IV network is unstable and easily degraded. We used this model to study whether bone marrow (BM) transplantation can rectify this podocyte defect by correcting the deficiency in Col4alpha3. Female C57BL/6 Col4alpha3(-/-) (-/-) mice were transplanted with whole BM from male wild-type (+/+) mice. Control female -/- mice received BM from male -/- littermates. Serum urea and creatinine levels were significantly lower in recipients of +/+ BM compared with those of -/- BM 20 weeks post-transplant. Glomerular scarring and interstitial fibrosis were also significantly decreased. Donor-derived cells were detected by in situ hybridization (ISH) for the Y chromosome, and fluorescence and confocal microscopy indicated that some showed an apparent podocyte phenotype in mice transplanted with +/+ BM. Glomeruli of these mice showed small foci of staining for alpha3(IV) protein by immunofluorescence. alpha3(IV) mRNA was detectable by reverse transcription-polymerase chain reaction and ISH in some mice transplanted with +/+ BM but not -/- BM. However, a single injection of mesenchymal stem cells from +/+ mice to irradiated -/- recipients did not improve renal disease. Our data show that improved renal function in Col4alpha3(-/-) mice results from BM transplantation from wild-type donors, and the mechanism by which this occurs may in part involve generation of podocytes without the gene defect.
Animals↗
Adult leydig cell tumors of the testis caused by germline fumarate hydratase mutations.
CONTEXT: Leydig cell tumors (LCTs) are the most common non-germ-cell neoplasms of the testis. LCTs are often hormonally active and can result in precocious virilization or in adult feminization. We identified an LCT in an affected individual from a kindred with hereditary leiomyomatosis and renal cell cancer (HLRCC) and a germline fumarate hydratase (FH) mutation (N64T). OBJECTIVE: Our objective was to investigate the role of FH mutations in predisposition to LCTs. DESIGN: We tested for pathogenic effects of the N64T mutation and screened an additional 29 unselected adult LCTs for FH alterations. We also tested these LCTs for mutations in two genes, the LH/choriogonadotropin receptor (LHCGR) and the guanine nucleotide-binding protein alpha (GNAS) that had been implicated in LCT tumorigenesis. RESULTS: No mutations were found in GNAS, and one tumor had a LHCGR somatic substitution. In addition to the HLRCC case with the N64T germline FH mutation, we identified one other LCT with a previously unreported FH mutation (M411I). Both LCTs from these patients showed loss of the wild-type FH allele. Immunohistochemical and in situ hybridization analyses demonstrated activation of the hypoxia/angiogenesis pathway not only in the tumors belonging to the FH mutation carriers but also in several other mutation-negative LCTs. CONCLUSIONS: Our study shows that some LCTs are caused by FH mutations and represents one of the first reports of germline mutations in any type of adult testicular tumor.
Base Sequence↗
Stem cell plasticity and tumour formation.
Stem cell plasticity refers to the ability of certain stem cells to switch lineage determination and generate unexpected cell types. This review applies largely to bone marrow cells (BMCs), which appear to contribute positively to the regeneration of several damaged non-haematopoietic tissues. This beneficial effect on regeneration may be a direct result of BMCs giving rise to organ parenchymal cells. Alternatively, it could be due to BMCs fusing with existing parenchymal cells, or providing paracrine growth factor support, or contributing to neovascularisation. In the context of oncology, BMC derivation of the tumour stroma and vasculature has profound biological and therapeutic implications, and there are several examples of carcinomas seemingly being derived from BMCs.
Animals↗
Reduced gene expression of intestinal alpha-defensins predicts diarrhea in a cohort of African adults.
