Stefan-Martin Brand

Patients with Fabry Disease after Enzyme Replacement Therapy Dose Reduction Versus Treatment Switch

Because of the shortage of agalsidase-beta in 2009, many patients with Fabry disease were treated with lower doses or were switched to agalsidase-alfa. This observational study assessed end-organ damage and clinical symptoms during dose reduction or switch to agalsidase-alfa. A total of 105 adult patients with Fabry disease who had received agalsidase-beta (1.0 mg/kg body weight) for ≥1 year were nonrandomly assigned to continue this treatment regimen (regular-dose group, n=38), receive a reduced dose of 0.3-0.5 mg/kg (dose-reduction group, n=29), or switch to 0.2 mg/kg agalsidase-alfa (switch group) and were followed prospectively for 1 year. We assessed clinical events (death, myocardial infarction, severe arrhythmia, stroke, progression to ESRD); changes in cardiac, renal, and neurologic function; and Fabry-related symptoms (neuropathic pain, hypohidrosis, diarrhea, and disease severity scores). Organ function and Fabry-related symptoms remained stable in the regular-dose group. In contrast, estimated GFR decreased by about 3 ml/min per 1.73 m(2) (P=0.01) in the dose-reduction group, and the median albumin-to-creatinine ratio increased from 114 (0-606) mg/g to 216 (0-2062) mg/g (P=0.03) in the switch group. Furthermore, mean Mainz Severity Score Index scores and frequencies of pain attacks, chronic pain, gastrointestinal pain, and diarrhea increased significantly in the dose-reduction and switch groups. In conclusion, patients receiving regular agalsidase-beta dose had a stable disease course, but dose reduction led to worsening of renal function and symptoms. Switching to agalsidase-alfa is safe, but microalbuminuria may progress and Fabry-related symptoms may deteriorate.

Serum-Mediated Inhibition of Enzyme Replacement Therapy in Fabry Disease

Fabry disease (FD) is a progressive multisystemic disorder, treatable with recombinant enzyme replacement therapy (agalsidase). However, recent studies suggest an endogenous inhibition of agalsidase in patients with FD, as reported for other lysosomal storage diseases. To assess the clinical consequences of serum-mediated agalsidase inhibition in affected patients, we determined the agalsidase inhibition status of 168 patients (68 male) with FD and compared outcomes of inhibition-positive patients with those of inhibition-negative patients. The assessment included clinical events during time on agalsidase, determination of renal and cardiac function, and evaluation of FD-related symptoms. The frequency of serum-mediated agalsidase inhibition was 40% in agalsidase-treated males. Inhibition did not depend on the compound initially used (agalsidase-α or -β). Agalsidase inhibition was associated with higher lyso-globotriaosylceramide levels and worse disease severity scores in patients. Compared with agalsidase inhibition-negative men, agalsidase inhibition-positive men showed greater left ventricular mass (P=0.02) and substantially lower renal function (difference in eGFR of about -30 ml/min per 1.73 m(2); P=0.04), which was confirmed by a longitudinal 5-year retrospective analysis. Additionally, affected patients presented more often with FD-typical symptoms, such as diarrhea, fatigue, and neuropathic pain, among others. Therefore, patients with poor clinical outcome on agalsidase should be tested for agalsidase inhibition. Future studies are warranted to determine if affected patients with FD benefit from acute reduction of anti-agalsidase antibodies or long-term immune modulation therapies to suppress agalsidase inhibition and to identify mechanisms that minimize antibody generation against agalsidase.

Multifocal White Matter Lesions Associated with the D313Y Mutation of the α-Galactosidase A Gene

White matter lesions (WML) are clinically relevant since they are associated with strokes, cognitive decline, depression, or epilepsy, but the underlying etiology in young adults without classical risk factors still remains elusive. Our aim was to elucidate the possible clinical diagnosis and mechanisms leading to WML in patients carrying the D313Y mutation in the α-galactosidase A (GLA) gene, a mutation that was formerly described as nonpathogenic. Pathogenic GLA mutations cause Fabry disease, a vascular endothelial glycosphingolipid storage disease typically presenting with a symptom complex of renal, cardiac, and cerebrovascular manifestations. We performed in-depths clinical, biochemical and genetic examinations as well as advanced magnetic resonance imaging analyses in a pedigree with the genetically determined GLA mutation D313Y. We detected exclusive neurologic manifestations of the central nervous system of the “pseudo”-deficient D313Y mutation leading to manifest WML in 7 affected adult family members. Furthermore, two family members that do not carry the mutation showed no WML. The D313Y mutation resulted in a normal GLA enzyme activity in leukocytes and severely decreased activities in plasma. In conclusion, our results provide evidence that GLA D313Y is potentially involved in neural damage with significant WML, demonstrating the necessity of evaluating patients carrying D313Y more thoroughly. D313Y might broaden the spectrum of hereditary small artery diseases of the brain, which preferably occur in young adults without classical risk factors. In view of the existing causal therapy regime, D313Y should be more specifically taken into account in these patients.

Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis.

Background Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions. Methodology/Principal Findings The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein. Conclusion/Significance The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.

Soluble Adenylyl Cyclase in Vascular Endothelium Gene Expression Control of Epithelial Sodium Channel-α, Na+/K+-ATPase-α/β, and Mineralocorticoid Receptor

The Ca2+- and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element–binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element–mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element–mediated promoter activity and affected cAMP response element–binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-α and Na+/K+-ATPase-α. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use. # Novelty and Significance {#article-title-48}The Ca2+- and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element–binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element–mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element–mediated promoter activity and affected cAMP response element–binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-&agr; and Na+/K+-ATPase-&agr;. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use.

Dose-Response of High-Intensity Training (HIT) on Atheroprotective miRNA-126 Levels

Aim: MicroRNA-126 (miR-126) exerts beneficial effects on vascular integrity, angiogenesis, and atherosclerotic plaque stability. The purpose of this investigation was to analyze the dose-response relationship of high-intensity interval training (HIIT) on miR-126-3p and -5p levels. Methods: Sixty-one moderately trained individuals (females = 31 [50.8%]; 22.0 ± 1.84 years) were consecutively recruited and allocated into three matched groups using exercise capacity. During a 4-week intervention a HIIT group performed three exercise sessions/week of 4 × 30 s at maximum speed (all-out), a progressive HIIT (proHIIT) group performed three exercise sessions/week of 4 × 30 s at maximum speed (all-out) with one extra session every week (up to 7 × 30 s) and a low-intensity training (LIT) control group performed three exercise sessions/week for 25 min <75% of maximum heart rate. Exercise miR-126-3p/-5p plasma levels were determined using capillary blood from earlobes. Results: No exercise-induced increase in miR-126 levels was detected at baseline, neither in the LIT (after 25 min low-intensity running) nor the HIIT groups (after 4 min of high-intensity running). After the intervention, the LIT group presented an increase in miR-126-3p, while in the HIIT group, miR-126-3p levels were still reduced (all p < 0.05). An increase for both, miR-126-3p and -5p levels (all p < 0.05, pre- vs. during and post-exercise) was detected in the proHIIT group. Between group analysis revealed that miR-126-3p levels after LIT and proHIIT increased by 2.12 ± 2.55 and 1.24 ± 2.46 units (all p < 0.01), respectively, compared to HIIT (−1.05 ± 2.6 units). Conclusions: LIT and proHIIT may be performed to increase individual miR-126 levels. HIIT without progression was less effective in increasing miR-126.

Tissue-specific differences in the regulation of KIBRA gene expression involve transcription factor TCF7L2 and a complex alternative promoter system

KIBRA has been described as a key regulator of the Hippo signaling pathway, regulating organ size control, cell contact inhibition, cell growth, as well as tumorigenesis and cystogenesis. Since there is scarce information on KIBRA gene expression regulation, we analyzed the molecular basis of tissue-specific KIBRA expression in human kidney epithelial (IHKE, HPCT) and neuroblastoma (SH-SY5Y, SK-SN-SH) cells. We detected four novel and differentially used transcription start sites, two of which positioned in the first intron, generating two novel alternative exons. We identified one constitutively active core promoter (P1a) and three alternative promoters (P1b, P2, and P3), which were exclusively active in kidney cells. Transcription factor 7-like 2 (TCF7L2) selectively activated KIBRA at P1a, P2, and P3 in kidney cells. The two genetic variants −580C>T (p < 0.05) and −1691C>T (p < 0.01) significantly affected the transcriptional activity of the KIBRA core promoter. We propose a novel functional structure of the KIBRA gene and provide detailed insight into molecular cell type-specific KIBRA transcriptional regulation by TCF7L2, the Yes-associated protein 1 and TEA domain family member. Our findings provide a potential basis for future studies on malfunctioning KIBRA regulation in pathophysiological conditions such as cancer development.Key messageKIBRA expression is regulated by three independent, cell type-specific promotersTwo novel TSS were located within intron one resulting in two alternative exonsTSS utilization is cell type-specificTCF7L2, YAP1, and TEAD are involved in the differential KIBRA expression regulation

Renal function predicts long-term outcome on enzyme replacement therapy in patients with Fabry disease

