Carbonic Anhydrase IV Research Articles

Carbonic anhydrase 4 and crystallin alpha-B immunoreactivity may distinguish benign from malignant thyroid nodules in patients with indeterminate thyroid cytology

BackgroundThyroid nodules are present in 19%–67% of the population and carry a 5%–10% risk of malignancy. Unfortunately, fine-needle aspiration biopsies are indeterminate in 20%–30% of patients, often necessitating thyroid surgery for diagnosis. Numerous DNA microarray studies including a recently commercialized molecular classifier have helped to better distinguish benign from malignant thyroid nodules. Unfortunately, these assays often require probes for >100 genes, are expensive, and only available at a few laboratories. We sought to validate these DNA microarray assays at the protein level and determine whether simple and widely available immunohistochemical biomarkers alone could distinguish benign from malignant thyroid nodules. MethodsA tissue microarray (TMA) composed of 26 follicular thyroid carcinomas (FTCs) and 53 follicular adenomas (FAs) from patients with indeterminate thyroid nodules was stained with 17 immunohistochemical biomarkers selected based on prior DNA microarray studies. Antibodies used included galectin 3, growth and differentiation factor 15, protein convertase 2, cluster of differentiation 44 (CD44), glutamic oxaloacetic transaminase 1 (GOT1), trefoil factor 3 (TFF3), Friedreich Ataxia gene (X123), fibroblast growth factor 13 (FGF13), carbonic anhydrase 4 (CA4), crystallin alpha-B (CRYAB), peptidylprolyl isomerase F (PPIF), asparagine synthase (ASNS), sodium channel, non-voltage gated, 1 alpha subunit (SCNN1A), frizzled homolog 1 (FZD1), tyrosine related protein 1 (TYRP1), E cadherin, type 1 (ECAD), and thyroid hormone receptor associated protein 220 (TRAP220). Of note, two of these biomarkers (GOT1 and CD44) are now used in the Afirma classifier assay. We chose to compare specifically FTC versus FA rather than include all histologic categories to create a more uniform immunohistochemical comparison. In addition, we have found that most papillary thyroid carcinoma could often be reasonably distinguished from benign disease by morphological cytology findings alone. ResultsIncreased immunoreactivity of CRYAB was associated with thyroid malignancy (c-statistic, 0.644; negative predictive value [NPV], 0.90) and loss of immunoreactivity of CA4 was also associated with malignancy (c-statistic, 0.715; NPV, 0.90) in indeterminate thyroid specimens. The combination of CA4 and CRYAB for discriminating FTC from FA resulted in a better c-statistic of 0.75, sensitivity of 0.76, specificity of 0.59, positive predictive value (PPV) of 0.32, and NPV of 0.91. When comparing widely angioinvasive FTC from FA, the resultant c-statistic improved to 0.84, sensitivity of 0.75, specificity of 0.76, PPV of 0.11, and NPV of 0.99. ConclusionsLoss of CA4 and increase in CRYAB immunoreactivity distinguish FTC from FA in indeterminate thyroid nodules on a thyroid TMA with an NPV of 91%. Further studies in preoperative patient fine needle aspiration (FNAs) are needed to validate these results.

Results of a phase II trial of novel carbonic anhydrase IX radiotracer 18F-VM4-037 in renal cell carcinoma.

LBA400 Background: Carbonic Anydrase IX (CAIX) has been identified as a potential marker for clear cell renal cell carcinoma (ccRCC). We report the results of a phase II clinical trial of a novel small molecule radiotracer that binds to CAIX in clinically localized kidney tumors or biopsy proven metastases. Methods: Between October 2012 and May 2013, patients with kidney tumors underwent imaging with the 18F-VM4-037 radiotracer within 4 weeks of biopsy or surgery for kidney tumors. Imaging characteristics with the radiotracer were collected. All H&E pathology was centrally reviewed by a single pathologist. Immunohistochemistry of the surgical specimens for CAIX and Carbonic Anhydrase XII (CAXII) was reviewed by two experienced pathologists. Primary objectives were to evaluate the biodistribution of the radiotracer within tumor and non-tumor tissues as well to assess the safety of the radiotracer in patients. Results: 12 patients were enrolled and 11 received radiotracer and images were obtained. All patients tolerated the radiotracer well with no significant adverse events. 10 of the 11 patients had histologically confirmed malignancy. 1 patient had a complex cyst with no tumor found at surgery. Two patients had extrarenal disease and 9 had tumors in the kidney only. 100% had ccRCC and 4/11 had germline VHL mutations. Mean SUV for the entire group as 5.44 in the index lesions; in patients with histologically confirmed ccRCC the mean SUV was 5.91. 12 samples were stained for CAIX and CAXII including a biopsy obtained from a lung metastasis in one patient. The concordance between pathologists for CAIX IHC was 75%, for CAXII was 67%. Of the CAIX IHC slides, 67% had 3+ staining, 25% had 2+ and one specimen did not demonstrate CAIX staining. Only one specimen demonstrated 3+ staining for CAXII; 2 specimens had no detectable staining for CAXII and the remainder stained 1-2+ for CAXII. Conclusions: The 18F-VM4-037 was well tolerated and demonstrated modest signal uptake in tumors within the kidney. Immunohistochemistry staining for CAIX and CAXII was robust in nearly all tumors making correlation with radiotracer signal difficult. Larger studies are needed to evaluate the diagnostic performance of this radiotracer. Clinical trial information: NCT01712685.

