Hospital treatment costs associated with incident complications in patients with type 2 diabetes-real-world study based on electronic patient information systems.

BACKGROUND: Type 2 diabetes (T2D) and its complications cause a significant public health and economic challenge. To enable the optimal resource allocation across different prevention and treatment policies for the management of T2D-related complications, detailed cost estimates related to the complications of T2D are needed. Therefore, the objective of the study was to provide reliable and sufficiently detailed real-world estimates of costs associated with different T2D complications in a Finnish university hospital setting. METHODS: A cohort of T2D patients living in the catchment area of a university hospital during 2012 and 2016 was identified from the comprehensive national FinDM diabetes database for longitudinal assessment of T2D associated complication treatment costs. Data on patient-level events were extracted from the FinDM data and complemented with all accountable services and related detailed costing data gathered from the university hospital's electronic patient information systems by using unique personal identity codes. Patients were screened for their first diagnoses of complications using the same national quality registry definitions as in the FinDM database. Multivariable gamma regression model with a log link function was applied to study the association between baseline factors and complication costs. In addition, an interactive online tool was developed to create predicted costs for complication costs with selected baseline factors. RESULTS: A total of 27 255 prevalent and incident patients with T2D were identified from the national FinDM register. Finally, a total of 16 148 complication episodes for 7 895 patients were included in the cost analyses. The mean estimated one-year hospital treatment costs of T2D-related complication varied from 6 184 to 24 507 euros per complication. Regression analyses showed that coexisting conditions are significantly associated with initial and recurrent complication costs. CONCLUSIONS: The study shows updated Finnish cost estimates and their main cost drivers for T2D-related complications treated in the university hospital setting. The results of our study highlight the significance of guideline implementation, effective preventive treatments for T2D, as well as the importance of treatment adherence to avoid these costly complications.

Heat shock protein 90 is downregulated in calcific aortic valve disease.

BACKGROUND: Calcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD. METHODS: We compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD. RESULTS: We detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot. CONCLUSIONS: We describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90.

Increased mesenchymal podoplanin expression is associated with calcification in aortic valves.

BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve disease (CAVD) is a progressive disease starting from mild valvular sclerosis and progressing to severe aortic stenosis (AS) with calcified valves. The origin of the calcification is proposed to be mesenchymal cells which have differentiated towards an osteoblastic phenotype. Podoplanin is a glycoprotein expressed in the endothelium of lymphatic vessels and in osteoblasts and osteocytes, mesenchymal cells, as well as in many carcinomas and aortic atherosclerotic lesions. In CAVD, its expression has been evaluated only as a marker of the lymphatic vasculature. MATERIALS AND METHODS: We determined podoplanin expression in human aortic valves in four patient groups: control (C, n=7), aortic regurgitation (AR, n=8), aortic regurgitation and fibrosis (AR + f, n=15) and AS (n=49) by immunohistochemistry and quantitative real-time PCR (RT-PCR). RESULTS: Immunohistochemically, podoplanin expression was significantly increased in AR + f and AS groups when compared with the control and AR groups and the level of expression positively correlated with the extent of calcification and vascularity. Podoplanin mRNA levels were 1.7-fold higher in the AS group as compared with the control group (P=.05). Podoplanin-positivity was present not only in lymphatic vessel endothelium but also in osteoblasts, osteocytes, chondrocytes, macrophages and extracellular matrix. The majority of the podoplanin-positivity was in spindle cells with a myofibroblastic phenotype, often associated with calcifications. Tricuspid valves had more calcification-associated podoplanin than bi/unicuspid valves (median 1.52 vs 1.16, P<.001). CONCLUSIONS: CAVD is characterized by an increased expression of podoplanin; this is associated with the differentiation of valvular interstitial cells into calcium-producing, myofibroblast-like cells. In addition, tricuspid valves express relatively more podoplanin than bi/unicuspid valves.

Targeting vasoactive peptides for managing calcific aortic valve disease.

