Aim
The aim of this study was to investigate function, expression and localization/abundance of selected PDEs in biopsies from non-neoplastic appearing colonic mucosa obtained from patients with and without CRN. Thus, questioning if a possible predisposition to the cancer disease exists in non-neoplastic appearing colonic mucosa.
Study population
Patients referred for a colonoscopy on suspicion of CRC, were included and divided into two groups. The first group consisted of patients with present or history of CRN, while the second group was control patients (i.e. CTRL) with no present or history of CRN. Patients with incomplete colonoscopy, hemorrhagic diathesis, and inflammatory bowel disease or with previous sigmoid resection were excluded from the study. A total of 27 subjects were enrolled, hereof 12 CRN-subjects (4 women) and 15 CTRLs (7 women). For each patient, we noted age, body mass index (BMI), previous illnesses, medication, all signs of earlier colorectal disease and the findings during the colonoscopy. Age and BMI were well balanced between groups. Mean age (±SEM) for CTRL patients was 58 ± 3.7 and for CRN patients 66 ± 3.7 years. Mean BMI was 24.9 ± 1.5 in CTRL patients and 26.2 ± 0.9 in CRN patients. Eight patients from the CTRL and 6 patients from the CRN group had comorbidities such as ischemic heart disease, heart failure, hypertension, atrial fibrillation, diabetes, kidney failure, chronic obstructive lung disease, dyslipidemia, intermittent claudication, peptic ulcer disease, osteoporosis, rheumatic arthritis, polycystic ovary syndrome and pacemaker implantation. There were no apparent differences between the two groups in comorbidity. Eight patients in the CTRL group and 8 patients in the CRN group were using regular medications e.g. anti-thrombotic, ACE inhibitors, statins, levothyroxine, proton pump inhibitors, angiotensin II receptor antagonists, glucocorticoids, β-blockers, β-agonists, anti-histamines, xanthine oxidase inhibitors, diuretics, antifolate and anti-emetics. There were no apparent differences between the two groups in prescribed medications. None of the drugs has a direct effect on the PDE metabolism.
Ethics
The Scientific Ethical Committee of Copenhagen (H32013107) and The Danish Data Protection Agency approved the study protocol (BBH-2013-024, I-Suite no: 02342). The study was conducted by the Helsinki declaration. All patients participating gave written informed consent.
Biopsy extraction and processing
Six biopsies from each patient were obtained during endoscopy from non-neoplastic appearing colonic mucosa using standard biopsy forceps (Boston Scientific, Radial Jaw 4, outside diameter of 2.2 mm). Biopsies were obtained approximately 30 cm orally from the anal verge and at least 10 cm from endoscopically abnormal tissue (i.e. neoplasia). The biopsies were immediately transferred to an iced, oxygenized bicarbonate Ringer solution with the following composition (in mM): Na+ (140), Cl− (117), K+ (3.8), PO43− (2.0), Mg2+ (0.5), Ca2+ (1.0), glucose (5.5) and HCO3− (25). Before obtaining biopsies, the media pH was adjusted to 7.4 by gassing with 95% O2/5% CO2.
Chemicals
Theophylline, indomethacin, acetazolamide, zaprinast, cilostamide, rolipram, cAMP and cGMP were purchased from Sigma-Aldrich (Seelze, Germany). Sildenafil, db-cAMP, cGMP, and antibodies for PDE3B (cat. no.: SC-376823), PDE4A (cat. no.: SC-74428), PDE4B (cat. no.: SC-25812), PDE4D (cat. no.: SC-25097) and PDE5A (cat. no.: SC-32884) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for PDE3A (cat. no.: LS-A732) and PDE4C (cat. no.: LS-C185777) were purchased from LifeSpan BioSciences, Inc. (Seattle, WA, USA). Primer sequences were synthesized by TAGCopenhagen (Copenhagen, Denmark). The human PDE10 real-time PCR primers were purchased from Qiagen. Bumetanide was obtained from Leo Pharmaceuticals (Copenhagen, Denmark). All other chemicals were of analytical grade.
