Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy

Aims/hypothesis Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats.
Introduction
Type 1 diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells [1]. Treatment requires daily injections of insulin to normalise blood glucose levels. Tight glucose control delays, but does not prevent, the onset of complications, which increase morbidity and mortality. Transplantation therapy is limited by the scarcity of donors and chronic immunosuppressive regimens [2]. Physiological glucose control could be achieved by genetically engineering surrogate beta cells that are capable of synthesising, storing and secreting insulin in response to metabolic signals.
One approach to liver-directed gene therapy of diabetes is the transcriptional control of insulin expression without the induction of storage [3, 4], which is nonresponsive to minute-to-minute fluctuations in blood glucose. Another strategy is the delivery of beta cell transcription factors to the liver to induce pancreatic transdifferentiation. Studies have employed transiently expressing adenoviral vectors to deliver pancreatic and duodenal homeobox gene 1 (Pdx1) and neurogenic differentiation gene 1 (Neurod1)/betacellulin gene (Btc) [5–8] to streptozotocin (STZ)-induced diabetic mice and isolated human liver cells, which have resulted in liver-to-pancreas transdifferentiation and reversal of diabetes. However, conversion of significant portions of the liver to ‘true’ pancreatic tissue may lead to an increased risk of autoimmune attack and development of hepatitis, due to the exocrine differentiating capacity of PDX1 [7].
Most studies on the delivery of genes to the liver have employed adenoviral vectors [5–8] due to their ability to efficiently transduce non-dividing cells. However, the therapeutic gene remains episomal, often with consequent short-term gene expression and detrimental host immune responses [9]. Short-term euglycaemia has been achieved following delivery of insulin via an adeno-associated vector, with dose-dependent efficacy [10]. Lentiviral vectors (LV) are capable of stably integrating genes into the chromosomes of dividing and non-dividing cells [9, 11–13]. However, their ability to transduce quiescent hepatocytes in vivo remains low, as efficient transduction generally requires induction of cell cycling [14], which can be achieved by partial hepatectomy [15] or delivery of hepatotoxins [16, 17].
We have developed an improved method for high efficiency transduction of rat hepatocytes in vitro and describe a modified surgical technique for efficient delivery of LV to rat livers resulting in efficient transduction, without the requirement for cell division. We have used this technique to deliver lentivector-encoded furin-cleavable insulin (INS-FUR) [18], which enables cells to cleave proinsulin to mature insulin, in the livers of diabetic rats. This has resulted in permanent reversal of diabetes, insulin storage and restoration of glucose tolerance.
Materials and methods
Vector production INS-FUR cDNA [18] (proins.IfurIIfur.B10D; INS-FUR; gift from Genentech, San Francisco, CA, USA) was cloned into the multi-cloning site of the LV HIV/MSCV (HMD) [19] (a gift from J. Choi, Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA, USA) at the EcoRI site to produce the HMD/INS-FUR construct. This LV vector has an HIV/murine stem cell virus hybrid long terminal repeat as the promoter and an internal ribosomal entry site, allowing bicistronic expression of the genes encoding insulin and enhanced green fluorescent protein (EGFP) simultaneously. The vectors were produced by calcium phosphate precipitation cotransfection of three plasmids (10 μg HMD/INS-FUR or 10 μg pHR’CMVGFP [20] expressing EGFP alone, plus 20 μg pCMVΔR8.2 and 10 μg of the heterologous VSV-G envelope construct pCVSV-G; all other vector constructs were gifts from I. Verma, Salk Institute for Biological Sciences, CA, USA) into 293T cells [9]. Conditioned medium was collected 48 h after transfection, filtered and pelleted (50,000 g). Virus titre was determined by transducing 293T cells (5 × 105) with serially diluted vector stocks and quantifying numbers of EGFP-positive cells by flow cytometry as described [21]. Viral replication-competency was also assessed.
Transduction of hepatocytes in vitro Maintenance and experimental manipulation of male Wistar rats (250–300 g; Gore Hill Research Laboratories, Sydney, NSW, Australia) were performed in accordance with the NIH principles of laboratory care and regulations of the Australian Research Council and were approved by the University of Technology Sydney. Hepatocytes were isolated using a two-step EDTA/collagenase digestion. The portal vein (PV) and the abdominal aorta were cannulated and the liver was flushed in situ with warmed (40°C) Hanks’ buffered salt solution without Ca2+ and Mg2+ (Sigma-Aldrich, Castle Hill, NSW, Australia), plus 1 mmol/l EDTA for 5–7 min. This was followed by perfusion with 0.05% collagenase (C5138; Sigma-Aldrich) in Hanks’ buffered salt solution with Ca2+ and Mg2+ (Sigma-Aldrich) for 3–5 min. The hepatocytes were collected, filtered through sterile 250 and 100 μm mesh and washed three times in DMEM plus 10% FCS. Preparations containing greater than 85% viable cells were plated onto Matrigel (gift from C. Liddle, Westmead Hospital, Sydney, NSW, Australia) and cultured in DMEM supplemented with NaHCO3 (44 mmol/l), insulin (100 mU/l), glutamic acid (2.5 mmol/l), proline (1 mmol/l), ascorbic acid (56 mg/l), penicillin (100 mg/ml), nicotinamide (10 mmol/l), epidermal growth factor (20 ng/ml), selenium salts (0.1 μg/ml) and hydrocortisone (1 nmol/l). After 5 days, 1.5% DMSO together with CuSO4 (2.5 μg/l), FeSO4.7H2O (834 μg/l) and ZnSO4 (1.47 mg/l) was also added to the medium. Although Matrigel inhibits cell division, hepatocytes were also irradiated (caesium-137 source; total doses of 0, 10 or 20 Gy) 24 h after seeding and DNA synthesis was assessed by [3H]thymidine incorporation [22]. The positive control was hepatocytes plated on rat-tail collagen 1 (Collaborative Biomedical Products, Bedford, MA, USA).At 24 h after cell attachment, hepatocytes (5×105 per well) were transduced with pHR’CMVGFP LV (2.5 × 107 transduction units [TDU]) for 16 h in the presence of 8 μg/ml polybrene. Transduction efficiency was assessed at 2, 7, 14, 21 and 28 days by fluorescence microscopy and flow cytometry. EGFP-expressing cells were also stained for the liver-specific marker glucose-6-phosphatase.
Transduction of hepatocytes in vivo Rats were divided into four groups of 16 animals. For Group 1, the right adrenal vein was ligated and successively the infrahepatic vena cava (IVC), hepatic artery, PV and suprahepatic vena cava were clamped before pHR’CMVGFP or HMD LV (5 × 108 TDU) was infused through the PV. During this procedure, blood supply to the liver was stopped for 5 min and then recommenced for 2 min. This procedure of intervallic infusion in full flow occlusion (FFO) was repeated three times. Animals in Group 2 were treated similarly; except 0.9% saline was infused. A 70% partial hepatectomy was performed on animals in Group 3 by resecting the left lateral and median liver lobes. The right adrenal vein was ligated and LV was infused 24 h later by the procedure detailed for Group 1. A fourth group of animals was treated the same as Group 3, except 0.9% saline was infused. Livers were removed at 1, 2, 4 and 8 weeks. Transduction efficiency was assessed by flow cytometric quantification of EGFP-positive cells.To assess cell proliferation, 5-bromo-2′-deoxyuridine (100 mg/kg; Sigma) was injected intraperitoneally at 18, 28 and 48 h after surgery (partial hepatectomy or sham-operated) and into untreated animals (three animals per time-point). After 2 h, livers were excised and processed by conventional histological techniques. Incorporation of 5-bromo-2′-deoxyuridine was determined by immunohistochemistry.Diabetes was induced by an intraperitoneal injection of STZ (75 mg/kg body weight; Sapphire Science, Crows Nest, NSW, Australia). Animals received HMD/INS-FUR or empty HMD vector administered by FFO without partial hepatectomy. After treatment, animals were monitored for body weight and blood glucose. Animals were killed at 60 or 500 days and body tissues were sampled. To determine the proportion of EGFP-expressing cells that were hepatocytes, liver cells were isolated and stained with anti-cytokeratin 18 (CK18) antibody (Chemicon, Temecula, CA, USA) and R-phycoerythrin-conjugated secondary antibody (Chemicon). Numbers of insulin-positive cells were determined using an anti-insulin antibody (Sigma-Aldrich) and aminomethylcoumarin-conjugated secondary antibody followed by flow cytometric analysis.