Calcific aortic valve disease (CAVD) represents a spectrum of disease spanning from milder degrees of calcification of valve leaflets, i.e., aortic sclerosis, to severe calcification i.e., aortic stenosis (AS) with hemodynamic instability. The prevalence of CAVD is increasing rapidly due to the aging of the population, being up to 2.8% among patients over 75 years of age. Even without significant aortic valve stenosis, aortic sclerosis is associated with a 50% increased risk of myocardial infarction and death from cardiovascular causes. To date, there is no pharmacological treatment available to reverse or hinder the progression of CAVD. So far, the cholesterol-lowering therapies (statins) and renin-angiotensin system (RAS) blocking drugs have been the major pharmacological agents investigated for treatment of CAVD. Especially angiotensin receptor blockers (ARB)s and angiotensin convertase enzyme inhibitors (ACEI)s, have been under active investigation in clinical trials, but have proven to be unsuccessful in slowing the progression of CAVD. Several studies have suggested that other vasoactive hormones, including endothelin and apelin systems are also associated with development of AS. In the present review, we discuss the role of vasoactive factors in the pathogenesis of CAVD as novel pharmacological targets for the treatment of aortic valve calcification. Key messages Vasoactive factors are involved in the progression of calcific aortic valve disease. Endothelin and renin-angiotensin systems seem to be most prominent targets for therapeutic interventions in the view of valvular pathogenesis. Circulating vasoactive factors may provide targets for diagnostic tools of calcified aortic valve disease.

MicroRNA-125b and chemokine CCL4 expression are associated with calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a progressive pathological condition with no effective pharmacological therapy. To identify novel molecular pathways as potential targets for pharmacotherapy, we studied microRNA (miRNA) profiles of heavily stenotic aortic valves (AS). One of the most upregulated miRNAs in AS valves compared to control valves was miR-125b (1.4-fold; P < 0.05). To identify CAVD-related changes in gene expression, DNA microarray analysis was performed, including an intermediate fibro(sclero)tic stage of the disease. This revealed changes especially in genes related to inflammation and immune response, including chemokine (C-C motif) ligand 3 (CCL3) and 4 (CCL4). CCL3 mRNA level was increased 3.9-fold (P < 0.05) when AS valves were compared to control valves, and a 2.5-fold increase (P < 0.05) in CCL4 gene expression was observed when fibro(sclero)tic valves were compared to control valves. Both CCL3 and CCL4 localized to macrophages by immunofluorescence. To identify chemokine-miRNA target pairs, data from miRNA target prediction databases were combined with valvular miRNA and mRNA expression profiles. MiR-125b was computationally predicted to target CCL4, as confirmed experimentally in cultured human THP-1 macrophages. Collectively, miR-125b and CCL4 appear to be involved in the progression of CAVD and may offer novel therapeutic and diagnostic strategies related to this disease.

Expression and Localization of Granzymes and Perforin in Human Calcific Aortic Valve Disease.

BACKGROUND AND AIM OF THE STUDY: Calcified aortic valve disease (CAVD) is an actively regulated disease that shares pathophysiological hallmarks with atherosclerosis. One of these common features is extracellular matrix (ECM) remodeling, which consists of a dynamic degradation and deposition of the ECM composition. Granzymes (Grs) are ECM- degrading and pro-apoptotic proteases that have been detected in atherosclerotic lesions, but their role in CAVD remains unknown. METHODS: The expression of granzymes and perforin was characterized in heavily stenotic valves (n = 20) and control valves (n = 6) using quantitative RT-PCR and immunohistochemistry. RESULTS: Quantitative RT-PCR revealed that levels of granzymes A, B, H, K and M mRNA were 4.9-fold (p < 0.001), 7.1-fold (p < 0.001), 4.6-fold (p < 0.001), 4.7-fold (p < 0.001) and 2.8-fold (p = 0.069) higher, respectively, in stenotic aortic valves than in control valves. Perforin mRNA levels were 3.6-fold (p < 0.001) higher in stenotic valves than in control valves. Granzyme A immunohistochemical positivity was observed in mast cells and lymphocytes, granzyme H in mast cells but not in lymphocytes, and granzyme K in lymphocytes but not in mast cells. A statistical analysis was also performed to investigate the effect of statin treatment on granzyme expression, but no differences were found when compared to non-statin-treated patients. CONCLUSIONS: The data acquired showed that CAVD is characterized by an increased expression of granzymes A, B, H, K, and perforin.