Carbonic Anhydrase 4 (CA4) and Crystallin Alpha-B (CRYAB) Immunoreactivity May Distinguish Benign From Malignant Thyroid Nodules in Patients With Indeterminate Thyroid Cytology

Carbonic Anhydrase 4 (CA4) and Crystallin Alpha-B (CRYAB) Immunoreactivity May Distinguish Benign From Malignant Thyroid Nodules in Patients With Indeterminate Thyroid Cytology

Regulation of zinc transporter 3 and carbonic anhydrases 2 and 14 mRNA expression in the retina of rats affected by low dietary zinc

We looked at how zinc transporter 3 ZnT-3) mRNA expression in the rat retina is affected by low dietary zinc. Groups of 12 four-week-old male Sprague Dawley rats were fed on a low-zinc diet for 2, 4 or 6 weeks. Half of each group was then fed with a normal-zinc content diet and the other half was given a low-zinc content diet. The expression of ZnT-3, carbonic anhydrase 2 (CA2) and 14 (CA14) were detected by RT-PCR. After the rats were fed a low-zinc content diet for 2 weeks, their retina CA2 and CA14 mRNA levels were decreased, and the ZnT-3 mRNA was increased compared with the control rats. After they were fed a low-zinc diet for 4 weeks, ZnT-3, CA2 and CA14 mRNA levels decreased significantly. Then, after being changed back to a normal diet for 2 weeks, the rats had ZnT-3, CA2 and CA14 mRNA levels recovery in the retina.

Proteomic Interactions in the Mouse Vitreous-Retina Complex

PurposeHuman vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina. MethodsVitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software. ResultsWe identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor. ConclusionsOur analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.

Osteocytes: Regulating the Mineral Reserves?