Microscopic analyses Livers and pancreases were collected 60 and 500 days after vector infusion. Islet hormones were identified by peroxidase immunohistochemistry using diaminobenzidine as substrate. Primary antibodies against insulin (BioGenex, San Ramon, CA, USA), glucagon (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and somatostatin (Santa Cruz Biotechnology) were used. All cells in ten random fields were scored. Data were expressed as the number of positive cells per mm2 of tissue.For electron microscopy, tissue was fixed and processed using uranyl acetate block-staining and lead citrate counter-staining of ultra-thin sections (80 nm). For insulin immunoelectronmicroscopy, a post-embedding immunogold procedure was used. The beta cell line Nit-1 was the positive control. Tissues and cells were embedded in LR white and labelling procedures were performed as previously described [23].
Functional studies Intravenous glucose tolerance tests were performed on fasted (6–8 h) animals. After infusion of glucose (0.5 g/kg body weight), blood samples were collected from the tail vein. Insulin content of pancreas and liver was determined after acid/ethanol extraction [23].Insulin and proinsulin in acid/ethanol extractions were separated on a Sephadex G50 superfine column (Sigma-Aldrich) [24]. Insulin in serum samples and column fractions was measured using an RIA for human insulin (Linco Research, St Charles, MO, USA), which has less than 1 and 6% cross-reactivity with rodent insulin and human proinsulin, respectively. Commercial RIAs (Linco Research) were used to measure human and rat C-peptide [25].To assess liver function, aspartate transaminase (AST) and alanine aminotransferase (ALT) were measured daily over the first 10 days and then monthly using commercial kits (Roche Diagnostics, Castle Hill, NSW, Australia).
PCR analyses For RT-PCR, liver, pancreas, spleen, kidney and lung were collected at 60 and 500 days after vector infusion and snap-frozen in liquid nitrogen. Total RNA was extracted using Trizol (Invitrogen Australia, Mt Waverly, VIC, Australia). Samples were treated with DNase I (Invitrogen). Real-time quantitative PCR was conducted using SYBR green I and a sequence detection system (ABI 7500 HT; Applied Biosystems, Scoresby, VIC, Australia). Optimal primer and cDNA template concentrations were determined by titration. Amplification efficiencies for each primer set were determined to be similar. Data were represented as differences between threshold cycle values (ΔCT) for the transcripts of interest and the internal standard, β-actin. The expression of the genes encoding human insulin and EGFP was examined in normal and HMD/INS-FUR-transduced liver cells using semi-quantitative RT-PCR. Oligonucleotide sequences are available on request.
Qualitative western blot analyses Nuclear and cytoplasmic extracts of liver and pancreas were prepared using a nuclear extraction Kit (Chemicon). Samples (1 μg protein) were separated by SDS-PAGE and transferred to nitrocellulose. Primary antibodies against PDX1 and NEUROD1 (Santa Cruz) were used.
Statistical analyses Differences between groups were determined by Student’s paired t test or, if there were more than two groups, by one-way analysis of variance after log transformation of data. Data are expressed as means ± standard errors (SEM).
Results
Efficient transduction of hepatocytes Isolation of hepatocytes by dual perfusion of the PV and abdominal aorta resulted in increased cell viability as compared with perfusion of the PV alone (85.7 ± 6.2% vs 67.5 ± 7.8%, respectively, p = 0.03). The cells were morphologically normal, as assessed by light and electron microscopy, with maintenance of gap junctions between cells and limited vacuolation. Hepatocytes grown on Matrigel in the optimised medium, with or without irradiation, did not undergo cell division, as assessed by [3H]thymidine incorporation. Cells grown on collagen, which enhances cell growth, reached a maximum growth rate at day 2 and this plateaued between days 5 and 6 (data not shown). Expression of EGFP was seen 48 h post-transduction and persisted for over 1 month (Fig. 1a). Up to 87 ± 1.2% of the irradiated cells expressed EGFP (Fig. 1b). Most EGFP-positive cells (80.2 ± 10.7%) were also positive for glucose-6-phosphatase, which suggested a hepatocyte lineage (data not shown). Growth of hepatocytes on collagen did not increase transduction efficiency. Delivery of HMD/INS-FUR to hepatocyte cultures resulted in low constitutive insulin expression (0.2 pmol per 3 × 105 cells) and no insulin storage; therefore transplantation of hepatocytes transduced in vitro was not performed.
Fig. 1EGFP expression in hepatocytes in vitro and in vivo. a EGFP expression in primary hepatocytes in vitro at indicated times post transduction with pHR’CMVGFP. Original magnification: 400×. The insets show untransduced hepatocytes. b Flow cytometric analysis of irradiated (10 Gy), non-dividing primary hepatocytes 14 days post-transduction with pHR’CMVGFP. Negative control cells show background fluorescence only. EGFP-positive cells constituted 87 ± 1.2% of all cells following transduction. Graphs representative of three independent experiments. c Flow cytometric analysis of liver cells isolated from a normal control rat and a rat transduced with the HMD LV vector 1 month previously. EGFP-positive cells constituted 60 ± 3.2% of all liver cells. The results are representative of three experiments. d Representative flow cytometry dot-plot showing the proportion of EGFP-expressing cells that were hepatocytes (CK-18-positive: 58 ± 3.1%) from an animal that had been transduced with the HMD/INS-FUR LV 2 months previously and e the proportion of insulin-positive (57 ± 2.8%) hepatocytes. Graphs are representative of three experimentsWe delivered pHR’CMVGFP to the livers of diabetic rats by FFO and compared the transduction efficiency in non-hepatectomised and partially-hepatectomised animals. The total wet weights of liver 48 h after partial hepatectomy were similar to those of non-hepatectomised rats (14.3 ± 0.4 and 14.7 ± 0.8 g, respectively). We also determined whether FFO stimulated cell division. In control rats, 0.1 ± 0.06% of hepatocytes were proliferating at 18 h and this rate did not significantly alter at any time point. At 18 h in animals that had undergone FFO, 0.3 ± 0.2% of hepatocytes were proliferating, compared with 0.4 ± 0.3% at 28 h and 0.1 ± 0.04% at 48 h. These values were not significantly different to those recorded for control animals. By comparison, in animals that had undergone a partial hepatectomy, 12.8 ± 2.4% of hepatocytes were proliferating at 18 h. This value increased to 21.4 ± 4.3% at 28 h and then decreased to 7.4 ± 1.2% at 48 h.To determine transduction efficiency, liver cells were isolated after transduction with pHR’CMVGFP and EGFP-positive cells were assessed by flow cytometry. The maximal number of EGFP-expressing cells was detected 2 weeks after transduction (57.0 ± 2%) and was not significantly different after 2 months (58.2 ± 3.5%). In animals that were partially hepatectomised prior to transduction, there was no significant difference in numbers of EGFP-expressing hepatocytes (59.2 ± 4%) after 2 months compared with non-hepatectomised animals. Use of the HMD vector also resulted in high levels of transduction (Fig. 1c). Cells expressing EGFP/INS-FUR were almost exclusively hepatocytes, as verified by CK18 and insulin labelling (Fig. 1d,e).
Transduction with HMD/INS-FUR reversed STZ-diabetes  Blood glucose levels of STZ-diabetic rats decreased to subnormal levels immediately after transduction with HMD/INS-FUR vector (Fig. 2a). By day 5, blood glucose levels of insulin-transduced animals were not different to non-diabetic controls and reversal of diabetes was maintained for 500 days (experimental endpoint). STZ-diabetic rats treated with HMD alone remained hyperglycaemic until day 60, when they were killed. The body weights of rats treated with HMD/INS-FUR were not significantly different to those of non-diabetic controls until day 125, lower between days 135 and 275, becoming not significantly different again after day 300 (Fig. 2b). The body weights of rats treated with the empty vector alone continued to fall from day 50 to 60. Throughout the experimental period, there was no elevation of liver enzymes in animals treated with HMD/INS-FUR (AST: 38.4 ± 6, ALT: 26.9 ± 5 U/l) compared with control animals (AST: 44.2 ± 16, ALT: 19.1 ± 7).
