Copy number architectures define treatment-mediated selection of lethal prostate cancer clones.

Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient evolutionary relationships of metastases that evade treatment, we perform genome-wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 167 metastatic regions from 11 organs harvested post-mortem from 10 men who died from prostate cancer. We identify diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number changes, we confirm a common clone of origin across metastases and diagnostic biopsies, and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. These autosome-defined clusters are characterized by cluster-specific AR gene architectures, and in two index cases are topologically more congruent than by chance (p-values 3.07 × 10-8 and 6.4 × 10-4). Integration with anatomical sites suggests patterns of spread and points of genomic divergence. Here, we show that copy number boundaries identify treatment-selected clones with putatively distinct lethal trajectories.

Combined CT-guided high-dose-rate brachytherapy (CT-HDRBT) and transarterial chemoembolization with irinotecan-loaded microspheres improve local tumor control and progression-free survival in patients with unresectable colorectal liver metastases compared with mono-CT-HDRBT.

Purpose: To compare the effectivity and toxicity of monotherapy with computed tomography-guided high-dose-rate brachytherapy (CT-HDRBT) vs. combination therapy of transarterial chemoembolization with irinotecan (irinotecan-TACE) and CT-HDRBT in patients with large unresectable colorectal liver metastases (CRLM) with a diameter of > 3 cm. Material and methods: Forty-four retrospectively matched patients with unresectable CRLM were treated either with mono-CT-HDRBT or with a combination of irinotecan-TACE and CT-HDRBT (n = 22 in each group). Matching parameters included treatment, disease, and baseline characteristics. National Cancer Institute Common Terminology Criteria for Adverse Events (version 5.0) were used to evaluate treatment toxicity and the Society of Interventional Radiology classification was applied to analyze catheter-related adverse events. Statistical analysis involved Cox regression, Kaplan-Meier estimator, log-rank test, receiver operating characteristic curve analysis, Shapiro-Wilk test, Wilcoxon test, paired sample t-test, and McNemar test. P-values < 0.05 were deemed significant. Results: Combination therapy ensued longer median progression-free survival (PFS: 5/2 months, p = 0.002) and significantly lower local (23%/68%, p < 0.001) and intrahepatic (50%/95%, p < 0.001) progress rates compared with mono-CT-HDRBT after a median follow-up time of 10 months. Additionally, tendencies for longer local tumor control (LTC: 17/9 months, p = 0.052) were found in patients undergoing both interventions. After combination therapy, aspartate and alanine aminotransferase toxicity levels increased significantly, while total bilirubin toxicity levels showed significantly higher increases after monotherapy. No catheter-associated major or minor complications were identified in each cohort. Conclusions: Combining irinotecan-TACE with CT-HDRBT can improve LTC rates and PFS compared with mono-CT-HDRBT in patients with unresectable CRLM. The combination of irinotecan-TACE and CT-HDRBT shows satisfying safety profiles.

Clinical testing of transcriptome-wide expression profiles in high-risk localized and metastatic prostate cancer starting androgen deprivation therapy: an ancillary study of the STAMPEDE abiraterone Phase 3 trial.

Metastatic and high-risk localized prostate cancer respond to hormone therapy but outcomes vary. Following a pre-specified statistical plan, we used Cox models adjusted for clinical variables to test associations with survival of multi-gene expression-based classifiers from 781 patients randomized to androgen deprivation with or without abiraterone in the STAMPEDE trial. Decipher score was strongly prognostic (p<2×10-5) and identified clinically-relevant differences in absolute benefit, especially for localized cancers. In metastatic disease, classifiers of proliferation, PTEN or TP53 loss and treatment-persistent cells were prognostic. In localized disease, androgen receptor activity was protective whilst interferon signaling (that strongly associated with tumor lymphocyte infiltration) was detrimental. Post-Operative Radiation-Therapy Outcomes Score was prognostic in localized but not metastatic disease (interaction p=0.0001) suggesting the impact of tumor biology on clinical outcome is context-dependent on metastatic state. Transcriptome-wide testing has clinical utility for advanced prostate cancer and identified worse outcomes for localized cancers with tumor-promoting inflammation.