Osteocytes are increasingly recognized as multifunctional cells that help orchestrate bone and mineral metabolism— and are currently one of the hottest topics in the field. In addition to their putative role as detectors/transducers of mechanical strain in bone, osteocytes are a major source of sclerostin, a key inhibitor of Wnt signaling and bone formation, and appear to be important regulators of osteoclast function via their production of NF‐kB ligand (RANKL) and osteoprotegerin. It has also long been believed that osteocytes could remove or even replace mineralized bone around their lacunae and canaliculi. Thanks to a number of studies and reviews in recent years, this once‐ controversial concept of “osteocytic osteolysis” is now verymuch back on the agenda. In this issue of the Journal of Bone and Mineral Research, Kogawa and colleagues present evidence that sclerostin could act to stimulate the dissolution of bone mineral by osteocytes by increasing their expression of some of the same resorptive machinery used by osteoclasts. The authors show that treatment of cultured MLO‐Y4 and primary human osteocyte‐like cells with recombinant human sclerostin increased the expression of mRNA for a number of resorptionmarkers, most notably carbonic anhydrase 2 (CA2). The authors found that sclerostin caused both intracellular and extracellular acidification in cultures of the osteocyte‐like cells, albeit in a non–dose‐dependentmanner. The classic CA inhibitor, acetazolamide decreased the sclerostin‐ stimulated extracellular acidification in MLO‐Y4 cells, whereas antisense knockdown of CA2 led to an extracellular alkalinization, with elimination of the effect of acetazolamide. Sclerostin additionally caused a slight stimulation of calcium release from mineralized layers of primary osteocyte‐like cells, an effect that was also blocked by acetazolamide. Stimulation of mRNA for CA2, tartrate‐resistant acid phosphatase (TRAP), chloride channel 7 (CLCN7), and cathepsin K by sclerostin in the MLO‐Y4 cell line was inhibited by small interfering RNA (siRNA) constructs for Lrp4, Lrp5, or Lrp6, suggesting the possible involvement of Wnt signaling. Last, sclerostin treatment increased the plan area of osteocyte lacunae by about 25% in cultured samples of trabecular bone taken from patients undergoing hip surgery, an effect that was again abrogated by acetazolamide. The concept of osteocytic osteolysis was first mooted in 19th century, although is perhaps now most closely associated with Belanger. He suggested that either parathyroid hormone (PTH) or a low‐calciumdiet can induce osteocytes to enlarge their lacunae. In its broadest sense, the term osteocytic osteolysis implies dissolution of not only the mineral component of bone adjacent to lacunae and canaliculi but of the collagenous matrix, too. These ideas provoked vigorous rebuttals but began to gain traction again following reports that osteocyte lacunar area wasmarkedly increased in rats continuously infused with PTH for 4 weeks and in mice infused with prednisolone for 3 weeks. Moremodest increases in lacunar sizewere also observed inmice subjected to ovariectomy. More recently, some of the coauthors of the paper by Kogawa and colleagues have presented a comprehensive study of the effects of lactation on osteocyte lacunae in mice. Lactation was shown to be associated with increases in lacunar area of about 20–40% in tibias and lumbar vertebrae; these increases were reversed with 7 days of cessation of lactation. The effects were mimicked by PTH‐related protein (PTHrP) and abrogated by targeted deletion of PTH receptor 1. Increased expression of mRNAs for osteoclast‐specific markers (including cathepsin K, which cleaves collagen) was also noted in “osteocyte‐enriched” bones of lactating animals. These reversible changes in osteocyte lacunar size have been referred to as “remodeling,” although this process clearly differs from bone remodeling by osteoclasts and osteoblasts, which involves obvious turnover of substantial quantities of both mineral and organic matrix. Despite suggestions that removal and replacement of collagenous matrix around osteocytes might occur in extreme pathophysiological situations, such as in the ultra‐high turnover bone of egg‐ laying hens, there seems to be little wider evidence for such a process. Removal of mineral alone might be sufficient to account for the enlargement of osteocyte lacunae observed by light or scanning electron microscopy if the resulting thin layer of demineralized collagenous matrix shrinks back against the lacunar walls during dehydration for specimen preparation. In vivo, such a layer of demineralized (hydrated) matrix adjacent to the osteocyte could presumably serve as a scaffold to facilitate

Analysis of expression of membrane-bound tumor markers in ductal carcinoma in situ of the breast: paving the way for molecular imaging

Achieving radicality during breast conserving surgery for pure ductal carcinoma in situ (DCIS) of the breast and invasive cancer surrounded by DCIS is challenging. Molecular imaging holds promise here, when applied as a tool for image-guided surgery of DCIS. Tissue microarrays containing 24 pure DCIS and 63 DCIS with adjacent invasive breast cancer cases were stained by immunohistochemistry for a panel of membrane-bound targets. GLUT1 expression was present in 60.9%, IGF1-R in 55.2% HER2 in 28.7%, MET in 18.4%, EGFR in 16.1%, CD44v6 in 69%, carbonic anhydrase XII (CAXII) in 24.1% and Mammaglobin in 14.9% of DCIS cases. No expression differences between pure DCIS and DCIS with adjacent cancer were observed. Further, HER2 and EGFR expression were correlated with high grade DCIS (p=0.001) and CAXII with low grade DCIS (p=0.027). A putative panel containing HER2, EGFR, GLUT1 and IGF1-R had a detection rate of 90.2% for DCIS and 78.3% for adjacent breast cancer. We found that membrane-bound targets are more frequently expressed in DCIS than in invasive breast cancer, but that single membrane proteins are too infrequently expressed to serve as single imaging targets for the detection of DCIS. However, a panel of markers consisting of IGF1-R, CD44v6, GLUT1, EGFR, and HER2 was found to be positive in 96.3% of DICS based on marker expression in the adjacent invasive breast cancer as described earlier. This implies that detection of DCIS based on marker expression in the adjacent invasive breast cancer during breast conserving surgery should be possible with a panel of molecular imaging tracers targeting CD44v6, GLUT1, HER2, IGF1-R, and EGFR.

Abstract 1702: Metabolic heterogeneity of HER2-positive breast cancers.