Fig. 2Stable expression of INS-FUR ameliorated STZ-induced hyperglycaemia. Blood glucose concentrations (a) and body weights (b) of non-diabetic control rats (black circles) and STZ-diabetic rats treated with HMD/INS-FUR (white squares) or HMD alone (black triangles). Values are means±SEM (n = 5)
HMD/INS-FUR transduction stimulated expression of pancreatic hormones We examined pancreatic hormone-producing cells in the livers of rats transduced with LV after 2 months. Figure 3a shows extensive staining for insulin in normal pancreatic islets. By comparison, pancreatic beta cells were rarely observed in STZ-diabetic animals (1.5 ± 1.4 cells) (Fig. 3b). Normal liver was negative for insulin (Fig. 3c), glucagon and somatostatin (not shown). No pancreatic hormones were detected in livers of animals treated with empty vector. Following transduction with HMD/INS-FUR, significant numbers of hepatocytes were converted into insulin-positive cells (Fig. 3d). There was also evidence of some pancreatic transdifferentiation, as glucagon- and somatostatin-positive cells were detected (Fig. 3e,f). Cells positive for pancreatic hormones were distributed throughout the livers of animals transduced with HMD/INS-FUR and not restricted to regions adjacent to the PV.
Fig. 3Expression of pancreatic hormones following reversal of STZ-induced hyperglycaemia. Photomicrographs of anti-insulin staining of (a) normal pancreas, (b) STZ-diabetic HMD/INS-FUR vector-treated pancreas, (c) normal liver and (d) STZ-diabetic HMD/INS-FUR vector-treated liver at 2 months. Photomicrographs of anti-glucagon (e) and anti-somatostatin (f) staining in STZ-diabetic HMD/INS-FUR vector-treated liver at 2 months. Positive cells appear brown. Original magnification: 200×. In a total of 1,200 ± 61 cells the number of insulin-positive cells (585.6 ± 73.8 cells/mm2) was fivefold higher than the number of glucagon-positive cells (109.9 ± 37.1 cells/mm2) and 11-fold higher than the number of somatostatin-positive cells (52.2 ± 8.1 cells/mm2). The number of EGFP-positive cells was 854 ± 80.9 (data not shown). Therefore, some EGFP-positive cells did not store pancreatic hormones. No pancreatic hormones were detected in liver from animals treated with empty vector or from control animals. g Transmission electron micrograph showing secretory vesicles with dense granules surrounded by a pale halo (bar: 500 nm). Inset, vesicle 320 nm in diameter. h Immuno-electron micrographs showing localisation of insulin in liver 2 months after the transduction of an STZ-diabetic rat with HMD/INS-FUR and (i) insulin-secreting granules in Nit-1 beta cells (positive control); bar: 400 nmElectron microscopy revealed cytoplasmic granules (270–330 nm in diameter) in liver tissue from animals treated with HMD/INS-FUR (Fig. 3g). Immunoelectron microscopy of insulin-producing cells in the liver of STZ-diabetic rats transduced with HMD/INS-FUR was comparable to those observed in Nit-1 beta cells (Fig. 3h,i). Inflammatory cell infiltration and necrosis within the livers of transduced animals were not observed. Insulin-containing granules were absent in rats treated with empty vector and in untreated animals.
HMD/INS-FUR transduction induced hepatic insulin production Insulin storage was 4,165 ± 180 pmol per liver in HMD/INS-FUR-treated animals, compared with 10,418 ± 210 pmol in normal rat pancreas (n = 3). STZ-treated rat pancreas only contained 255±110 pmol insulin. Separation of insulin from proinsulin in liver extracts from animals treated with HMD/INS-FUR yielded two distinct peaks. The first represented proinsulin and partially processed material and the second larger peak corresponded to fully processed insulin (Fig. 4).
Fig. 4Stable expression of INS-FUR resulted in production of mature human insulin. Extracts of rat livers were separated by gel permeation chromatography and assayed for insulin. Arrows indicate mobility of mature human insulin (I) (5,800 Da) and proinsulin (pI) (9,400 Da). Results are representative of three independent experiments
HMD/INS-FUR transduced animals displayed regulated insulin secretion There was no significant difference between the ability of STZ-diabetic animals transduced with the HMD/INS-FUR vector and untreated animals to normalise blood glucose levels after delivery of a bolus of intravenous glucose (Fig. 5a). Levels of human C-peptide and human insulin in serum samples from HMD/INS-FUR-treated animals (Fig. 5b,c) were comparable to levels of rat C-peptide and insulin recorded for non-diabetic animals. Negligible levels of rat C-peptide were detected in serum from HMD/INS-FUR-treated STZ-diabetic animals (Fig. 5c).
Fig. 5Plasma glucose (a), insulin (b) and C-peptide (c) levels after IVGTT. IVGTTs remained normal for animals maintained for 500 days (data not shown). White squares, HMD/INS-FUR-treated animals (60 days) expressing human insulin and human C-peptide; black diamonds, normal control animals expressing rat insulin and rat C-peptide; black triangles, rat C-peptide in HMD/INS-FUR-treated animals. Values are means±SEM (n = 5)
HMD/INS-FUR transduction stimulated expression of beta cell transcription factors EGFP expression was only detected in liver tissue of pHR’CMVGFP- (Fig. 6a) and HMD-treated (data not shown) rats. Similarly, INS-FUR expression was restricted to the livers of HMD/INS-FUR-treated rats (Fig. 6a). Insulin was not detected in livers from diabetic control animals and diabetic animals injected with HMD alone (data not shown). Mean ratios of insulin : β-actin (1.000 ± 0.069) and EGFP : β-actin (1.088 ± 0.062) in HMD/INS-FUR liver, determined by semi-quantitative PCR, were not significantly different. Transduction of STZ-diabetic rats with the HMD/INS-FUR vector induced sustained expression of several pancreatic transcription factors (Fig. 6b). Normal liver and untreated diabetic liver (not shown) were negative for all pancreatic transcription factors, while normal pancreas was positive.
Fig. 6Beta cell transcription factors and pancreatic hormones expressed in transduced rat livers. a RT-PCR analysis of transcription of the genes encoding EGFP or INS-FUR in animals transduced with pHR’CMVGFP or HMD/INS-FUR LV, respectively: liver (lane 1), pancreas (lane 2), spleen (lane 3), kidney (lane 4) and brain (lane 5) at 60 days. Results for HMD/INS-FUR transduced animals at 500 days were identical (not shown). b RT-PCR analysis of transcription of the genes for: (1) beta cell transcription factors (Pdx1, Neurod1, Neurog3, Nkx2-2, Nkx6-1, Pax4, Pax6); (2) the pancreatic endocrine hormones somatostatin (Sst), glucagon (Gcg) and pancreatic polypeptide (Ppy); (3) GLUT2 (Slc2a2) and glucokinase (Gck); (4) rat insulin 1 and 2 (Ins1, Ins2); (5) insulin proconvertase PC2 (Pc2); (6) the exocrine marker p48 (P48); and (7) β-actin (Actb) in normal liver (lane 1), liver transduced with HMD alone at 60 days (lane 2), liver transduced with HMD/INS-FUR LV at 60 days (lane 3) and 500 days (lane 4), and normal pancreas (lane 5). c Western blot analysis for PDX1 and NEUROD1 in normal pancreas (lane 1), liver transduced with HMD/INS-FUR LV at 60 days (lane 2), liver transduced with HMD alone at 60 days (lane 3) and normal liver (lane 4). PDX1 and NEUROD1 generated bands at 43 and 50 kDa, respectivelyUnexpectedly, both Pdx1 and Neurod1 expression were detected by RT-PCR in empty vector-transduced livers; however, protein production was not detected (Fig. 6c). HMD/INS-FUR-transduced livers also expressed the genes for neurogenin 3 (Neurog3) and Nkx2-2 (Nkx2-2). Glucagon and somatostatin were present in liver transduced with HMD/INS-FUR (confirmed by immunohistochemistry; Fig. 3e,f). Pancreatic polypeptide was not expressed. The transduction of diabetic animals with HMD/INS-FUR did not result in complete pancreatic transdifferentation of liver tissue since expression of the rat insulin genes (Ins1, Ins2) and the genes encoding the insulin prohormone convertase 2 (Pc2, also known as Pcsk2) and the exocrine marker p48 (Ptf1a, also known as P48) were not detected (Fig. 6b). Quantitative real time PCR analysis of the expression levels of beta cell transcription factors Pdx1 (4.3 ± 0.9), Neurod1 (5.7 ± 1.1) and Neurog3 (7.4 ± 1.8) in HMD/INS-FUR-treated livers at 500 days indicated that they were lower (p < 0.001) than levels expressed in normal pancreatic tissue (Pdx1: 12.5 ± 2.3, NeuroD1: 16.8 ± 1.4, Neurog3: 18.5 ± 2.2).