Spatio-temporal analysis of prostate tumors in situ suggests pre-existence of treatment-resistant clones.

The molecular mechanisms underlying lethal castration-resistant prostate cancer remain poorly understood, with intratumoral heterogeneity a likely contributing factor. To examine the temporal aspects of resistance, we analyze tumor heterogeneity in needle biopsies collected before and after treatment with androgen deprivation therapy. By doing so, we are able to couple clinical responsiveness and morphological information such as Gleason score to transcriptome-wide data. Our data-driven analysis of transcriptomes identifies several distinct intratumoral cell populations, characterized by their unique gene expression profiles. Certain cell populations present before treatment exhibit gene expression profiles that match those of resistant tumor cell clusters, present after treatment. We confirm that these clusters are resistant by the localization of active androgen receptors to the nuclei in cancer cells post-treatment. Our data also demonstrates that most stromal cells adjacent to resistant clusters do not express the androgen receptor, and we identify differentially expressed genes for these cells. Altogether, this study shows the potential to increase the power in predicting resistant tumors.

Accumulation of copy number alterations and clinical progression across advanced prostate cancer.

BACKGROUND: Genomic copy number alterations commonly occur in prostate cancer and are one measure of genomic instability. The clinical implication of copy number change in advanced prostate cancer, which defines a wide spectrum of disease from high-risk localised to metastatic, is unknown. METHODS: We performed copy number profiling on 688 tumour regions from 300 patients, who presented with advanced prostate cancer prior to the start of long-term androgen deprivation therapy (ADT), in the control arm of the prospective randomised STAMPEDE trial. Patients were categorised into metastatic states as follows; high-risk non-metastatic with or without local lymph node involvement, or metastatic low/high volume. We followed up patients for a median of 7 years. Univariable and multivariable Cox survival models were fitted to estimate the association between the burden of copy number alteration as a continuous variable and the hazard of death or disease progression. RESULTS: The burden of copy number alterations positively associated with radiologically evident distant metastases at diagnosis (P=0.00006) and showed a non-linear relationship with clinical outcome on univariable and multivariable analysis, characterised by a sharp increase in the relative risk of progression (P=0.003) and death (P=0.045) for each unit increase, stabilising into more modest increases with higher copy number burdens. This association between copy number burden and outcome was similar in each metastatic state. Copy number loss occurred significantly more frequently than gain at the lowest copy number burden quartile (q=4.1 × 10-6). Loss of segments in chromosome 5q21-22 and gains at 8q21-24, respectively including CHD1 and cMYC occurred more frequently in cases with higher copy number alteration (for either region: Kolmogorov-Smirnov distance, 0.5; adjusted P<0.0001). Copy number alterations showed variability across tumour regions in the same prostate. This variance associated with increased risk of distant metastases (Kruskal-Wallis test P=0.037). CONCLUSIONS: Copy number alteration in advanced prostate cancer associates with increased risk of metastases at diagnosis. Accumulation of a limited number of copy number alterations associates with most of the increased risk of disease progression and death. The increased likelihood of involvement of specific segments in high copy number alteration burden cancers may suggest an order underlying the accumulation of copy number changes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00268476 , registered on December 22, 2005. EudraCT  2004-000193-31 , registered on October 4, 2004.

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

Fusion transcript detection using spatial transcriptomics.