Tezosentan inhibits uptake of proinflammatory endothelin-1 in stenotic aortic valves.

BACKGROUND AND AIM OF THE STUDY: Aortic valve stenosis (AS) is an actively regulated pathobiological process which has an inflammation origin, and manifests as an accumulation of lipids and, ultimately, calcification of the aortic valve tissue. Increased plasma levels of the proinflammatory factor endothelin-1 (ET-1) have been reported in AS. Moreover, increased tissue levels of ET-1 and its ET(A) receptor, which mediates the fibrotic and proliferative effects of ET-1, have been reported in stenotic aortic valves. The study aim was to determine whether endothelin receptor antagonism has an effect on the supposed receptor-mediated uptake of ET-1 to aortic valves when ET-1 may be involved in the pathogenesis of AS. METHODS: By using valve tissue explants in culture, it was determined whether the ET(A)-ET(B) receptor antagonist tezosentan was capable of reducing the uptake of 125I-labeled ET-1 to human aortic valves. Aortic valves were obtained from 16 patients (11 males, five females; mean age 71 +/- 11.2 years) and from two donors without AS (as controls) at the time of aortic valve or aortic root surgery. Valve tissue samples were cultured in ET-1 (10 nmol/l), in the presence or absence of tezosentan (10 nmol/l). RESULTS: ET-1 uptake was found to be pronounced in the calcified areas of the valve, and tezosentan markedly reduced the receptor-mediated uptake of 125I-labeled ET-1. The inhibitory effect was most evident in the well-calcified part of the valve. The gene expression levels of the ET receptors ET(A) and ET(B) were unaltered in human aortic valves during a four-day exposure to the antagonist. CONCLUSION: The ability of the ET(A)-ET(B) receptor antagonist tezosentan to inhibit ET-1 uptake in valve tissue suggests that continuous ET antagonist therapy might serve as new strategy to slow down the pathophysiological processes of AS.

Increased thrombospondin-2 in human fibrosclerotic and stenotic aortic valves.

BACKGROUND: Active involvement of extracellular matrix (ECM) and its composition regulating factors may have a central role in the pathogenesis of calcific aortic valve disease (CAVD). Thrombospondins (TSPs) are highly conserved matricellular proteins regulating inflammation, angiogenesis and ECM remodeling. These processes are strongly associated with progression of aortic valve stenosis (AS). However, the expression of TSPs in CAVD is not known. METHODS: We characterized the expression of TSPs 1-4 in human aortic valves by real-time quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Control valves (n=8), thickened and stiffened fibro(sclero)tic valves (n=8), and calcified AS valves (n=24) were compared. Furthermore, potential factors regulating TSP-2 expression was studied by western blotting and gel mobility shift assay in another set of control (n=10) and AS (n=20) valves. RESULTS: TSP-2 mRNA levels were increased 4.9-fold (P=0.037) and 4.8-fold (P=0.001) in fibro(sclero)tic and stenotic valves, respectively, whereas the expression of other TSPs did not change significantly. All TSPs 1-4 were detected from aortic valves by immunohistochemistry. Positive TSP-2 immunostaining was seen in the valvular myofibroblasts and patchily in endothelial cells. Semiquantitative analysis of TSP-2 staining indicated increased immunoreactivity for TSP-2 in neo vessels of fibro(sclero)tic and calcified aortic valves. Finally, when compared to controls, AS was associated with significant down regulation of Akt-pathway and diminished binding activity of nuclear factor-κB (NF-κB). CONCLUSIONS: We report for the first time that TSPs 1-4 are expressed in human aortic valves. CAVD is characterized by myofibroblastic proliferation and neovascularization associated upregulation of TSP-2 expression, as well as inactivation of Akt and NF-κB.

Statin treatment and gene expression of anti-atherogenic factor C-type natriuretic peptide system in stenotic aortic valves.