Discussion
This is the first report of permanent amelioration of STZ-induced hyperglycaemia using LV delivery of INS-FUR. Transduced animals processed, stored and secreted insulin in a glucose-regulated manner. Remission of diabetes, due to the secretion of insulin from residual beta cells, was discounted as insulin-positive pancreatic beta cells were rarely observed and levels of rat C-peptide were negligible. This is also the first report showing that transduction of INS-FUR alone can induce expression of several beta cell transcription factors resulting in hepatocyte differentiation along a pancreatic lineage.
All previous reports of liver–pancreas transdifferentiation have been described in cells that endogenously express beta cell transcription factors [23, 26] or have been engineered to express these factors, leading to varying degrees of transdifferentiation [5–8, 27, 28]. Following immediate reduction in blood glucose due to transgene expression, induction of long-term euglycaemia in the present study was probably related to partial hepatic-to-pancreas transdifferentiation triggered by the lentiviral intrahepatic delivery method of INS-FUR or from expression of stably integrated transgene in the host genome. The HMD/INS-FUR transgene alone, which constitutively expresses insulin, could not mimic the glucose-sensitive kinetics demonstrated by IVGTT, nor could it have solely induced insulin storage. Evidently, the hepatic delivery of the LV was a key factor, for expression of HMD alone triggered expression of Pdx1 and Neurod1, if only at the mRNA level. It is possible that the use of B10-insulin may have been another factor. This insulin is superior to normal insulin in biological activity [29] and has some mitogenic properties [30–32]. However, there was no evidence of excessive mitogenic activity or tumour development, while liver function tests remained normal throughout, with no lymphocytic infiltrates. Parallels can be drawn to the partial pancreatic transdifferentiation observed in Huh7ins cells, which endogenously produce NEUROD1 [23] and PDX1 (C. Tao, University of Technology Sydney; unpublished results). This cell line was transfected with normal human insulin under the control of a constitutive promoter. After this, secretory granules developed, which stored di-arginyl insulin and secreted it in a glucose-responsive manner, correcting diabetes. However, in Huh7ins cells the expression of fully processed native insulin was absent. Similarly, in the present study, pancreatic transdifferentiation was incomplete, as genes encoding transcription factors such as NKX6-1 and PAX6, which occur late in the hierarchy; were not expressed, nor were the rodent insulin genes or the genes for PC2 pancreatic polypeptide or exocrine markers. Therefore, the problems encountered with non-selective pancreatic transdifferentiation, produced after expression of PDX1 alone [5–7, 33, 34], were not encountered.
How the hepatic delivery of INS-FUR induced the expression of Pdx1 and other beta cell transcription factors requires further investigation. Liver and pancreas derive from common progenitor cells. Transdifferentiation between liver and pancreas is controlled by only a few transcription factors [35, 36]. Pdx1 is one such master switch gene. In a recent study, liver cells producing pancreatic markers (insulin and PDX1) were induced and localised to areas adjacent to or within injured tissue areas [37]. Lentiviral transduction may represent a cellular insult, making progenitor cells permissive to a pancreatic developmental shift.
The mechanisms that prevented both complete pancreatic transdifferentiation and expression of endogenous rat insulin are yet to be elucidated. Studies on the development of the rat pancreas indicate that chronologically glucagon and somatostatin appear prior to insulin [38]. The production of these pancreatic hormones may have prevented hypoglycaemia by providing counter-regulatory mechanisms. Complete transdifferentiation into true beta cells may require additional external factors, such as prolonged hyperglycaemia [39, 40]. If this is true, then the rapid reversion to euglycaemia following delivery of the insulin gene in our study may have prohibited complete liver-to-pancreas transdifferentiation. Additionally, the expression of human insulin may have exerted a negative feedback mechanism to inhibit endogenous insulin expression.
Unlike other studies that have engineered beta cell neogenesis from liver cells [5–7], the insulin-containing cells were not isolated to the area surrounding the portal circulation. Insulin storage was 40% of the insulin content of a rat pancreas, satisfying clinical requirements for insulin independence [41]. Corroborating previous studies using INS-FUR, granules predominately stored fully processed insulin [18, 42, 43]. EGFP and insulin were produced at equivalent levels in liver tissue transduced with HMD/INS-FUR. The majority of cells that expressed EGFP and insulin were also CK18-positive, indicative of a hepatocyte lineage for transduced cells.
Despite the lack of the central polypurine tract, LV pHR’CMVGFP efficiently transduced non-dividing hepatocytes in vitro, in the absence of cell division. We obtained an improvement in transduction efficiency of up to 87% in vitro, compared with 68% obtained by Zahler et al. [44]. Increased transduction efficiency may be attributable to several factors, including: (1) the isolation of hepatocytes by perfusion of both the PV and abdominal aorta as opposed to the PV alone, the most widely employed procedure [45]; (2) the optimisation of growth medium; (3) the use of LV at high multiplicity of infection (50:1); and/or (4) the use of the R8.2 packaging plasmid, which encodes accessory HIV proteins. The use of the attenuated plasmid R8.91, used by Zahler et al., resulted in a 5% reduction in transduction efficiency (B. Ren, unpublished observations). Similarly, delivery of INS-FUR in the HMD hybrid vector, which lacks the central polypurine tract [19], to the portal circulation of STZ-diabetic rats by FFO resulted in up to 60% transduction of liver cells, without inducing cell division and with transgene expression being confined to the liver. The FFO technique, which employs repeated vector infusion, is probably responsible for the increased transduction efficiency and is more clinically applicable than the widely used perfusion method, where the IVC is cannulated, thereby increasing the risk of IVC narrowing and pelvic limb thrombosis [46, 47].
This novel approach offers potential as a beta cell replacement strategy with diabetic patients serving as their own donors. Current studies in our laboratory using nonobese diabetic mice, indicate that this procedure was able to reverse autoimmune diabetes for 5 months with no evidence of autoimmune destruction of insulin-secreting hepatocytes (B. Ren, unpublished observations), giving hope that this procedure will be useful for reversal of autoimmune diabetes. There are distinct advantages to the use of LV for transduction of hepatocytes. LV stably integrates into the genome of non-dividing cells, including hepatocytes, and could provide long-lasting expression of therapeutic genes [48, 49]. Whilst LV integration was not specifically examined in this study, the longevity of INS-FUR and EGFP expression provides some evidence for this. Further, human hepatocytes may be more susceptible to LV transduction [50] than rodent cells. LV are probably superior to adenoviral vectors where problems of short-term expression of genes and immunological responses have been reported [9]. Adeno-associated vectors are extremely efficient at transducing mouse livers; however, a recent study using adeno-associated viral 8-mediated delivery of Pdx1 did not result in correction of STZ-diabetes in mice [28]. The unique results reported in this study point to possible benefits of gene therapy using LV.

gene therapy
type 1 diabetes
lentiviral vector
hepatocytes
beta cell transcription factors
insulin storage
wistar rats


Comparison of two arthroscopic pump systems based on image quality

The effectiveness of arthroscopic pump systems has been investigated with either subjective measures or measures that were unrelated to the image quality. The goal of this study is to determine the performance of an automated pump in comparison to a gravity pump based on objective assessment of the quality of the arthroscopic view. Ten arthroscopic operations performed with a gravity pump and ten performed with an automated pump (FMS Duo system) were matched on duration of the surgery and shaver usage, type of operation, and surgical experience. Quality of the view was defined by means of the presence or absence of previously described definitions of disturbances (bleeding, turbidity, air bubbles, and loose fibrous tissue). The percentage of disturbances for all operations was assessed with a time-disturbance analysis of the recorded operations. The Mann–Whitney U test shows a significant difference in favor of the automated pump for the presence of turbidity only (Exact Sig. [2*(1-tailed Sig.)] = 0.015). Otherwise, no differences were determined (Exact Sig. [2*(1-tailed Sig.)] > 0.436). A new objective method is successfully applied to assess efficiency of pump systems based on the quality of the arthroscopic view. Important disturbances (bleeding, air bubbles, and loose fibrous tissue) are not reduced by an automated pump used in combination with a tourniquet. The most frequent disturbance turbidity is reduced by around 50%. It is questionable if this result justifies the use of an automated pump for straightforward arthroscopic knee surgeries using a tourniquet.