Abstract Breast cancers are at least five molecularly distinct diseases with reported metabolic differences. Basal-like breast cancers are typically more glycolytic as compared to luminal-like breast cancers. However, HER2-positive breast tumors have variable FDG-PET maximum uptake values. We hypothesized that HER2-positive breast cancer cell lines would reflect the metabolic heterogeneity reported in breast cancer tumors. In the current study, a panel of HER2-positive breast cancer cell lines (N = 8) was profiled by their metabolic properties. To evaluate glycolytic dependency for ATP production, ATP-synthase dependent oxygen consumption rates were normalized to extracellular acidification rates using the Seahorse Bioscience XF analyzer. The glycolytic dependency was highly variable (12-fold range); ER-negative lines including AU565 and HCC1954 were the most glycolytic. Consistent with these results, HER2-positive/ER-negative cell lines produced more lactate compared to HER2-positive/ER-positive cell lines (1.5 versus 1.2 lactate produced/glucose consumed, p = 0.02). The glycolytic phenotypes of HER2-positive/ER-negative breast cancer cell lines were similar to that of basal-like breast cancer cell lines. For the HER2-positive lines, the highest rates of glutamine consumption were in ER-positive lines BT474 and HCC1419. Differential expression of mitochondrial glutaminase 2 (GLS2, p = 0.02), but not cytosolic glutaminase (GLS) or glutamine synthetase (GLUL), may contribute to higher rates of glutamine consumption by HER2-positive/ER-positive breast cancers. Consistent with higher rates of mitochondrial respiration, ER-positive/HER2-positive cells were also associated with higher expression of carbonic anhydrase 12 (CA12) and lower expression of the lactate transporter MCT1 (SLC16A1). Metabolic heterogeneity of HER2-positive cells was only evident in normoxic conditions. Per the “Pasteur effect,” all cell lines cultured under hypoxia (1% O2) were equally glycolytic (average = 1.8 lactate production/glucose consumption ratio) with correspondingly reduced rates of glutamine uptake. We conclude that the heterogeneity observed in FDG-PET may be influenced by fundamental metabolic differences between HER2-positive breast cancers, in particular, for well-oxygenated regions of the tumor. Citation Format: Jennifer B. Dennison, Gordon B. Mills. Metabolic heterogeneity of HER2-positive breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1702. doi:10.1158/1538-7445.AM2013-1702

Alterations in digestive enzyme activities during the development of diet-induced enteritis in Atlantic salmon, Salmo salar L.

This study followed the time-related changes in intestinal digestive physiology of post-smolt Atlantic salmon (Salmo salar L.; initial weight 500–600g) during the development of soybean meal induced enteritis. Intestinal tissue and content (chyme) were sampled after 1, 2, 3, 5, 7, 10, 14, 17, and 21days of dietary exposure to a 20% extracted soybean meal-containing (SBM) diet. Fish fed a fishmeal-based control diet sampled on day 0 (prior to SBM feeding) served as controls. From the different intestinal regions, histomorphological evaluation was conducted, activities of the pancreatic enzymes trypsin, chymotrypsin, elastase and lipase, as well as leucine aminopeptidase (LAP) were determined in chyme, and the brush border membrane (BBM) enzymes LAP and maltase were measured in homogenates of the intestinal wall. Epithelial cell functional differentiation was visually assessed using immunohistochemical expression of carbonic anhydrase 12 (CA-12). Histological signs of SBM-induced inflammation were observed in the distal intestine (DI) from day 5. At each given sampling time, pancreatic enzyme activities generally decreased aborally along the intestinal tract. Within each intestinal region, pancreatic protease activities tended to increase with exposure time to the SBM diet, with the greatest activity increases observed in the DI. The magnitude of the decrease in activity between the most proximal region and the most distal decreased with SBM exposure. DI tissue weight and BBM maltase and LAP activities were significantly reduced from day 1–2 and continued to decrease progressively with increasing SBM exposure time. Immunohistochemical staining of CA-12 also diminished over time starting at day 5. By day 7, very little CA-12 staining was observed. The data suggests an immediate and generalized physiological response to a change in diet and/or feed intake in all regions of the intestine, as well as a more localized functional loss in the DI that progressed with the development of SBM-induced inflammation from day 5. The diet change appeared to cause a rapid pancreatic response with increased secretion of all the enzymes investigated, while the longer term responses of the pancreatic proteases differed from that of lipase.

Clinical Impact and Reliability of Carbonic Anhydrase XII in the Differentiation of Malignant and Tuberculous Pleural Effusions

To assess the practical utility of pleural fluid carbonic anhydrase XII (CAXII) quantification for differential diagnosis of effusions. Fluid was collected prospectively from fifty patients presenting with lymphocytic pleural effusions for investigation and CAXII was quantified by ELISA. Pleural fluid CAXII concentrations were significantly higher in lung cancer patients (n=30) than in tuberculous controls (n=20). The sensitivity and specificity of this biomarker were 60%and 75%, respectively. CAXII measurement was not inferior to cytological examination in the diagnosis and exclusion of pleural effusions from lung cancer patients (sensitivity 60% vs. 57%; specificity 75% vs. 100%; positive predictive value 77%; negative predictive value 54%). In patients with negative cytology, it offered a sensitivity of 54%. Pleural fluid CAXII is elevated in pleural effusions from lung cancer patients. Measurement of CAXII may be used in the future as a valuable adjunct to cytology in the diagnostic assessment of patients with pleural effusions related to lung cancer, especially when cytological examination is inconclusive.