BACKGROUND: Human defensin (HD) 5 and HD6, both Paneth cell alpha-defensins, contribute to the antimicrobial barrier against intestinal infection. We have previously demonstrated that levels of both HD5 and HD6 mRNA were reduced in adults living in urban Zambia, compared with those in adults living in London. The aim of the present study was to determine, during 2 years of follow-up, whether alpha-defensin expression in Zambian adults is related to susceptibility to diarrhea. METHODS: We analyzed intestinal biopsy samples from a longitudinal cohort study conducted in 83 Zambian adults by quantitative reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry, and in situ hybridization, and we measured the incidence of diarrhea. RESULTS: Levels of HD5 and HD6 mRNA in Paneth cells varied between participants, over time, and seasonally and were strongly correlated with mucosal architecture. Gene expression was almost exclusively restricted to Paneth cells. The median (interquartile range) HD5 mRNA level was 6.0 (5.6-6.7) log10 transcripts/microg of total RNA among 18 participants who experienced diarrhea within 2 months after biopsy-sample collection, compared with 6.8 (6.2-7.3) log10 transcripts/microg of total RNA among 94 participants who did not (P=.006). A similar observation was made for HD6. CONCLUSIONS: These data indicate that intestinal alpha-defensin expression is dynamic and seasonal and suggest that susceptibility to intestinal infection is related to alpha-defensin expression.
Adult↗
Bone marrow-derived cells do not contribute significantly to collagen I synthesis in a murine model of renal fibrosis.
Interstitial fibroblasts play a central role in kidney fibrosis. Their origin is debated, with recent data indicating a contribution of bone marrow (BM)-derived cells to the expanded population of interstitial cells after kidney damage in animals and humans. This study investigated whether these BM-derived cells would respond appropriately to a fibrotic drive by producing collagen. A transgenic mouse that expresses both luciferase and beta-galactosidase reporter molecules under the control of a 17-kb promoter and enhancer element of the gene encoding the alpha2 chain of the collagen I was used. Male transgenic BM was transplanted into female wild-type C57BL/6 mice (n=14), and unilateral ureteric obstruction was performed later to induce renal fibrosis. In the obstructed kidney of the BM-chimeric female mice, a mean of 8.6% of smooth muscle actin-positive interstitial cells were Y chromosome positive. Increased collagen I mRNA in the obstructed kidney was detected by in situ hybridization. No luciferase activity was detected by enzyme assays in tissue homogenates of BM recipients, and very few luciferase mRNA transcripts were seen, mainly in tubular cells. beta-Galactosidase activity was not a useful reporter molecule because it could not be distinguished from enhanced endogenous beta-galactosidase activity in the obstructed kidney. These results indicate that BM-derived interstitial cells do not make a significant contribution to collagen I synthesis in the context of renal injury.
Animals↗
Bone marrow-derived stromal cells express lineage-related messenger RNA species.
Evidence has emerged that bone marrow cells have a greater degree of plasticity than previously thought. However, there has been a call to establish proof that these bone marrow-derived cells function appropriately in their new environment. We have already shown that the bone marrow contributes to myofibroblasts in multiple organs and that this is exacerbated by injury and occurs in a mouse tumor model. Here, we provide evidence that these cells are functioning appropriately by showing that bone marrow-derived myofibroblasts are expressing mRNA for the alpha(1) chain of type I (pro)collagen using a new customized technique. This provides evidence that the bone marrow-tumor stroma axis is functionally relevant and may therefore subsequently be exploited to develop new strategies for anticancer therapy.
Animals↗
Identification of blottin: a novel gastric trefoil factor family-2 binding protein.
The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.
Alkaline Phosphatase↗
Isolation of gut SP cells does not automatically enrich for stem cells.
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ATP Binding Cassette Transporter, Subfamily G, Mem↗
Effects of mouse and human lipocalin homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin on gastrointestinal mucosal integrity and repair.