Introduction
During arthroscopic surgery, the joint is continuously irrigated with saline fluid which is pressurized to create joint distension. The saline fluid flow removes disturbances in the arthroscopic view such as bleeding, air bubbles or synovial fluid. This improves the visibility of the arthroscopic view, which is an essential condition to perform an operation safe and fast. In this light, the quality of arthroscopic view has been defined as good or optimal if no disturbances are present [11]. In clinical practice, disturbances cannot always be prevented and maintaining a clear view is sometimes difficult [7, 10]. In a recent study performed by the authors, post-procedure analysis of 20 routinely performed arthroscopic knee procedures showed that 6% (SD 4%) of the total operation time was solely dedicated to obtaining a clear view. Since the number of arthroscopic procedures is large and still growing (60,000 per year in the Netherlands [8], and 1.7 million meniscectomies alone per year worldwide [3]), reducing the operating time by optimizing the view would be desirable.
The quality of the arthroscopic view is dependent on a number of factors such as the type of pump system, instruments, and condition of the joint [11]. In this study, the focus is on the type of pump system. The classical gravity pump and (automated) volumetric pumps are available for irrigation [10]. The main differences between these pumps are that the gravity pump causes a pressure due to a height difference caused by an elevated fluid bag and is manually controlled, whereas the volumetric pumps cause a fluid flow and have some type of automated pressure control. Since the automated pumps are regulated, in theory they should perform better in irrigation in the sense that the percentage of disturbed view over time should be less. In literature, the effectiveness of automated pump systems has been investigated [2, 4–6]. However, only subjective measures were used, such as visual clarity on a three-point scale or measures that were unrelated to the image quality, such as the number of fluid bags. The goal of this study is to determine the performance of an automated pump in comparison to the gravity pump based on objective assessment of the quality of the arthroscopic view. Therefore, a quantitative time-disturbance analysis was performed with uniquely described definitions for the different types of disturbances [11].
Methods
Only routinely performed procedures were included such as meniscectomy, cyst removal, debridement of a partial rupture of the anterior cruciate ligament, and diagnostic arthroscopy. Cruciate ligament reconstructions were not included. The patients were not notified of the recordings, because the operation was not affected and the patients’ identities could not be traced from the videos. No power analysis could be performed for this study, because no information was available on the variation of the disturbances. In accordance with other studies, it was decided to use ten operations for each of the two pump systems (20 in total) [2, 4]. The matching criteria were the number of operations, the duration of the operations, the duration of shaver usage, the type of operation, and the working experience of the surgeons. Matching of the shaver duration was performed, because some disturbances were expected to occur more frequently when using the shaver. Besides, a shaver is often used to clarify the view by temporarily increasing the flow. All surgeons who performed the operations had the same education and comparable working experience.
Ten arthroscopic knee procedures for which the gravity pump was used were recorded at 6 days within a time frame of 2 months. From the operations that met the inclusion criteria, ten were selected with randomization tables. Ten arthroscopic knee procedures performed with an automated pump were matched postoperatively to eliminate factors other than the irrigation pump systems that influence the arthroscopic view (Table 1).
Table 1Matching of the two pump groups was performed based on the number of operations, the total duration of all operations, and the share of shaver usageOperationTime (min:s:frames)Shaver time (s)Gravity pump Cyst removal17:55:11114 Meniscectomy26:03:15363 Meniscectomy33:15:09288 Meniscectomy19:56:10416 Meniscectomy13:37:18313 Meniscectomy17:55:13508 Meniscectomy12:10:15153 Meniscectomy15:09:02150 Debridement partial rupture of anterior cruciate ligament16:30:03290 Diagnostic7:12:03 Total duration 179:45:2443:15:0 Shaver time as percentage of total duration (%)24.1Automated pump Meniscectomy30:37:11410 Meniscectomy13:49:15120 Meniscectomy16:07:12122 Meniscectomy22:10:01404 Meniscectomy15:08:16237 Meniscectomy11:05:19290 Meniscectomy27:35:00325 Meniscectomy14:16:13149 Debridement partial rupture of anterior cruciate ligament17:41:10284 Diagnostic7:08:21 Total duration 175:40:1839:01:0 Shaver time as percentage of total duration (%)22.2
The recordings of operations with a gravity pump were performed in the day care centre of Academic Medical Center (Amsterdam, The Netherlands). The recordings of operations with an automated pump were performed in the day care centre of the Sint Maartenskliniek (Nijmegen, The Netherlands). The latter hospital utilizes the automated FMS Duo pump (FMSGroup, Nice, France). Table 2 shows the details on the standard operation setting for both hospitals using the different pumps. Most disturbances are more or less independent of the quality of the camera, the light source, and the arthroscope. It was assumed that these devices were functioning properly at the time of recording, because all operations were executed in acknowledged hospitals, in which the technical service department routinely inspects the equipment. A tourniquet was routinely applied at the start of the arthroscopic knee operations with a vacuum level of 300 mmHg. Drills, vaporizing devices, microfracturing, arthroscopic drills or meniscal repair devices were not used.
Table 2Standard operation setting for the gravity and the automated pumpGravity pumpAutomated pumpPortalsAnteromedial and anterolateralAnteromedial, anterolateral, and superomedialPump settingsPreset pressure of 49 mmHg (elevated saline fluid bag at 0.66 m)Suction pressure of −500 mmHgIndicated setting for knee arthroscopy is 50, which implies a preset pressure of 113 mmHg and a continuous flow of 90 ml/minInstrument setØ 4 mm arthroscope (Karl Storz, Gemany)Probe and punches (Dyonics, UK)Ø 4.5 or 5.5 mm full radius shaver (Dyonics, UK)Ø 4 mm arthroscope (Karl Storz, Gemany)Probe and punches (Dyonics, UK)Ø 4.5 or 5.5 mm full radius shaver (Dyonics, UK)
The comparison of the pumps was conducted by assessing the quality of the arthroscopic view per operation. Extending the definition of arthroscopic image quality by Tuijthof et al. [11] to the setting of irrigation performance of the pump systems, it is proposed that the pump for which the least percentage of disturbances occurs is the most effective. Seven disturbances of the arthroscopic view have been uniquely described and defined [11]. Not all of them can be prevented or removed by irrigation. Therefore, a subset of these disturbances was chosen for comparison of the pump systems: bleeding, turbidity (caused by synovial fluid and debris, Fig. 1), air bubbles, and loose fibrous tissue. The same recording equipment and quantitative time-disturbance analysis was performed as described in Tuijthof et al. [11]. Therefore, the recordings of the operations were analyzed frame by frame to assess the presence of the four disturbances in each frame. In addition to the total operation time, each operation was divided into phases to assess dominant prevalence of disturbances in a particular phase: (1) creation of portals, (2) joint inspection with or without a probe, (3) cutting, and (4) shaving. Phase 1 was the time required to achieve access to the joint by means of the arthroscope; thus the period from the first frame of the recorded digital video until the frame where a clear sharp arthroscopic view is presented for the first time. Added with this period is the time required to create the second and third portal: the period from the frame where the tip of a needle is seen for the first time until the frame where the tip of an instrument occurs. Phases 3 and 4 were defined as the frames in which the specific instrument was present in the arthroscopic view. The remainder of the operation time was indicated as Phase 2 inspection.
Fig. 1Three images of the disturbance turbidity, which consists of synovial fluid and small particles
The 20 digital videos were analyzed by two testers who each assessed an equal number of operations of each group. Their tester agreement was determined prior to the time-disturbance analyzes by means of the adjusted kappa [1, 9]. For each of the four disturbances, the adjusted kappa was larger than 0.80 indicating good agreement. Statistical analysis was performed with SPSS 12.0.2 (SPSS Inc., Chicago, IL, USA). The presence of a normal distribution for both datasets was determined with the Kolmogorov–Smirnov and Shapiro–Wilk tests. With the help of a one-way analysis of variance test (ANOVA) significant statistical differences were assessed in case the data were normally distributed. Otherwise, the nonparametric Mann–Whitney U test (Wilcoxon rank sum test) was applied.
Results
Almost 3 h of digital video material has been analyzed for both groups (Table 1). The mean operation time is around 18½ min for the gravity pump, and 17½ min for the automated pump. In each pump group, the total time of shaver usage is around 23% (Table 1). The Kolmogorov–Smirnov and Shapiro–Wilk tests show that the datasets are not distributed normally. As a consequence, the results of the percentages of each disturbance of the total operation time are presented with the median, minimum and maximum (Fig. 2). The disturbances show a substantial variation per operation. Turbidity was the dominant disturbance in both pump groups (median of 18% of the total operation time for the gravity pump versus 8% for the automated pump). The Mann–Whitney U test shows a significant difference for the presence of Turbidity (Exact Sig. [2*(1-tailed Sig.)] = 0.015) between both pump groups in favor of the automated pump. No significant differences are found for the other three disturbances (Exact Sig. [2*(1-tailed Sig.)] > 0.436) (Fig. 2).