BACKGROUND: Fusion transcripts are involved in tumourigenesis and play a crucial role in tumour heterogeneity, tumour evolution and cancer treatment resistance. However, fusion transcripts have not been studied at high spatial resolution in tissue sections due to the lack of full-length transcripts with spatial information. New high-throughput technologies like spatial transcriptomics measure the transcriptome of tissue sections on almost single-cell level. While this technique does not allow for direct detection of fusion transcripts, we show that they can be inferred using the relative poly(A) tail abundance of the involved parental genes. METHOD: We present a new method STfusion, which uses spatial transcriptomics to infer the presence and absence of poly(A) tails. A fusion transcript lacks a poly(A) tail for the 5' gene and has an elevated number of poly(A) tails for the 3' gene. Its expression level is defined by the upstream promoter of the 5' gene. STfusion measures the difference between the observed and expected number of poly(A) tails with a novel C-score. RESULTS: We verified the STfusion ability to predict fusion transcripts on HeLa cells with known fusions. STfusion and C-score applied to clinical prostate cancer data revealed the spatial distribution of the cis-SAGe SLC45A3-ELK4 in 12 tissue sections with almost single-cell resolution. The cis-SAGe occurred in disease areas, e.g. inflamed, prostatic intraepithelial neoplastic, or cancerous areas, and occasionally in normal glands. CONCLUSIONS: STfusion detects fusion transcripts in cancer cell line and clinical tissue data, and distinguishes chimeric transcripts from chimeras caused by trans-splicing events. With STfusion and the use of C-scores, fusion transcripts can be spatially localised in clinical tissue sections on almost single cell level.

MetaCNV - a consensus approach to infer accurate copy numbers from low coverage data.

BACKGROUND: The majority of copy number callers requires high read coverage data that is often achieved with elevated material input, which increases the heterogeneity of tissue samples. However, to gain insights into smaller areas within a tissue sample, e.g. a cancerous area in a heterogeneous tissue sample, less material is used for sequencing, which results in lower read coverage. Therefore, more focus needs to be put on copy number calling that is sensitive enough for low coverage data. RESULTS: We present MetaCNV, a copy number caller that infers reliable copy numbers for human genomes with a consensus approach. MetaCNV specializes in low coverage data, but also performs well on normal and high coverage data. MetaCNV integrates the results of multiple copy number callers and infers absolute and unbiased copy numbers for the entire genome. MetaCNV is based on a meta-model that bypasses the weaknesses of current calling models while combining the strengths of existing approaches. Here we apply MetaCNV based on ReadDepth, SVDetect, and CNVnator to real and simulated datasets in order to demonstrate how the approach improves copy number calling. CONCLUSIONS: MetaCNV, available at https://bitbucket.org/sonnhammergroup/metacnv, provides accurate copy number prediction on low coverage data and performs well on high coverage data.

Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity.

Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies.

Retinal vascular resistance in arterial hypertension.

PURPOSE: Retinal microcirculation represents an easily accessible, non-invasive, in-vivo possibility to assess early microvascular changes. In addition to the assessment of functional (e.g. retinal capillary flow, RCF) and retinal arteriolar structural parameters (e.g. wall-to-lumen-ratio, WLR) we now suggest a new parameter reflecting the resistance in small retinal arterioles (RVR). MATERIAL AND METHODS: In 45 normotensive (NT) subjects and 123 patients with hypertension stage 1 (HT) we assessed RCF, WLR, arteriolar diameter, lumen diameter and wall cross section area in the retinal circulation by using scanning laser Doppler flowmetry (SLDF). Mean arterial pressure (MAP) was measured immediately before the SLDF measurement and retinal vascular resistance was calculated (RVR = MAP/RCF). In a separate study the test-retest reliability was determined in 6 volunteers from our clinical staff by assessing RVR three times within six weeks. RESULTS: The analysis of the volunteers revealed a coefficient of variation for RVR of 7.75 ± 2.11% and Cronbach´s alpha was 0.90. WLR, a marker of vascular remodeling did not differ between NT and HT. In contrast, RCF and inner diameter of the retinal arterioles (ID) were significantly lower (RCF: p = .045 and ID: p = .001) in the HT group than in the NT group and RVR was significantly higher in the HT group than in the NT group (p < .001). In both groups we found no correlation of RVR with age, but a significant correlation of RVR with WLR (NT: r = 0.34, p = .006; HT: r = 0.25, p = .01), indicating that the RVR reflects vascular remodeling in the retinal circulation. CONCLUSION: Our data showed an increased retinal vascular resistance in hypertensive patients compared to non-hypertensive patients prior to the occurrence of structural retinal vascular remodeling. The correlation between RVR and WLR indicates that RVR is a reliable, non-invasive and early-sensitive marker of vascular remodeling in early hypertension.