Valproic Acid Downregulates RBP4 and Elicits Hypervitaminosis A-Teratogenesis—A Kinetic Analysis on Retinol/Retinoic Acid Homeostatic System

BackgroundValproic acid (VPA) is an antiepileptic and anti-migraine prophylactic drug. VPA exhibits two severe side effects, namely acute liver toxicity and teratogenicity. These side effects are usually seen at the genetic and somatic levels. The cited action mechanisms involve inhibition of histone deacetylase, hypofolatenemia, hyperhomocysteinemia, and reactive oxidative stress. The proteomic information associated with VPA teratogenicity is still unavailable. We hypothesized that proteomic analysis might help us identify functional proteins that could be relevantly affected by VPA, and this phenomenon could be very sensitive in early embryonic stage, resulting in VPA teratogenicity.Methodology/Principal FindingsProteomic analysis on the chicken embryos at Hamburger and Hamilton (HH) stage 28 showed that there were significant downregulations of ovotransferrins, carbonic anhydrase-2, retinol binding protein-4 (RBP4), NADH cytochrome b5 reductase 2 (CYB5R2), apolipoprotein A1, and protein SET, together with upregulation of 60S ribosomal protein L22. Among these, RBP4 was the most significantly downregulated (−32%). Kinetic analysis suggested that this situation could trigger hypervitaminosis A (+39.3%), a condition that has been well known to induce teratogenesis..Conclusions/SignificanceThis is the first report showing that VPA dowregulates RBP4. Our finding not only has led to a possible mechanism of VPA teratogenesis, but also has initiated new preventive strategies for avoiding VPA teratogeneis.

Identification of uterine ion transporters for mineralisation precursors of the avian eggshell.

BackgroundIn Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation.ResultsA total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters.ConclusionsOur Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.

Thermoreversible Hyaluronan Hydrogels Support Mesenchymal Stem Cells Discogenic Differentiation in Vitro and Ex Vivo

Introduction Hyaluronan is a proteoglycan widely present in the nucleus pulposus (NP) of the intervertebral disk (IVD). Recently, thermoreversible hyaluronan (HA) based hydrogels, which are injectable, minimally invasive, allow for encapsulation and maintenance of viable cells and promote re-differentiation of NP cells, have been developed.1 While it is known that hyaluronan is recognized by mesenchymal stem cells (MSCs)2 and can induce anabolic pathways, growth factors may further support MSC differentiation in HA-based hydrogels. Therefore, the objectives of the present work were: (i) to assess whether the addition of growth factors3 could support MSC differentiation towards the disk cell phenotype in HA based hydrogels and (ii) to determine if MSC preconditioning (culture under “chondrogenic” conditions) would improve the survival rate and differentiation potential of MSC supplemented to an IVD cultured ex vivo. Materials and Methods Hydrogel Thermoreversible HA-based hydrogels were prepared by grafting a thermoreversible segment poly (N-isopropylacrylamide) (pNIPAM) on a functionalized HA backbone, as previously described.1 In vitro Human bone marrow-derived MSCs (P2-P4) were cultured for 7 days in HA-pNIPAM hydrogel under hypoxic conditions (5% O2) in chondrogenic medium, with or without recombinant human growth and differentiation factor-5 (rhGDF-5) supplementation. Cell viability, DNA, and glycosaminoglycans (GAG) content were quantified by Live-dead, Picogreen, and 1,9 dimethylmethylene blue assays, respectively. Gene expression analysis was performed to assess the disc-like differentiation of MSCs. The expression of collagen type I and II (COL1 and COL2) aggrecan (ACAN), cytokeratin-19, sox-9, cluster of differentiation 24 (CD24), hyaluronan synthase I and II (HAS1 and HAS2), forkhead box protein F1 (FOXF1) and carbonic anhydrase 12 (CA12) was analyzed with 18s as endogenous control. Ex Vivo Bovine caudal IVDs were excised and nucleotomy performed through the endplate (Fig. 1A,B). MSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or precultured (under hypoxic conditions and with supplementation of GDF-5) for 1 week in HA-pNIPAM and then supplied to the IVDs. After 1 week of culture, gene expression analyses were performed using the same markers as for the in vitro study. Statistics After confirming the normality of the data distribution, one-way analysis of variance with Bonferroni posthoc was performed ( p < 0.05). Results MSC viability was maintained>90% over 1 week of culture in the HA-pNIPAM hydrogel both in vitro and ex vivo. DNA and GAG did not show significant variation over the 7 days of in vitro culture. Gene expression revealed 10–100-fold upregulation of the transcription factor SOX9 and 100–1000, fold upregulation of COL2, while COL1 was only slightly enhanced. The expression of the potential NP cell marker KRT19 was also increased. With the addition of rhGDF-5, an improved COL2/COL1 expression ratio was found. No significant differences were found for the other genes. Ex vivo cultures of nucleotomized discs supplemented with MSCs in HA-pNIPAM confirmed the positive effect of the hydrogel and the IVD environment on MSC differentiation towards the disc-like phenotype (Fig. 1C), with upregulation of collagen type II ( p < 0.05), CD24, and FOX-F1. No significant differences were found for the other genes. Preculture of MSCs before the supplementation in the IVD did not enhance MSC differentiation. Figure 1 Bovine IVD after nucleotomy: (A) top view and (B) view of the nucleotomized area. (C) Gene expression of human MSCs after 1 week of culture in bovine IVDs normalized to the mRNA levels at day 0 of culture: human MSCs were either suspended in HA-pNIPAM and directly supplied to bovine IVDs (white bars) or precultured for 1 week in HA-pNIPAM hydrogel and then supplied to bovine IVDs (grey bars);* p < 0.05, outliers. Conclusion The thermoreversible hyaluronan-based hydrogel was able to induce human MSC differentiation toward a “disc-like” phenotype without the addition of growth factors, although GDF-5 supplementation resulted in an improved gene expression profile. Preculture of MSCs before the supplementation in the IVD is not required with the thermoreversible hyaluronan-based hydrogel. Therefore, this type of hydrogel could be an appropriate carrier for MSCs supplementation to compromised discs. Investigations are ongoing to evaluate its biomechanical behavior under loading. Acknowledgements This study was partially supported by a NASS Research Grant. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Peroglio M, et al. European Spine Journal (Epub ahead of print) Zhu H et al. Stem cells 2006; 24:928–935 Gantenbein-Ritter B, et al. European Spine Journal 2011;20(6):962–971