Fig. 2Results of the share of disturbances in the arthroscopic view as percentage of the total operation time for each operation and each of the two groups. The results are presented with the median, minimum, and maximum numbers, because the datasets were not distributed normally. *The Mann–Whitney U test shows a significant difference for Turbidity (Exact Sig. [2*(1-tailed Sig.)] = 0.015)
Not all of the four operation phases were present in the 20 operations. Phases 3 and 4 were absent in both diagnostic arthroscopies. Additionally, punches were not used in five operations in the gravity pump group, and three in the automated pump group. As was expected the creation of portals was the shortest phase (10% for the gravity pump versus 5% for the automated pump), and inspection was the longest phase (59% for the gravity pump versus 51% for the automated pump). Punches were used more frequently in the automated group (7% for the gravity pump versus 21% for the automated pump). Analyzing each of the four operation phases separately shows a significant difference in favor of the automated pump for Turbidity in Phase 2 (Exact Sig. [2*(1-tailed Sig.)] = 0.019). For all other disturbances and operation phases no significant differences are determined (Exact Sig. [2*(1-tailed Sig.)] > 0.165). The Mann–Whitney U test within each of the pump groups shows a significant difference for loose fibrous tissue (Fig. 3). In detail, the gravity pump shows the following results: Phase 1 versus 3 (Exact Sig. [2*(1-tailed Sig.)] = 0.002), Phase 1 versus 4 (Exact Sig. [2*(1-tailed Sig.)] = 0.010), and Phase 2 versus 3 (Exact Sig. [2*(1-tailed Sig.)] = 0.002). The automated pump shows these results: Phase 1 versus 2 (Exact Sig. [2*(1-tailed Sig.)] = 0.017), Phase 1 versus 3 (Exact Sig. [2*(1-tailed Sig.)] = 0.001), and Phase 2 versus 3 (Exact Sig. [2*(1-tailed Sig.)] = 0.022).
Fig. 3Results of the share of disturbances in the arthroscopic view as percentage of the phase time for each operation in the gravity pump group (a), and the automated pump group (b). The results are presented with the median, minimum, and maximum numbers, because the datasets were not distributed normally. *The Mann–Whitney U test shows only a significant difference for loose fibrous tissue in both pump groups
Discussion
The objective of this study was to compare two types of irrigation pump systems (the classic gravity pump and a widely used automated pump) based on their effectiveness in maintaining a clear arthroscopic view. Comparison of the effectiveness of pump systems has been performed [2, 4–6]. However, none of these studies measured the arthroscopic image quality objectively in detail. Besides the objective comparison, the time-disturbance analysis gave quantitative values for the frequency of four disturbances.
Turbidity was the dominant disturbance in both pump groups being present in a considerable percentage of the operation time (Fig. 2). The results show that for routinely performed arthroscopic knee operations, the automated pump was significantly more effective in reducing the presence of turbidity with 50% on average. This finding can be explained by the continuous flow of 90 ml/min that is activated by the automated pump as opposed to the lower flow cause by leakage along the portals when using the gravity pump and a two-portal technique. No difference was determined for the other three disturbances. The low percentage of bleeding can be attributed to the use of a tourniquet in all operations. In order to determine the effectiveness of pump systems for this disturbance, analysis of shoulder surgeries would be more convenient.
The found variation in the duration of disturbances per operation could have been caused by the condition of the knee joints, because this factor could not be completely eliminated with precise matching. With this knowledge, a future suggestion is to analyze a larger number of patients per pump group to determine possibly other significant differences in pump performance.
Comparison of the operation phases independently showed similar results for the inspection phase compared to the total operation time, probably because this was the longest phase. Since the creation of portals was a relatively short phase, and the use of punches was absent for almost half of the operations, no significant differences were found. However, analysis of both groups internally showed that the presence of loose fibrous tissue was generally significantly higher in when an instrument was used (Phases 3 and 4). This is in accordance with the general expectation and confirms the applicability of this method for quantification of disturbances in the view.
Concluding, this study shows that important disturbances as bleeding, air bubbles, and loose fibrous tissue are not affected by a pump system when performing a routine knee arthroscopy with a tourniquet. The most frequent disturbance turbidity is reduced by around 50% when using an automated pump. It is questionable if this justifies the use of an automated pump for straightforward arthroscopic knee surgeries using a tourniquet, since the purchase of an automated pump is more costly.

pump
image quality
irrigation
arthroscopy
knee joint
video analysis


Validity, reliability, and responsiveness of the Kansas City Cardiomyopathy Questionnaire in anemic heart failure patients

Background While generic health status measures quantify the impact of all patients’ diseases on their health-related quality of life, disease specific measures focus on only one of the many conditions that a patient may have. If a patient has two diseases with similar clinical manifestations, they may respond differently to a disease-specific instrument if one of their conditions improves while the other worsens or remains stable, thus undermining the instruments in that patient population. We sought empirical evidence of the reliability and validity (including responsiveness) of the Kansas City Cardiomyopathy Questionnaire (KCCQ), a disease-specific measure for heart failure (HF), among HF patients with and without anemia, a condition that has similar symptoms to HF.
Introduction
The quantification of patients’ health status (their symptoms, function, and health-related quality of life) is becoming increasingly important as an endpoint in clinical trials [1, 2], as a means of monitoring patients over time [3], and even as a marker of health care quality [4–6]. Measuring patients’ health status is most commonly accomplished by direct solicitation from patients themselves, using either generic or disease-specific health status questionnaires [7]. Whereas generic health status measures seek to quantify the health-related quality of life limitations of patients’ ‘overall health’, including all combinations of diseases and ailments, disease-specific measures focus upon a specific disease process. Accordingly, an advantage of disease-specific questionnaires is that they can be both more sensitive to clinical change and more interpretable than general health status questionnaires. Because disease-specific instruments tend to be more actionable to clinicians, they can be useful in monitoring patients’ clinical status over time [8]. An unresolved challenge in the use of disease-specific measures, however, is whether they perform well when patients have other conditions that may also impact their symptoms, function, and quality of life in ways that are similar to the specific disease of interest. While this is not a problem with generic health status measures, if a co-occurring disease manifests similar effects to a primary disease of interest, and if one of the conditions worsens while the other remains stable or improves, then it is unknown whether the disease-specific measure will accurately measure the status of the disease it is designed to quantify. This can undermine the original validity, reliability and responsiveness of the disease-specific measure. One potential setting in which this type of confounding may occur is in the setting of heart failure and anemia.
Heart failure (HF) affects over 5 million Americans and is responsible for enormous health care costs and resource utilization [9]. While several disease-specific measures for patients with HF have been created to quantify HF patients’ health status [10–13], the psychometric properties of these instruments have all been demonstrated in patients whose predominant medical condition was heart failure. Recently, however, anemia has emerged as an important co-occurring condition that is prognostically important in HF [14–21]. Importantly, the symptoms of anemia may mimic those of HF. Specifically, chronic anemia manifests itself in patients as “fatigue, loss of stamina, breathlessness, and tachycardia, particularly with exertion” [22], coupled with the development of “gradual fatigue and a reduced exercise tolerance” [23]. The symptoms of anemia [24] are therefore remarkably similar to those of HF, for which breathlessness, reduced exercise tolerance, and fatigue are hallmarks of the disease [22, 23]. Because of the overlap in symptomatic manifestations of both diseases, it is not clear whether heart failure-specific health status assessments, such as the Kansas City Cardiomyopathy Questionnaire (KCCQ) which captures patients’ perspectives of their disease rather than physiologic or anatomic characteristics (e.g. their hematorit or left ventricular function), would perform similarly in patients with and without anemia. For example, if a patient’s heart failure symptoms improve from an increased use of diuretics, would there be an improvement in their KCCQ scores if their anemia did not change? Consequently, it is unknown whether the psychometric properties of a disease-specific HF instrument would be similar in anemic and nonanemic HF patients, given the potential for both conditions to induce similar symptoms in patients. The purpose of this analysis was to provide empirical evidence supporting the reliability and validity (including responsiveness) of the KCCQ in anemic HF patients. We sought to compare the psychometric properties, including reliability (internal consistency and test–retest) and validity (including responsiveness to clinical change), in anemic and nonanemic HF patients.