Retinal capillary rarefaction in patients with untreated mild-moderate hypertension.

BACKGROUND: Microvascular rarefaction influences peripheral vascular resistance, perfusion and metabolism by affecting blood pressure and flow pattern. In hypertension microvascular rarefaction has been described in experimental animal studies as well as in capillaroscopy of skin and biopsies of muscle tissue in patients. Retinal circulation mirrors cerebral microcirculation and allows non-invasive investigations. We compared capillary rarefaction of retinal vessels in hypertensive versus normotensive subjects. METHODS: In this study retinal capillary rarefaction in 70 patients with long time (more than 67 month of disease duration) and 64 patients with short time hypertension stage 1 or 2 has been compared to 55 healthy control subjects, who participated in clinical trials in our Clinical Research Center ( www.clinicaltrials.gov : NCT01318395, NCT00627952, NCT00152698, NCT01319344). Retinal vascular parameters have been measured non-invasively and in vivo in perfusion image by scanning laser Doppler flowmetry (Heidelberg Engineering, Germany). Capillary rarefaction was assessed by capillary area (CapA) (in pixel-number) and intercapillary distance (ICD) (in µm). Additionally retinal capillary flow (RCF) was measured. RESULTS: ICD was greater in the long time hypertensive group compared to healthy individuals (24.2 ± 6.3 µm vs 20.1 ± 4.2 µm, p = 0.001) and compared to short time hypertensive patients (22.2 ± 5.2 µm, p = 0.020). Long time hypertensive patients showed less CapA compared to healthy people (1462 ± 690 vs 1821 ± 652, p = 0.005). Accordingly, RCF was significantly lower in the long time hypertensive group compared to the healthy control group (282 ± 70 AU vs 314 ± 60 AU, p = 0.032). Our data indicate a lower level of retinal capillary density in hypertensive patients, especially in those with long time hypertension. CONCLUSION: Patients with hypertension stage 1 or 2 showed retinal capillary rarefaction in comparison to healthy normotensive subjects. Retinal capillary rarefaction was intensified with duration of disease.

Disturbed microcirculation in the hands of patients with systemic sclerosis detected by fluorescence optical imaging: a pilot study.

BACKGROUND: Utilising fluorescence optical imaging (FOI), the distribution of an intravenously applied colouring agent indocyanine green (ICG) can be analysed with the potential to identify malperfusion by little to no tissue enhancement. Systemic sclerosis (SSc) is characterised by the presence of digital ulcers reflecting progressive vasculopathy. The objective was to investigate the potential of FOI in the detection of disturbed microcirculation in the hands and fingers of patients with SSc and to link FOI findings to clinical signs of ischemia such as digital ulcers and pitting scars. METHODS: In this cross-sectional study, 63 patients with SSc and 26 healthy subjects were examined. FOI was performed in all 89 individuals and compared to clinical data and capillaroscopic findings assembled for the SSc cohort. RESULTS: Healthy subjects showed initial ICG signals in their fingertips in 93.6%, SSc patients in 78.5% (limited SSc) and 43.2% (diffuse SSc). Moreover, in SSc patients, FOI findings were significantly associated with a late capillaroscopic pattern, disseminated SSc features, a diffuse SSc subtype, and the presence of digital ulcers or pitting scars. Intra- and inter-reader reliability for FOI amounted to κ = 0.786 and κ = 0.834, respectively. CONCLUSIONS: FOI is able to detect areas of reduced microcirculation in patients with SSc with high association to capillaroscopic findings. The results pave the way for future FOI investigations into its role in the prediction of complications due to an impaired acral perfusion.