BACKGROUND & AIMS: The lipocalin superfamily, including the mouse and human homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin, show great functional diversity including roles in olfaction, transportation, and prostaglandin synthesis in mammals. Their potential role in maintaining gastrointestinal mucosal integrity and repair is, however, unclear. METHODS: Changes in 24p3/lcn2 expression in the mouse gut in response to various noxious agents were examined using Northern blot, in situ hybridization, and immunohistochemistry. Effects of recombinant 24p3/lcn2 on proliferation ([3H]-thymidine uptake), and restitution (cell-wounding migration) were assessed using human colonic HT29 and HCT116 cells. In addition, the effects of recombinant 24p3/lcn2 on the amount of gastric damage were assessed in rats treated with indomethacin (20 mg/kg) and restraint. RESULTS: Marked up-regulation of expression of 24p3/lcn2 was seen throughout the gut in response to indomethacin or dextran sodium sulfate treatment. Expression was increased particularly in the surface epithelial cells and infiltrating inflammatory cells. Proliferation and restitution assays in the presence of recombinant wild-type sequence neutrophil gelatinase-associated lipocalin, wild-type cys(98)-24p3/lcn2, and mutant ala98-24p3/lcn2 showed that all 3 peptides caused a 3- to 4-fold increase in promigratory activity (P < .01 vs control) but did not influence proliferation. The administration of wild-type cys98-, or mutant ala98-24p3/lcn2 (25 and 50 microg/kg/h, respectively), given via the subcutaneous route, both caused similar reductions in the rat gastric damage model (60% reduction at highest dose, P < .01 vs control), although oral administration was ineffective. CONCLUSIONS: 24p3/lcn2 facilitates mucosal regeneration by promoting cell migration.
Acute-Phase Proteins↗
Calcium channel TRPV6 expression in human duodenum: different relationships to the vitamin D system and aging in men and women.
UNLABELLED: Intestinal absorption of calcium affects bone mineralization and varies greatly. In human duodenum, expression of the calcium channel TRPV6 was directly related to blood 1,25-dihydroxyvitamin D in men, but effects of age with lower median vitamin D receptor levels were more significant in women. INTRODUCTION: The TRPV6 calcium channel/transporter is implicated in animal studies of intestinal calcium absorption, but in humans, its role and relationship to differences in mineral metabolism is unclear. We aimed to characterize TRPV6 expression in human intestine including defining relationships to the vitamin D endocrine system. MATERIALS AND METHODS: TRPV6 transcript expression was determined in endoscopic mucosal biopsies obtained from normal duodenum. Expression was compared with that in ileum and with in situ hybridization in archival tissues and related to sequence variants in genomic DNA. TRPV6 expression was related in 33 subjects to other transcripts involved in calcium absorption including the vitamin D receptor (VDR) and to blood vitamin D metabolites including 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. RESULTS: TRPV6 transcripts were readily detected in duodenum but not in ileum. Expression was highest in villous epithelial cells. Sequence variants in the coding and upstream regions of the gene did not affect TRPV6 expression. The relationship between duodenal TRPV6 expression and 1,25(OH)(2)D differed in men and women. In men, linear regression showed a strong association with 1,25(OH)(2)D (r = 0.87, p < 0.01), which was unaffected by age. In women, there was no significant overall relationship with 1,25(OH)(2)D, but there was a significant decrease with age (r = -0.69, p < 0.001). Individual expression of TRPV6 and VDR was significantly correlated. The group of older women (>50) had lower median levels of both TRPV6 and VDR transcripts than younger women (p < 0.001 and 0.02, respectively). CONCLUSIONS: Duodenal TRPV6 expression is vitamin D dependent in men, but not in older women, where expression of TRPV6 and VDR are both reduced. These findings can explain, at least in part, the lower fractional calcium absorption seen in older postmenopausal women.
Adult↗
In situ hybridization combined with immunohistochemistry to localize gene expression.
This chapter describes methods that allow researchers to localize sites of gene expression at both the mRNA and protein levels, within a histological section of a single tissue or tissue microarrays. Identification of the cells within a tumor specimen that express a specific mRNA (assessed by hybridization in situ) or protein (assessed by immunohistochemistry) is a significant step toward understanding tumor behavior that is not possible using methods in which tissues are homogenized before analysis. Combined detection of mRNA and protein may permit effects of subtle regulatory processes such as translational repression to be observed and allow coexpression of genes to be detected. Application of such combined techniques to tissue microarrays of tumor tissues from cohorts of patients with known clinical outcome would allow the predictive value of specific patterns of expression to be tested retrospectively.