153 CARBONIC ANHYDRASE XII: POTENTIAL ROLE IN LYMPHATIC METASTASES OF RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2012153 CARBONIC ANHYDRASE XII: POTENTIAL ROLE IN LYMPHATIC METASTASES OF RENAL CELL CARCINOMA Nnenaya Agochukwu, Kiranpreet Khurana, Eric Kauffman, Brian Shuch, Vladimir Valera, Youfeng Yang, W. Marston Linehan, and Gennady Bratslavsky Nnenaya AgochukwuNnenaya Agochukwu Bethesda, MD More articles by this author , Kiranpreet KhuranaKiranpreet Khurana Cleveland, OH More articles by this author , Eric KauffmanEric Kauffman Bethesda, MD More articles by this author , Brian ShuchBrian Shuch Bethesda, MD More articles by this author , Vladimir ValeraVladimir Valera Bethesda, MD More articles by this author , Youfeng YangYoufeng Yang Bethesda, MD More articles by this author , W. Marston LinehanW. Marston Linehan Bethesda, MD More articles by this author , and Gennady BratslavskyGennady Bratslavsky Syracuse, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.203AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastasis in renal cell carcinoma (RCC) is a major predictor of survival, yet little is known about metastasis of RCC to the lymph nodes. Using matched pairs of cell lines derived from patients with a primary tumor of RCC origin and sites of lymph nodal metastasis also of RCC origin, we sought to investigate the potential differences between the lymph node metastasic site and the primary site in tumor cell lines derived from the same patients. METHODS The cell lines used for this analysis were obtained from the Urologic Oncology Branch of the NCI cell repository and included 3 pairs with the von Hippel- Lindau (VHL) mutation and 1 pair with a mutation in the Transcription factor E3 (TFE3) gene. Each pair represents one patient. Sequencing and fingerprinting were done to confirm that the origin of each pair of cell lines were from the same patient. Gene expression microarrays using an affymetrix platform followed by Real Time Polymerase Chain Reaction (RT-PCR) were done to confirm the differentially expressed genes. Western blot was subsequently done to confirm RT-PCR findings using a monocolonal antibody. RESULTS Sequencing and fingerprinting confirmed that the matched pair cell lines were from the same patient. Microarray gene expression array demonstrated differential expression of several genes with Carbonic Anhydrase XII (CAXII) overexpression in metastatic cell lines in 3 different probes with up to 4 fold difference. RT-PCR confirmed our microarray findings with increased expression of CAXII in the cell lines derived from sites of lymph nodal metastases. Western blot analysis confirmed increased expression of the CAXII protein for the metastatic cell lines when compared to cell lines derived from the primary tumors. CONCLUSIONS The present results demonstrate that CAXII has increased expression in metastatic RCC as compared to primary RCC from the matched cell lines. Such findings may be due to adaptation to the lymph node environment and provide an explanation for the mechanism that metastatic cells employ to survive in the lymph environment thereby avoiding the recognition or destruction by the immune cells. Further assays will be performed to better elucidate the role of CAXII in lymph node metastases. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e63 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nnenaya Agochukwu Bethesda, MD More articles by this author Kiranpreet Khurana Cleveland, OH More articles by this author Eric Kauffman Bethesda, MD More articles by this author Brian Shuch Bethesda, MD More articles by this author Vladimir Valera Bethesda, MD More articles by this author Youfeng Yang Bethesda, MD More articles by this author W. Marston Linehan Bethesda, MD More articles by this author Gennady Bratslavsky Syracuse, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