Detection of severe digital vasculopathy in systemic sclerosis by colour Doppler sonography is associated with digital ulcers.

Objective: Colour Doppler ultrasonography (CDUS) is very important in general vascular diagnostic procedures. Its role in determining the extent of vasculopathy in Systemic Sclerosis (SSc) needs further investigation. The aim of this study was to compare the presence of altered arteries with nailfold capillaroscopy and clinical signs of ischaemia, that is, digital ulcers or pitting scars (DU/PS). A feasible CDUS protocol is provided. Methods: Two thousand five hundred and twenty-eight arteries of the fingers, palms and wrists from 79 SSc patients (32 arteries per patient) were examined using CDUS. Furthermore, nailfold capillaroscopy, clinical and laboratory data were evaluated. Results: Narrowed or occluded lumens were seen in 39.8% of all assessable arteries (n = 2489) and 48.9% of all proper palmar digital arteries (n = 1564) but only 15.6% (P < 0.0001) of proximal arteries (n = 924). Fingerwise analyses presented significant coincidence of pathological CDUS findings and DU/PS (P = 0.0009). Pathological CDUS findings were also associated with elevated CRP concentrations, current or past smoking with ⩾20 pack-years, male gender and present or past DU/PS. Receiver operating characteristic curve analysis (area under the curve = 0.727) suggested a cut-off value of ⩾20% pathological vessels (sensitivity: 90.7%; specificity: 47.8%) for the presence of DU/PS. An examination protocol focusing on the right-hand digits II-V (proper palmar digital arteries) revealed similar results (area under the curve = 0.751; sensitivity: 93.0%; specificity: 43.5%). Conclusion: CDUS of hand and finger arteries allows measurement of the extent of SSc vasculopathy, which is associated with clinical signs of chronic malperfusion. A shortened examination protocol of CDUS (right-hand digits II-V; 15 min instead of 45 min examination time) could complement vascular diagnostics in SSc.

A randomised study of the impact of the SGLT2 inhibitor dapagliflozin on microvascular and macrovascular circulation.

BACKGROUND: The sodium-glucose cotransporter 2 inhibitor, dapagliflozin, has been shown to improve diabetic control and reduce blood pressure in patients with type 2 diabetes mellitus. Its effects on micro- and macrovascular structure and function have not yet been reported. METHODS: This was a prospective, single-centre, placebo-controlled, double-blind, randomised crossover phase IIIb study conducted between March 2014 and February 2015. After a 4-week run-in/washout phase, patients (N = 59) received 6 weeks of either dapagliflozin 10 mg or placebo once daily. They then underwent a 1-week washout before crossing over to the other treatment. Changes in retinal capillary flow (RCF) and arteriole remodelling were evaluated using scanning laser Doppler flowmetry, while micro- and macrovascular parameters in the systemic circulation were assessed using pulse wave analysis. RESULTS: Six weeks of dapagliflozin treatment resulted in improvements in diabetes control, including blood glucose and insulin resistance, and reduced office and 24-h ambulatory blood pressure values. RCF decreased from 324 AU at baseline to 308 AU after treatment with dapagliflozin (p = 0.028), while there was little difference after the placebo (318 AU; p = 0.334). Furthermore, the arteriole remodelling that was seen after the placebo phase was not evident after the dapagliflozin phase. Central systolic and diastolic blood pressure values were significantly lower after 6 weeks of dapagliflozin, by 3.0 and 2.2 mmHg, respectively (p = 0.035 and 0.020, respectively vs. baseline). CONCLUSIONS: Six weeks of dapagliflozin treatment resulted in numerous beneficial effects. In addition to achieving superior diabetes control and blood pressure, parameters associated with the early stages of vascular remodelling were also improved. Trial registration http://www.clinicaltrials.gov (NCT02383238).

Enhancing Nitric Oxide Bioavailability via Exogen Nitric Oxide Synthase and L-Arginine Attenuates Ischemia-Reperfusion-Induced Microcirculatory Alterations.