Methods
Patient population
The STudy of AneMia IN A Heart Failure Population (STAMINA-HFP) was a prospective registry of 1,090 HF patients who were initially enrolled in the outpatient clinics of 58 U.S. cardiology centers. The purpose of STAMINA-HFP was to estimate the incidence and prevalence of anemia in patients with HF and to describe the association of anemia with HF progression. As such, the protocol mandated an initial clinical history and physical exam (including assignment of the New York Heart Association [NYHA] classification), a measurement of patients’ hemoglobin, and an assessment of their health status, including the KCCQ. Enrolled patients were contacted by telephone within 1–2 days after their initial enrollment and every 3 months for a period of 1 year to assess their health status. In addition, data from outpatient visits during the year of observation were abstracted at each visit. To be eligible for participation, patients had to be >18 years of age, diagnosed with a history of symptomatic HF and willing to provide informed consent. Patients who had a prior organ transplant, were planning a major surgery, or were participating in another interventional trial were excluded. Data capture was completed in the STAMINA-HFP registry in July 2004. Patients participating in this study (n = 811) had to have complete baseline health status assessments and a measure of hemoglobin. They were slightly more likely to be white (75% vs. 68%, P = 0.03) and to not smoke (90% vs. 86%, P = 0.03) but had similar distributions of NYHA class, KCCQ scores, and hemoglobin as compared with the entire cohort of 1,090 patients. For the longitudinal assessments of test–retest reliability and responsiveness, a 3-month health status assessment was also required from these patients (n = 698). At the time of follow-up interviews, a patient global assessment of change question, asking patients to describe ‘how [they] think [their] condition has changed compared to the start of the study,’ was also asked at each follow-up interview. Responses were on a 7-point categorical response scale ranging from ‘markedly improved’ to ‘markedly worse.’ The initial 3-month assessment was used for this study because it was the closest assessment to the time of their anemia evaluation and because it had the shortest recall period with which to assess patients’ perspectives of clinical change of any of the other assessments.
The KCCQ is a 23-item, disease-specific measure that quantifies four clinically relevant domains of patients’ health status including physical limitations, symptoms (frequency, severity, and change over time), a heart failure-specific assessment of their quality of life and their perceived social limitations due to heart failure, and a self-efficacy domain (a measure of patients’ knowledge of how to best manage their disease). The four health status scales can be combined into a single, overall summary score [10]. Scores range from 0–100, where higher scores indicate better functioning, fewer symptoms, and better disease-specific quality of life. The KCCQs validity and reproducibility have been previously supported and recent studies have demonstrated that both cross-sectional variations [25, 26] and changes [27] in KCCQ scores are prognostic of subsequent mortality and HF hospitalizations. A mean difference between groups of patients and an intra-individual change over time of ≥5 points is considered clinically significant. This was established in two studies: a prospective study of 476 outpatients in which a mean change of five points in the KCCQ overall summary scale was observed in those patients who experienced a small, but clinically significant change in their heart failure [28] and in a prospective study of 1,358 HF patients in which a 5-point change was associated with an 11% change in the multivariable-adjusted hazard ratio of hospitalization and cardiovascular death [27].
Classification of anemia
Patients were classified as being anemic or not based upon their hemoglobin at the time of study enrollment. Thus, the patients’ hemoglobin value at baseline determined their anemia status throughout the remainder of the study. The World Health Organization (WHO) definition of anemia was used (hemoglobin <13 g/dl for men, <12 g/dl for women).
Statistical analysis
The patient population was divided into those with and without anemia, and baseline demographic and clinical characteristics were compared with chi-square tests for categorical variables and t tests for continuous variables.
To evaluate the construct validity of the KCCQ in anemic and nonanemic patients, comparisons across NYHA classifications from the baseline interviews were conducted. A two-way ANOVA with KCCQ overall summary score as the dependent variable and baseline anemia classification, NYHA, and an anemia-by-NYHA interaction term as independent variables was constructed to establish whether the association of NYHA and KCCQ was different in patients with and without anemia.
Both internal consistency and test–retest reliability estimates were compared among anemic and nonanemic patients. Internal consistency reliability was estimated using Cronbach’s alpha. This value was calculated independently for patients with and without anemia. Test–retest reliability was performed using a t test, by comparing the 3-month change in KCCQ scores for patients who responded on the global change in health question that their condition was stable over the previous 3 months. Three months was selected as the interval of analysis to maximize the number of patients included in the analyses, to leverage the existing data in which a 3-month interval was selected, and to replicate the time frame of the original validation studies evaluating the KCCQ [10].
To compare the responsiveness of the KCCQ in patients with and without anemia, the association of patients’ means change in KCCQ scores with their responses to the global health question were assessed using linear regression. In these linear regression analyses, the mean change in KCCQ scores was used as the dependent variable and baseline anemia status, global assessment of change, and an anemia-by-global assessment of change interaction term were independent variables. As a secondary analysis, patients’ perspectives of their 3-month change in status was categorized into improved (mildly–markedly), stable (no change), and worsened (mildly–markedly), and mean change in KCCQ scores were compared for those with and without anemia by these patient-perceived categories of change in health status. All analyses were conducted in SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). All statistical tests were two-sided and a P value <0.05 was used as the threshold to declare statistical significance.
Results
Patient characteristics
Overall, 811 patients completed the baseline clinical assessments, including the KCCQ interview, and were used to assess baseline construct validity and internal consistency reliability. Only 4% of the population had KCCQ scores of 100, suggesting a minimal ceiling effect of the instrument in this population. Among the patients who completed the baseline assessments, 268 (33%) met the WHO criteria for anemia. Baseline characteristics of the anemic and nonanemic patients are provided in Table 1. Compared to nonanemic patients, anemic patients were significantly more likely to be older than  (mean ± SD = 67 ± 13 vs. 63 ± 14 years, P < 0.001), non-Caucasian (30% vs. 23%, P = 0.03), and to have an ischemic HF etiology (46% vs. 38%, P = 0.04), diabetes (47% vs. 34%, P < 0.001), lower glomerular filtration rate (GFR) (50.8 ± 24.6 vs. 63.9 ± 23.1 ml/min/m2, P < 0.001), chronic renal insufficiency (33% vs. 15%, P < 0.001), higher serum creatinine (1.7 ± 1.5 vs. 1.3 ± 1.2 mg/dl, P < 0.001), worse NYHA class (44% vs. 32% class III or IV, P < 0.001), lower KCCQ scores (61 ± 23 vs. 65 ± 23, P = 0.009), and to be treated with diuretics (90% vs. 84%, P = 0.04).
Table 1Baseline clinical characteristics of the populationCharacteristicAnemic (n = 268)Nonanemic (n = 543)P valueAge (mean +/− SD)67.0 +/− 13.162.9 +/− 13.5<0.001Male (n, %)105 (39.2%)239 (44.0%)0.190Non-Caucasian (n, %)80 (29.9%)123 (22.7%)0.026No insurance (n, %)6 (2.2%)19 (3.5%)0.329Etiology of HF (n, %)0.038    Hypertensive 49 (18.3%)92 (17.0%)    Ischemic124 (46.3%)208 (38.4%)    Other95 (35.4%)242 (44.6%)Mean left ventricular ejection fraction (mean +/− SD)36.4 +/− 15.935.9 +/− 16.20.742Diabetes (n, %)125 (46.8%)185 (34.1%)<0.001COPD (n, %)51 (19.1%)87 (16.0%)0.273Chronic renal insufficiency (n, %)89 (33.3%)82 (15.1%)<0.001Current smoker (n, %)17 (6.3%)61 (11.2%)0.026Body mass index (mean +/− SD)30.3 +/− 7.331.1 +/− 7.40.181NYHA (n, %)0.001     I35 (13.1%)99 (18.2%)    II116 (43.3%)268 (49.4%)    III105 (39.2%)169 (31.1%)    IV12 (4.5%)7 (1.3%)Mean KCCQ overall summary score (mean +/− SD)60.8 +/− 22.865.3 +/− 23.20.009Current medications (n, %)    ACE inhibitor/ARB232 (86.6%)486 (89.5%)0.217    ß-blocker222 (82.8%)446 (82.1%)0.806    Diuretic240 (89.6%)458 (84.3%)0.044    Spironolactone76 (28.4%)132 (24.3%)0.214    Digoxin126 (47.0%)245 (45.1%)0.610Mean hemoglobin (mean +/− SD)11.4 +/− 1.114.1 +/− 1.3<0.001Mean creatinine (mean +/− SD)1.7 +/− 1.51.3 +/− 1.2<0.001Glomerular filtration rate (ml/min/m2, mean +/− SD)*50.8 +/− 24.663.9 +/− 23.1<0.001* Modification of diet in renal disease (MDRD) abbreviated formula
For the longitudinal analyses of test–retest reliability and responsiveness, 698 (86.1%) patients provided both baseline and 3-month assessments. No statistically significant differences in any of the patient characteristics listed in Table 1 were observed between those who did and did not participate in the 3-month interviews. Notably, no difference in anemia classification (33.0% among participants vs. 33.6% in those without follow-up, P = 0.89) or mean hemoglobin (13.2 ± 1.8 vs. 13.2 ± 2.1, P = 0.94) was observed between those with and without follow-up.