AIMS: Aortic valve calcification is an actively regulated process with endothelial dysfunction displaying hallmarks of atherosclerosis. C-type natriuretic peptide (CNP) system has been reported to have a role in the pathogenesis of vascular atherosclerosis and to be distinctly downregulated in aortic valve stenosis (AS). Here we studied gene expressions of CNP and is target receptor natriuretic peptide receptor type B (NPR-B) in human aortic valves. Furthermore, we compared gene expression of CNP system in patients with HMG-coenzyme-A reductase (statin) treatment to non-statin-treated patients in AS group. METHODS AND RESULTS: With the study population of 108 patients, we characterized expression of CNP and NPR-B in human aortic valves and compared normal control valves (n = 12) with valves obtained from patients with aortic regurgitation (AR, n = 16), AR with fibrosis (AR+fibr., n = 19) and AS (n = 61). By reverse transcription-polymerase chain reaction (RT-PCR), CNP mRNA levels were 89% lower (p = 0.022) in stenotic valves, when compared to AR group. Moreover, the mRNA levels of NPR-B, the target receptor of CNP, were 62% lower (p < 0.001) in stenotic valves when compared to control group and 54% lower (p = 0.002) in stenotic valves, when compared to AR group. There was no statistical significant difference in CNP and NPR-B levels in AS group when the statin-treated patients were compared to untreated patients. CONCLUSIONS: These results show for the first time that the gene expression of anti-atherogenic CNP system did not differ between statin-treated and non-statin-treated patients in AS. The research data supports the results of clinical trials with the same drug class.

(Pro)renin receptors and angiotensin converting enzyme 2/angiotensin-(1-7)/Mas receptor axis in human aortic valve stenosis.

BACKGROUND: There is increasing evidence that renin-angiotensin system (RAS) may play a major role in the actively regulated fibrocalcific process in aortic valve stenosis (AS), but the gene expression or function of (pro)renin receptor ((P)RR), prorenin and renin or angiotensin converting enzyme 2(ACE2)/angiotensin-(1-7)/Mas receptor axis in calcific aortic valve disease is not known. METHODS AND RESULTS: We characterized expression of (P)RR, ACE2 and Mas receptor as well as renin, prorenin and angiotensin II type 2 (AT(2)) receptors in human aortic valves, and compared normal control valves (n = 11) with valves obtained from patients with aortic regurgitation (AR, n = 14), AR with fibrosis (n = 20) and AS (n = 61). By immunohistochemistry (P)RR positive staining was seen in the valvular endothelial cells of control and in the neovessels of stenotic valves. By RT-PCR, renin mRNA levels were 72% (P = 0.001) and prorenin mRNA levels 64% lower (P = 0.002) in stenotic aortic valves compared to control valves. ACE2, Mas receptor and AT(2)-receptor mRNA levels were 69% (P < 0.001), 58% (P = 0.008) and 75% (P = 0.001) lower, respectively, in stenotic valves. ACE2 positive staining, existing to lesser extent in stenotic aortic valves, was localized mainly to stromal area in spongiosa layer in control valves. CONCLUSIONS: (P)RR, prorenin and renin are expressed in human aortic valves. We also report for the first time expression of ACE2/angiotensin-(1-7)/-Mas receptor axis in human aortic valve cusps. The downregulation of ACE2/angiotensin-(1-7)/-Mas receptor axis as well as AT(2)-receptors may promote fibrosis, proliferation and inflammation in patients with AS.

Apelin and its receptor APJ in human aortic valve stenosis.

BACKGROUND AND AIM OF THE STUDY: Aortic valve stenosis (AS) is an actively regulated pathobiological process that shows some hallmarks of atherosclerosis. Apelin and its receptor, APJ, are highly expressed in the heart, and the proposed effects of the apelin-APJ system are opposite to those of the angiotensin II-AT1-receptor pathway. The role of the apelin-APJ signaling pathway in calcified aortic valve disease is unknown. METHODS: The study involved the characterization and comparison of expression of apelin and APJ as well as angiotensin II receptors (AT1 and AT2) in the aortic valves of patients with normal valves (n = 6), aortic regurgitation (n = 9 AR), regurgitation and fibrosis/mild sclerosis (n = 14), and AS (n = 25). RESULTS: By employing the reverse-transcriptase polymerase chain reaction (RT-PCR), the gene expression of apelin (3.63-fold, p = 0.001) and the APJ receptor (2.70-fold, p = 0.01) were shown to be significantly up-regulated in stenotic valves when compared to controls. In addition, APJ receptor mRNA levels were higher (2.9-fold, p = 0.010) in the AR + sclerosis group when compared to controls. Using immunohistochemistry, apelin was shown to be localized in stenotic aortic valves to the valvular endothelial layer of the aortic valve, to vascular endothelial cells in neovessels, and to fibroblasts and macrophages adjacent to vessels in the stromal area. AT2-receptor mRNA levels were 90% (p < 0.001) lower in stenotic valves. In contrast, the gene expression of AT1-receptors did not differ significantly among the groups. CONCLUSION: Aortic valve stenosis is characterized by an up-regulation of the apelin-APJ signaling pathway, revealing a possible novel target for drug discovery in calcified aortic valve disease by suppressing chemotaxis, angiogenesis and osteoblast activity, all of which are well-documented phenomena in the disease process.