BACKGROUND: Nitric oxide (NO) is an important cytoprotective agent against ischemia and reperfusion injury (IRI). Enhancing NO bioavailability via exogen NO synthases (NOSs) and L-arginine promotes conversation to NO, circumventing the problem of nonfunctioning NOSs under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of endothelial, inducible and neuronal NOS, and L-arginine on reperfusion-induced microcirculatory alterations and hemodynamic adverse effects in the microvasculature of skeletal muscle. METHODS: Vascular pedicle isolated rat cremaster model was used that underwent 2 hours of warm ischemia followed by 1 hour of reperfusion. At 30 minutes before ischemia, normal saline (control group with/without ischemia), endothelial-, inducible-, and neuronal NOSs (2 IE) and L-arginine (50 mg/kg BW) were administered systemically (IV). Ischemia-reperfusion-induced microcirculatory alterations were measured after 1 hour of reperfusion. Mean arterial blood pressure and heart frequency were measured throughout the experiment to determine hemodynamic adverse effects. RESULTS: The isoforms of NOSs and L-arginine attenuated ischemia-reperfusion-induced vasoconstriction, improved red blood cell velocity, capillary flow, and leukocyte adherence to the endothelium wall. Hemodynamics was stable throughout the experiment. CONCLUSIONS: Enhancing NO bioavailability via exogen application of NOSs and L-arginine significantly attenuated ischemia-reperfusion-induced microcirculatory alterations in the microvasculature of skeletal muscle. Significant hemodynamic adverse effects were not present, thus demonstrating this approach might be useful for therapeutic intervention. This "pharmacologic preconditioning" could be an easy and effective interventional strategy to uphold conversation of L-arginine to NO under ischemic conditions.

Retinal Capillary Rarefaction in Patients with Type 2 Diabetes Mellitus.

PURPOSE: In diabetes mellitus type 2, capillary rarefaction plays a pivotal role in the pathogenesis of end-organ damage. We investigated retinal capillary density in patients with early disease. METHODS: This cross-sectional study compares retinal capillary rarefaction determined by intercapillary distance (ICD) and capillary area (CapA), measured non-invasively and in vivo by scanning laser Doppler flowmetry, in 73 patients with type 2 diabetes, 55 healthy controls and 134 individuals with hypertension stage 1 or 2. RESULTS: In diabetic patients, ICD was greater (23.2±5.5 vs 20.2±4.2, p = 0.013) and CapA smaller (1592±595 vs 1821±652, p = 0.019) than in healthy controls after adjustment for differences in cardiovascular risk factors between the groups. Compared to hypertensive patients, diabetic individuals showed no difference in ICD (23.1±5.8, p = 0.781) and CapA (1556±649, p = 0.768). CONCLUSION: In the early stage of diabetes type 2, patients showed capillary rarefaction compared to healthy individuals.

Effects of linagliptin on renal endothelial function in patients with type 2 diabetes: a randomised clinical trial.

AIMS/HYPOTHESIS: Endothelial dysfunction predicts cardiovascular damage and renal involvement. Animal experiments and human studies indicate an increased nitric oxide (NO) activity and endothelial NO synthase (NOS) expression in the early stage of type 2 diabetes. The aim of the study was to assess the effect of linagliptin on the endothelial function of the renal vasculature. METHODS: In this randomised, double-blind, parallel-group, investigator-initiated trial, 62 patients with type 2 diabetes were randomly assigned (by computer-generated random code) to receive linagliptin 5 mg (n = 30) or placebo (n = 32) for 4 weeks. The primary objective was to assess endothelial function of the renal vasculature, by constant-infusion input-clearance and urinary albumin/creatinine ratio (UACR), both before and after blockade of NOS with N G-monomethyl-L-arginine (L-NMMA). RESULTS: Treatment with linagliptin for 4 weeks reduced fasting, postprandial blood glucose and HbA1c, although not significantly; no change occurred with placebo. Renal plasma flow (RPF) did not change after linagliptin or placebo. After 4 weeks the absolute change in RPF due to L-NMMA was smaller in the linagliptin group than in the placebo group (-46.8 ± 34 vs -65.1 ± 36 ml/min, p = 0.045), indicating a lower basal NO activity after treatment with linagliptin. Consistently, the response of UACR to L-NMMA increased in the placebo group (p = 0.059) but not in the linagliptin group (p = 0.276), pointing to an upregulation of NO activity in the placebo group. No clinically meaningful safety concerns were evident. CONCLUSIONS/INTERPRETATION: Our data suggest that treatment with the dipeptidyl peptidase-4 inhibitor linagliptin for 4 weeks prevented the impairment of renal endothelial function due to hyperglycaemia in type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT01835678 FUNDING: : This study was funded by Boehringer Ingelheim.