Breast Neoplasms↗
Up-regulation of delta-like 4 ligand in human tumor vasculature and the role of basal expression in endothelial cell function.
The Notch signaling pathway and the delta-like 4 ligand (DLL4) play key roles in embryonic vascular development. Many of the pathways involved in embryonic vascular development also play important roles in tumor angiogenesis. In this study, we assessed the expression of DLL4 in primary renal cancer and investigated the biological function of DLL4 in primary endothelial cells. Using real-time quantitative PCR and in situ hybridization, we showed that the expression of DLL4 was up-regulated within the vasculature of clear cell-renal cell carcinoma almost 9-fold more than normal kidney and was correlated with the expression of vascular endothelial growth factor (VEGF). The expression of DLL4 in endothelial cells was up-regulated by VEGF and basic fibroblast growth factor synergistically, and by hypoxia through hypoxia-inducible factor 1alpha. Down-regulation of DLL4 expression with RNA interference led to decreased expression of HEY1 and EphrinB2, and the inhibition of endothelial cell proliferation, migration, and network formation, all of which are important processes in tumor angiogenesis. The inhibition of proliferation occurred via the induction of cell cycle arrest in G0-G1 by increased expression of p21 and decreased phosphorylation of retinoblastoma. We conclude that an optimal window of the DLL4 expression is essential for tumor angiogenesis and that selective modulation of the DLL4 expression within human tumors may represent a potential novel antiangiogenic therapy.
Adaptor Proteins, Signal Transducing↗
Proliferation of bone marrow-derived cells contributes to regeneration after folic acid-induced acute tubular injury.
Studies of tissue from recipients of bone marrow transplantation or organ allograft suggest that bone marrow-derived cells (BMDC) may differentiate into a variety of nonhematologic tissues, including renal tubular epithelium. The aims of this study were to examine whether BMDC contribute to recovery after acute renal injury and to assess the effects of cytokine mobilization on regeneration. Female mice (6 wk old) were lethally irradiated and transplanted with male bone marrow (BM) cells and later assigned into control, folic acid-treatment, and folic acid-treatment with granulocyte-colony stimulating factor (G-CSF), and control with G-CSF. Tritiated thymidine was given 1 h before death. Kidney sections were stained for a tubular epithelial marker, Y chromosome (in situ hybridization), periodic acid-Schiff staining, and subjected to autoradiography. Renal tubular epithelial cells in S-phase were scored as female (indigenous) or male (BM-derived). This is the first report to show that BMDC can respond by engrafting the renal tubules and undergo DNA synthesis after acute renal injury. BMDC contributed to the renal tubular epithelial cell population, although most (90%) renal tubular regeneration came from female indigenous cells. Some evidence was found for cell fusion between indigenous renal tubular cells and BMDC, but this was infrequent and the significance and consequences of cell fusion in the kidney are unresolved. G-CSF treatment nearly doubled the frequency of thymidine-labeled BM-derived tubular cells and might facilitate the recovery of renal tubular epithelium.
Animals↗
Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion.
In adults, bone marrow-derived cells (BMDC) can contribute to the structure of various non-haematopoietic tissues, including skin. However, the physiological importance of these cells is unclear. This study establishes that bone marrow-derived epidermal cells are proliferative and, moreover, demonstrates for the first time that BMDC can localize to a known stem cell niche: the CD34-positive bulge region of mouse hair follicles. In addition, engraftment of bone marrow cells into the epidermis is significantly increased in wounded skin, bone marrow-derived keratinocytes can form colonies in the regenerating epidermis in vivo, and the colony-forming capacity of these cells can be recapitulated in vitro. In some tissues this apparent plasticity is attributed to differentiation, and in others to cell fusion. Evidence is also provided that bone marrow cells form epidermal keratinocytes without undergoing cell fusion. These data suggest a functional role for bone marrow cells in epidermal regeneration, entering known epidermal stem cell niches without heterokaryon formation.
Animals↗