Pretreatment
Up to 4 biopsies from each patient were mounted in Mini-Ussing-Air-Suction-Chambers (MUAS chambers) for measuring transepithelial electrolyte transport captured as short circuit currents (SCC). The method used has previously been described in detail [21, 22]. Correction for media resistance between tips of potential electrodes was carried out just before mounting of biopsies, thus circumventing challenges of edge damage in measuring correct active ion transport. The time between the biopsy procedure and mounting of up to 4 biopsies into MUAS chambers was about 45 min during which the samples were kept in oxygenated-Ringer solution at approximately 0 °C. Following approximately 15 min of equilibration, baseline SCC and slope conductance values were recorded. Amiloride (20 μM) was subsequently added to the luminal side of the MUAS chamber to inhibit epithelial sodium channels and thereby reducing cell energy expenditure. Indomethacin (13 μM) was added to the basolateral side to inhibit endogenous synthesis of cNTs. Experiments were resumed after an additional 20 min equilibration period. If stable SCC was not achieved during pretreatment, the biopsy was excluded from further study.
Cyclic-AMP-related PDEs and inhibitors
In one part of our functional study, db-cAMP (16 μM) was applied on the basolateral side of biopsies. After 10 min, biopsies were then exposed to cilostamide (PDE3 inhibitor, 1 μM) or rolipram (PDE4 inhibitor, 1 μM). PDE3 and PDE4 primarily degrade cAMP [23, 24], and their inhibition elicited a rise in SCC. Then a dose of theophylline (400 μM, added bilaterally), a competitive and nonselective PDE-inhibitor raising intracellular levels of cNT [25], was applied to give maximal inhibition of PDE activity and maximal cNT-induced SCC. Finally each experiment was terminated after adding bumetanide (25 μM), ouabain (200 μM) and acetazolamide (250 μM), alone or in combination to the basolateral side of the biopsies.
Cyclic-GMP investigation
In a second part of the functional study, cGMP (16 μM) was applied to biopsies. Previous reports have provided data showing more consistent responses when cGMP is applied vs db-cGMP [21]. After 10 min, samples were exposed to sildenafil (1 μM) or zaprinast (1 μM), both PDE5 and PDE10 inhibitors [26]. However, when no changes in SCC were observed, concentrations were increased stepwise to reach a final concentration of maximally 500 μM for the inhibitors and 50 μM for cGMP. Each experiment was terminated following the basolateral addition of bumetanide (25 μM), ouabain (200 μM) and acetazolamide (250 μM), alone or in combination.
RNA extraction from biopsies
One biopsy from each patient was obtained for RNA-analysis. The mean wet weight of each biopsy was 5.8 mg. Thus we were only able to perform qPCR on a selected number of PDE isoforms. Biopsies were immediately transferred to RNA Later (Life Technologies, Naerum, Denmark). Biopsies were homogenized using a TissueLyser II (Qiagen, Copenhagen, Denmark), and RNA was extracted using NucleoSpin RNA® (Macherey-Nagel, Düren, Germany). RNA concentration and purity were determined using a NanoDrop® ND-1000 (NanoDrop Technologies, Wilmington, DE, USA), and the latter as the A260/A280 and A260/A230 absorbance ratios.
cDNA synthesis
RNA was converted to cDNA using the iScript™ cDNA Synthesis Kit (BioRad, Copenhagen, Denmark) according to the manufacturers protocol.