Construct validity
Figure 1 illustrates the relationship between the overall KCCQ score by NYHA class in anemic and nonanemic patients. Mean KCCQ scores tracked strongly with NYHA class (mean summary score ± SE for the entire population = 80.3 ± 1.6, 68.0 ± 1.0, 50.9 ± 1.2, and 45.6 ± 5.4 for NYHA classes I–IV respectively, P < 0.0001). While the mean KCCQ scores for the patients with NYHA class IV differed substantially, this was likely due to the small numbers of patients in these subgroups, resulting in a large standard deviation of scores (KCCQ scores in anemic [n = 12] and nonanemic [n = 7] patients = 39.5 ± 20 vs. 56.2 ± 28). Furthermore, the relationship between KCCQ and NYHA did not differ significantly between anemic and nonanemic patients (P = 0.38 for interaction).
Fig. 1KCCQ overall summary score by NYHA class in anemic and nonanemic patients
Reliability
The internal consistency reliability (Cronbach’s alpha) of the KCCQ overall summary score for the entire population was 0.93. In patients who were anemic and nonanemic, the Cronbach alphas were 0.92 and 0.93, respectively.
The mean (±SE) KCCQ overall summary score change in patients who reported no change over 3 months in their clinical condition (n = 257) was −1.2 ± 0.7. Among anemic patients (n = 73) the mean change was −2.8 ± 1.4, and for nonanemic patients (n = 184) the mean change was −0.5 ± 0.8. These mean 3-month changes were similar between anemic and nonanemic patients (P = 0.14). Importantly, the change between and within both groups was less than five points over time and, thus, not considered clinically significant. The intraclass correlation coefficient for the KCCQ overall summary score was 0.885 in the overall cohort and did not differ among those with and without anemia (0.865 for anemic patients and 0.892 for nonanemic patients [P = 0.11]).
Responsiveness
To compare the sensitivity of the KCCQ to patients’ perceptions of change, the mean change in KCCQ scores across each category of change were compared in anemic and nonanemic patients. Figure 2 describes the means and 95% confidence intervals of change in KCCQ scores across crude categories of improved (mild to marked), no change, and worsening (mild to marked) of their overall clinical status during the first 3 months of the study. No significant differences by anemia status were detected within any of the three categories of reported health change. In regression analysis, mean 3-month KCCQ change was found to be linearly related to reported health change, with a slope of −2.7 points (95% CI = [−3.6, −1.9]) per 1 step down on the Likert-scale response. Nonlinearity was tested using cubic splines and was found to be nonsignificant (P = 0.68). The association was consistent in both anemic and nonanemic patients (slope estimates = −3.4 [95% CI = (−5.0, −1.8)] vs. −2.5 [95% CI = (−3.4, −1.6)], P-value = 0.33).
Fig. 2Three-month KCCQ change by reported health change and anemia status
Discussion
We report empirical evidence to support the reliability and validity (including responsiveness) of the KCCQ in HF patients with anemia. We found similar associations between the KCCQ overall summary score and NYHA classification, similar internal consistency and test–retest reliability in stable patients, and similar responsiveness of the KCCQ to patients’ perceptions of 3-month clinical change. In light of the potential overlap in symptoms between anemia and HF, we tested and rejected the hypothesis that the psychometric properties of the KCCQ might differ between HF patients with and without anemia. Thus, the KCCQ could be a valid, reliable, and responsive outcome for clinical trials of anemia treatment in HF.
To date, there has been a paucity of literature examining attribution of symptoms to one of a spectrum of potentially co-occurring diseases in patient-reported health status assessments. A common recommendation for the design of clinical trials is to include both disease-specific and generic measures of health status. These recommendations are predicated upon the desire to capture the impact of treatments on a specific disease of interest, as well as the overall impact of treatment on patients’ health outside the condition of interest. The latter intended to capture side effects or other unanticipated complications of therapy.
However, since patients are often only aware of the symptoms that they experience, and not the underlying pathophysiology that is responsible for those symptoms, it is unclear whether a distinct pathophysiology (e.g. anemia) could confound the psychometric properties of a disease-specific health status measure quantifying a disease with similar clinical manifestations (e.g. HF). While a review of the literature and an independent investigation of Rijken et al. demonstrated that co-occurring diseases are associated, as expected, with worse scores on general health status measures, they did not examine the influence of co-occurring diseases on disease-specific instruments [29]. To examine the relative performance of generic and disease-specific instruments, Ren and colleagues demonstrated, in both cross-sectional [30] and longitudinal [31] analyses, that disease-specific measures outperformed generic measures in terms of their content and discriminative validity among veterans with multiple comorbidities. However, these latter studies did not focus upon the ability of a disease-specific measure to be a psychometrically valid representation of the disease of interest when another co-occurring illness may manifest similar symptoms.
One research situation in which one disease has similar symptoms to another is depression and heart failure. Numerous studies have documented that depressed patients have worse health status than nondepressed patients [32–35], yet both depression and heart failure can manifest symptoms of fatigue. Interestingly, Rumsfeld et al. were able to demonstrate that depression was associated with worse KCCQ scores over time, regardless of baseline scores, and that among nondepressed patients, poorer health status was associated with a greater likelihood of becoming depressed [36]. These studies did not address whether the KCCQ was equally valid in both depressed and nondepressed patients, but did show important interrelationships between diseases that can have similar clinical manifestations.
Several potential limitations of this article should be considered in interpreting our findings. First, validation is always difficult to establish given the absence of a criterion standard for a patient-centered outcome such as health status. We selected the NYHA classification because it is a commonly used metric with which to quantify HF patients’ health status. Yet the reproducibility and interobserver variability of NYHA assessments is known to be lower than directly soliciting such data from patients themselves. [37]. A second concern is that our assessments of change in HF came from a global patient-reported estimate of change. At best this is a crude assessment and much more detailed examinations of clinical change in the KCCQ have been conducted [28]. Although the quantitative assessments of change in KCCQ scores may be different than in previous studies, there is no a priori reason to suspect that these patient-reported assessments of global change would differ by patients’ anemia status. Therefore, finding similar associations between 3-month change in KCCQ scores and patients’ global assessments of change between those with and without anemia supports the fact that the responsiveness of the KCCQ to clinical change is similar in both populations of patients. In addition, concluding that no differences exist in the performance characteristics of the KCCQ in anemic and nonanemic patients could be incorrect due to a type II error. However, the point estimates for all of our analyses were extremely similar and included more than 800 patients, which is greater than six times as large as the original study documenting the reliability and validity (including responsiveness) of the KCCQ [10]. Finally, 12% of patients were lost to follow-up and we cannot exclude the introduction of a potential bias from failing to include these patients. Supporting the generalizability of our study, however, is the similar degree of association between KCCQ scores and NYHA classification, the similar estimates of internal consistency and test–retest reliability, and similar responsiveness observed in this population as seen in other studies testing the psychometric properties of the KCCQ [10, 28].
In summary, we provide empirical evidence to support the ability of a disease-specific measure of heart failure patients’ health status, the KCCQ, regardless of the presence of anemia. As such, we believe that patient-reported health status outcomes are a valid, reliable, and sensitive means to quantify the impact of anemia treatment on HF-specific outcomes. Given that the improvement in patients’ health status is a primary goal of treatment, being able to accurately capture HF patients’ health status in trials of anemia therapy are important. The KCCQ should be a valid and sensitive measure for accomplishing these goals.