CAXII Is a Sero-Diagnostic Marker for Lung Cancer

To develop sero-diagnostic markers for lung cancer, we generated monoclonal antibodies using pulmonary adenocarcinoma (AD)-derived A549 cells as antigens by employing the random immunization method. Hybridoma supernatants were immunohistochemically screened for antibodies with AMeX-fixed and paraffin-embedded A549 cell preparations. Positive clones were monocloned twice through limiting dilutions. From the obtained monoclonal antibodies, we selected an antibody designated as KU-Lu-5 which showed intense membrane staining of A549 cells. Based on immunoprecipitation and MADLI TOF/TOF-MS analysis, this antibody was recognized as carbonic anhydrase XII (CAXII). To evaluate the utility of this antibody as a sero-diagnostic marker for lung cancer, we performed dot blot analysis with a training set consisting of sera from 70 lung cancer patients and 30 healthy controls. The CAXII expression levels were significantly higher in lung cancer patients than in healthy controls in the training set (P<0.0001), and the area under the curve of ROC was 0.794, with 70.0% specificity and 82.9% sensitivity. In lung cancers, expression levels of CAXII were significantly higher in patients with squamous cell carcinoma (SCC) than with AD (P = 0.035). Furthermore, CAXII was significantly higher in well- and moderately differentiated SCCs than in poorly differentiated ones (P = 0.027). To further confirm the utility of serum CAXII levels as a sero-diagnostic marker, an additional set consisting of sera from 26 lung cancer patients and 30 healthy controls was also investigated by dot blot analysis as a validation study. Serum CAXII levels were also significantly higher in lung cancer patients than in healthy controls in the validation set (P = 0.030). Thus, the serum CAXII levels should be applicable markers discriminating lung cancer patients from healthy controls. To our knowledge, this is the first report providing evidence that CAXII may be a novel sero-diagnostic marker for lung cancer.

Identification of cell surface-specific markers to target human nucleus pulposus cells: Expression of carbonic anhydrase XII varies with age and degeneration

Back pain is a major cause of disability, affecting millions of people worldwide. One cause of axial back pain is degeneration of the nucleus pulposus (NP) of the intervertebral disc. This study was undertaken to investigate associations of NP cells with cell surface-specific proteins that differ from proteins in closely related cell types, i.e., intervertebral disc anulus fibrosus (AF) cells and articular cartilage (AC) chondrocytes, in order to identify potential surface molecules for directed delivery of therapeutic agents. We conducted a complementary DNA microarray analysis of 16 human samples from 6 donors, followed by gene list reduction using a systematic approach. Genes that were more highly expressed in NP than AC cells, contained transmembrane domains, and appeared attractive for targeting were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). As a viable candidate, carbonic anhydrase XII (CAXII) was analyzed at the protein level by immunohistochemistry and functional study. Microarray results demonstrated a clear divide between the AC and AF and between the AC and NP samples. However, the transcriptomic profile of AF and NP samples displayed a greater intersubject similarity. Of the 552 genes with up-regulated expression in NP cells, 90 contained transmembrane domains, and 28 were quantified by RT-PCR. Most intense CAXII labeling was observed in the NP of discs from young subjects and in degenerative tissue. CAXII may be considered for detection or targeting of degenerating disc cells. Furthermore, CAXII may be involved in pH regulation of NP cells. Its potential for directed delivery of regenerative factors and its functional role in NP cell homeostasis warrant further investigation.