Primer design
Primers against genes of interest and against β-actin [21] were designed using Primer3 (http://frodo.wi.mit.edu/primer3/input.htm) based on sequences obtained from Ensembl (http://www.ensembl.org/). The primer sequences were synthesized by TAGCopenhagen (Copenhagen, Denmark): PDE3A forward (5′-CAGAGTGAATCCCGTCACTTC-3′) reverse (5′-TGGTCCAAGTGGAAGAAACTC-3′); PDE3B forward (5′-TATGATCACCCAGGGAGGAC-3′) reverse (5′-GTTGTATTCTGGGCGAGAAAG-3′); PDE4A forward (5′-TGAAGACCGATCAAGAAGAGC-3′) reverse (5′-GTAATCCGACACGCAAAAGAT-3′); PDE4B forward (5′-ATCTCACGCTTTGGAGTCAAC-3′) reverse (5′-TTAAGACCCCATTTGTTCAGG-3′); PDE4C forward (5′-GCGATATCTTCCAGAACCTCA-3′) reverse (5′-CTTGTCACCTTCTTGGTCTCC-3′); PDE4D forward (5′-TTTCACGGTGGCACATACAT-3′) reverse (5′-GTGGACAAAATTTGCTTGGAG-3′); PDE5A forward (5′-TTGTGCAGAACTTCCAGATGA-3′) reverse (5′-TTTAGAGCAGCAAACATGCAC-3′); β-Actin forward (5′-ACCCAGCACAATGAAGATCA-3′) reverse (5′-CGTCATACTCCTGCTTGCTG-3′).
Dilution series of cDNA from HEK293 cells were run to verify acceptable amplification efficiencies and specificities by standard and dissociation curves for all primer sets.
qPCR analysis
cDNA was amplified on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using Fast SYBR® Green Master Mix (Applied Biosystems) by the manufacturer’s manual. All samples were run in triplicates with β-actin primers on all plates. Results were analysed using SDS 2.3 (Applied Biosystems), and expression was calculated by the 2-ΔCT method.
Immunohistochemical localization and quantification
One colon biopsy from each patient was put aside in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), embedded in paraffin, and cut into 4 μm sections. Sections were boiled in citric buffer (pH 6 or 9) in a microwave oven for 15 min, followed by preincubation in 2% BSA for 10 min, and overnight incubation at 4 °C with primary antibodies. Subsequently, the sections were incubated for 40 min with biotinylated secondary antibody immunoglobulins, followed by a preformed avidin and biotinylated horseradish peroxidase macromolecular complex (code number PK-4000; Vector Laboratories) for 30 min. Three percent hydrogen peroxide was added after the second antibody layer. Finally, 3,3-diaminobenzidine (KEM-EN-TEC, catalog number 4170) was added for 15 min, followed by a 2 min incubation in 0.5% copper sulfate (Merck; catalog number 2790) diluted in Tris buffer containing 0.05% Tween 20. Counterstaining was performed with Mayer’s hemalun.
Images for quantification were recorded using a Zeiss Axioplan 2 plus microscope (Jena, Germany) fitted with a Photometrics CoolSNAP camera (Tucson, AZ, USA) and analysis was performed using Image-Pro Plus 7.0 software. A quantification of coloring was performed by to blinded investigators as a marker for protein abundance in biopsies (Nctrl = 7, NCRN = 8). Images for quantification were recorded at 20x magnification and the area measured represented 176000 μm2 of tissue. The area of stained structures was measured by selecting a colored region of interest. Automatically, areas with same color were measured - one image from each biopsy was measured. Mean ± SEM for samples in each group were calculated. Images for localization were recorded using a Zeiss Axio10 Imager A1 microscope (Jena, Germany) fitted with a Zeiss AxioCam ICc 3 camera (Jena, Germany) and analysis was performed using Image-Pro 9.1 software. Only mucosal layers were analyzed.
Statistical analysis
Values are expressed as the mean ± SEM. The mean value was calculated and used when identical experiments were performed on several biopsies from the same patient. The statistical significance of differences between two groups was tested using Student’s t-test, provided that the variances of the groups were similar. For functional data, the Mann–Whitney U-test was applied in the case of unequal variance. For qPCR data, data with unequal variance were log transformed. When the variance of log transformed data was unequal, the Mann–Whitney U-test was applied. In qPCR experiments, when calculating statistics for multiple groups, one-way ANOVA with Tukey’s multiple comparisons test was used. A p-value less than 0.05 were considered statistically significant. Statistical analysis was done using SigmaPlot 12.3 for Windows, Systat Software Inc. (USA/Canada).