Increase in tissue endothelin-1 and ETA receptor levels in human aortic valve stenosis.

AIMS: Aortic valve stenosis (AS) is an actively regulated process like atherosclerosis, which is accompanied by changes e.g. in endothelin-related genes. However, the role of endothelin peptides in AS is unknown. METHODS AND RESULTS: We characterized the expression of the endothelin system in aortic valves of patients with normal valves (n = 12), regurgitation, and fibrosis (n = 6) and AS (n = 18) by reverse-transcriptase-polymerase chain reaction and immunohistochemistry. The number of endothelin-1 (ET-1) positive cells was higher in AS than in control valves, while levels of ET-1 mRNA did not differ between groups. Endothelin receptor-A (ET(A)) mRNA levels were upregulated in stenotic valves (4.3-fold, P = 0.032) associated with a remarkable increase in number of ET(A)-immunopositive cells. ET(B)-receptor mRNA levels did not change during disease progression. Endothelin-converting enzyme-1 (ECE-1) mRNA levels were 42% lower (P = 0.007) in stenotic valves. Finally, because ET-1 and ECE-1 have binding site for activator protein-1 (AP-1), we measured AP-1 DNA binding by gel shift assays, which showed significantly lower (76%, P = 0.003) activity in AS. CONCLUSION: AS is characterized by distinct upregulation of ET-1 and its target receptor ET(A), promoting growth, inflammation, and fibrosis. These findings suggest therapeutic potential for ET(A)-receptor antagonists in aortic valve calcification.

Distinct downregulation of C-type natriuretic peptide system in human aortic valve stenosis.

BACKGROUND: Aortic valve calcification is an actively regulated process that displays hallmarks of atherosclerosis. Natriuretic peptides (A-, B-, and C-type natriuretic peptides [ANP, BNP, and CNP]) have been reported to have a role in the pathogenesis of vascular atherosclerosis, but their expression in aortic valves is not known. Here, we characterized and compared expression of natriuretic peptide system in aortic valves of patients with normal valves (n=4), aortic regurgitation (n=11), regurgitation and fibrosis (n=6), and aortic valve stenosis (n=21). METHODS AND RESULTS: By reverse-transcription polymerase chain reaction, all 3 natriuretic peptides were found to be expressed in aortic valves. CNP mRNA levels were 92% lower (P<0.001) in stenotic valves, whereas no significant changes in the expression of ANP and BNP genes were found compared with valves obtained from patients with aortic regurgitation. CNP was localized by immunohistochemistry with specific CNP (32-53) antibody to valvular endothelial cells and myofibroblasts. Gene expression of furin, which proteolytically cleaves proCNP into active CNP, was 54% lower in aortic valve stenosis (P=0.04). Moreover, natriuretic peptide receptor-A and natriuretic peptide receptor-B mRNA levels were 78% and 76% lower, respectively, in stenotic valves. In contrast, gene expression of corin, a proANP- and proBNP-converting enzyme, and natriuretic peptide receptor-C did not differ between groups. CONCLUSIONS: We show that natriuretic peptides, their processing enzymes, and their receptors are expressed in human aortic valves. Aortic valve stenosis is characterized by distinct downregulation of gene expression of CNP, its processing enzyme furin, and the target receptors natriuretic peptide receptor-B and natriuretic peptide receptor-A, which suggests that CNP acts as a paracrine regulator of the aortic valve calcification process.