Background Renal and cardiac involvement is responsible for substantial morbidity and mortality in Fabry disease (FD). We analysed the incidence of FD-related renal, cardiac and neurologic end points in patients with FD on long-term enzyme replacement therapy (ERT). Methods A retrospective analysis of prospectively collected data from two German FD centres was performed. The impact of renal and cardiac function at ERT-naïve baseline on end point development despite ERT was analysed. Results Fifty-four patients (28 females) receiving ERT (mean 81 ± 21 months) were investigated. Forty per cent of patients were diagnosed with clinical end points before ERT initiation and 50% of patients on ERT developed new clinical end points. In patients initially diagnosed with an end point before ERT initiation, the risk for an additional end point on ERT was increased {hazard ratio [HR] 3.83 [95% confidence interval (CI) 1.61-9.08]; P = 0.0023}. A decreased glomerular filtration rate (eGFR) ≤75 mL/min/1.73 m2 in ERT-naïve patients at baseline was associated with an increased risk for cardiovascular end points [HR 3.59 (95% CI 1.15-11.18); P = 0.0273] as well as for combined renal, cardiac and neurologic end points on ERT [HR 4.77 (95% CI 1.93-11.81); P = 0.0007]. In patients with normal kidney function, left ventricular hypertrophy at baseline predicted a decreased end point-free survival [HR 6.90 (95% CI 2.04-23.27); P = 0.0018]. The risk to develop an end point was independent of sex. Conclusions In addition to age, even moderately impaired renal function determines FD progression on ERT. In patients with FD, renal and cardiac protection is warranted to prevent patients from deleterious manifestations of the disease.

Differential response to endothelial epithelial sodium channel inhibition ex vivo correlates with arterial stiffness in humans.

Objectives: Recently, the nanomechanical properties (i.e. stiffness) of endothelial cells have been identified as crucial for appropriate endothelial function. One major determinant of endothelial stiffness is the endothelial sodium channel (EnNaC). EnNaC-dependent stiffening leads to reduced nitric oxide release, which is a hallmark for endothelial dysfunction. In the current study, we hypothesized that endothelial function is directly linked to the overall function of the arterial system. Methods: Sixty-four human ex-vivo arterial samples were collected from femoral bypass or vein-stripping procedures. Nanomechanical characteristics of ex-vivo endothelium from isolated arterial side branches were determined using atomic force microscopy. The endotheliums potential to respond to EnNaC inhibition by amiloride was defined as endothelial amiloride index. In addition, patients’ arterial stiffness was determined by pulse wave velocity (PWV). Results: Fifty-three percentage of the ex-vivo samples responded ‘classically’ to amiloride with endothelial softening, whereas 47% of the patients’ samples did not. Interestingly, a lack of endothelial softening in the presence of amiloride in vitro was observed with higher frequency among samples obtained from individuals with elevated PWV. Further, an increased PWV was associated with impaired renal function and endothelial dysfunction (higher levels of von Willebrand factor). Conclusions: Here, we report differential responses of human ex-vivo vessels to amiloride. Although the mechanism of differential amiloride response is still unknown, the data indicate that drug action on endothelial function could differ strongly among patients, especially in those with a vascular end-organ damage determined by PWV.

Uric acid and essential hypertension: the endothelial connection.

E levated uric acid concentrations are commonly observed in patients with hypertension and chronic kidney disease [1,2], coronary artery disease [3], chronic heart failure [4], acute myocardial infarction and stroke [5]. Of note, hyperuricemia has been associated with an increased risk for incident hypertension independent of traditional hypertension risk factors [2]. Strong evidence for a causal relationship between elevated uric acid plasma levels and hypertension has been provided by earlier research of Feig and Johnson [6,7]. In a screening of 125 children (6–18 years), they detected serum uric acid concentrations more than 5.5 mg/dl in 89% of patients with essential hypertension and in 30% of patients with secondary hypertension [6]. Subsequently, they demonstrated that allopurinol (200 mg twice daily for 4 weeks) lowered uric acid levels in adolescents with newly diagnosed hypertension from a mean 7.0 mg/dl to 4.2 mg/dl. Blood pressure during allopurinol treatment was consistently reduced by 6.9 mmHg for SBP and 5.1 mmHg for DBP (vs placebo, 2.0 mmHg SBP/ 2.4 mmHg DBP) [7]. Beyond these epidemiologic and interventional observations, we have learnt that uric acid is a biologically active molecule [8,9]. In contrast to the replicated associations of increased uric acid levels with cardiovascular disease, uric acid has been suggested to be one of the major antioxidants in human plasma [10,11]. At physiological concentrations, the uric acid anion urate has the ability to scavenge oxygen radicals and may protect the erythrocyte membrane from lipid oxidation [10]. However, uric acid does not scavenge superoxide, and the uric acid antioxidative activity depends on a hydrophilic environment such as human plasma [10,11]. In addition, it has been postulated that uric acid may turn into a prooxidant risk factor under certain inflammatory conditions such as atheromatous plaque formation