Overexpression of carbonic anhydrase XII in tissues from resectable non‐small cell lung cancers is a biomarker of good prognosis

The pattern of protein expression in tumors is under the influence of nutrient stress, hypoxia and low pH, which determines the survival of neoplastic cells and the development of tumors. Carbonic anhydrase XII (CAXII) is a transmembrane enzyme that catalyzes the reversible hydration of cell-generated carbon dioxide into protons and bicarbonate. Hypoxic conditions activate its transcription and translation and enhanced expression is often present in several types of tumors. The aim of our study was to assess the prognostic significance of CAXII tumor tissues expression in patients with NSCLC. Five hundred fifty-five tumors were immunostained for CAXII on tissue microarrays (TMA) and the results were correlated with clinicopathological parameters and outcome of patients. CAXII overexpression was present in 105/555 (19%) cases and was associated with tumors of lower grade (p = 0.015) and histological type (p < 0.001), being significantly higher in squamous cell carcinoma. High CAXII expression correlated with better overall and disease-specific survival of patients with resectable NSCLC in univariate (p < 0.001) and multivariate survival analyses (p < 0.001). In conclusion, this is the first study demonstrating that a high CAXII tumor tissue expression evaluated on TMAs is related to a better outcome in a large series of patients with resectable NSCLC.

Specific features of the intestinal mucosa of obese Zucker rats

Metabolic syndrome is a group of obesity-related metabolic abnormalities that increase an individual’s risk of developing type 2 diabetes and cardiovascular disease. The obese Zucker rats (OZR) may represent a valuable animal model for studying several aspects of this increasingly prevalent problem in worldwide. In fact, the genetically obese (fa/fa) Zucker rats, due a recessive mutation of the leptin receptor gene (lepr), exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity and oxidative stress. Here, we report the preliminary results from our current studies aimed to investigate different metabolic markers in the OZR intestinal mucosa, compared with their lean counterparts (LZR). Starting from the important role attributed to carbohydrates in regulating the critical equilibrium of the intestinal environment, we applied lectin histochemistry to visualize the glycosylation pattern expressed in the OZR intestinal mucosa. The investigation was mainly focused to identification and in situ characterization of sialylated and fucosylated glycomponents which were directly demonstrated with SNA, MAL II, LTA, and UEA lectin binding. In addition, in order to look for additional and complementary information about sialic acid acetylation degree and sites, PNA and DBA lectin histochemistry was combined with sialidase predigestion, potassium hydroxide deacetylation, and differential periodate oxidation. As a parallel study, the distributional patterns of carbonic anhydrase (CA), the enzyme which is differently expressed in the gastrointestinal tract with several functions, such as regulation of cellular and extracellular acid-base homeostasis, salt absorption and fluid balance, were visualized. The immunohistochemical localization of the CA isoenzymes CAIV, CA IX, CA XII, and CA XIV was performed with the relevant specific antibodies. The complex of the data obtained suggest a marked modulation of the sialoglycoconjugate expression in the OZR intestinal epithelium, when compared with the LZR, to be considered as an interesting topic for further investigations.

Autosomal recessive hyponatremia due to isolated salt wasting in sweat associated with a mutation in the active site of Carbonic Anhydrase 12

Genetic disorders of excessive salt loss from sweat glands have been observed in pseudohypoaldosteronism type I (PHA) and cystic fibrosis that result from mutations in genes encoding epithelial Na+ channel (ENaC) subunits and the transmembrane conductance regulator (CFTR), respectively. We identified a novel autosomal recessive form of isolated salt wasting in sweat, which leads to severe infantile hyponatremic dehydration. Three affected individuals from a small Bedouin clan presented with failure to thrive, hyponatremic dehydration and hyperkalemia with isolated sweat salt wasting. Using positional cloning, we identified the association of a Glu143Lys mutation in carbonic anhydrase 12 (CA12) with the disease. Carbonic anhydrase is a zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide to form a bicarbonate anion and a proton. Glu143 in CA12 is essential for zinc coordination in this metalloenzyme and lowering of the protein-metal affinity reduces its catalytic activity. This is the first presentation of an isolated loss of salt from sweat gland mimicking PHA, associated with a mutation in the CA12 gene not previously implicated in human disorders. Our data demonstrate the importance of bicarbonate anion and proton production on salt concentration in sweat and its significance for sodium homeostasis.

Role of Carbonic Anhydrase IV in the Bicarbonate-Mediated Activation of Murine and Human Sperm

HCO3 − is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO3 − containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO3 − stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO3 − enters spermatozoa either directly by anion transport or indirectly via diffusion of CO2 with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO2 with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV−/− mice we found a 32.13% reduction in total CA activity in the latter. The CA IV−/− sperm also have a reduced response to CO2. While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO2 application, beat frequency of CA IV−/− sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO3 −, which is necessary for stimulation of sAC and hence early activation of spermatozoa.