Improvement in Retinal Capillary Rarefaction After Valsartan Treatment in Hypertensive Patients.

Decreased capillary density influences vascular resistance and perfusion. The authors aimed to investigate the influence of the renin-angiotensin receptor blocker valsartan on retinal capillary rarefaction in hypertensive patients. Retinal vascular parameters were measured noninvasively and in vivo by scanning laser Doppler flowmetry before and after 4 weeks of treatment with valsartan in 95 patients with hypertension stage 1 or 2 and compared with 55 healthy individuals. Retinal capillary rarefaction was determined with the parameters intercapillary distance (ICD) and capillary area (CapA). In hypertensive patients, ICD decreased (23.4±5.5 µm vs 21.5±5.6 µm, P<.001) and CapA increased (1564±621 vs 1776±795, P=.001) after valsartan treatment compared with baseline. Compared with healthy normotensive controls (ICD 20.2±4.2 µm, CapA 1821±652), untreated hypertensive patients showed greater ICD (P<.001) and smaller CapA (P=.019), whereas treated hypertensive patients showed no difference in ICD (P=.126) and CapA (P=.728). Therapy with valsartan for 4 weeks diminished capillary rarefaction in hypertensive patients.

Early Signs of End-Organ Damage in Retinal Arterioles in Patients with Type 2 Diabetes Compared to Hypertensive Patients.

BACKGROUND: Eutrophic and hypertrophic remodeling are major vascular hallmarks for hypertension and diabetes-associated microvascular end-organ damage in peripheral arterioles. The aim of this study is to compare retinal arterioles of diabetic, hypertensive, and healthy individuals. METHODS: Retinal parameters were assessed in 99 patients with T2DM, 158 hypertensive, and 149 healthy individuals. WT and CA of retinal arterioles (80-140 µm) were measured noninvasively and in vivo by scanning laser Doppler flowmetry (Heidelberg Engineering, Germany). RESULTS: After adjustment for values differing between the groups (age, BMI, gender, HDL cholesterol and serum creatinine, systolic office BP), patients with T2DM showed no significant difference in WT (14.2 ± 3), and CA (4199 ± 1107) in comparison with hypertensive patients (WT = 13.3 ± 4, p = 0.18, CA = 3862 ± 1546, p = 0.10) and healthy individuals (WT = 13.1 ± 3, p = 0.55, CA = 3864 ± 1216, p = 0.86). However, the subgroup of patients with diabetes duration of more than 60 months showed greater WT (14.9 ± 4, p = 0.04) and CA (4557 ± 1137, p = 0.02) than the hypertensive group and greater WT (p = 0.04) and CA (p = 0.03) than the healthy group, which is consistent with hypertrophic remodeling. CONCLUSION: In the early stage of T2DM no hypertrophic remodeling was seen in retinal arterioles. However, hypertrophic remodeling was found in diabetic patients with more than 60 months duration of disease.

Adherence to Antihypertensive Medication in Treatment-Resistant Hypertension Undergoing Renal Denervation.

BACKGROUND: Adherence to medication has been repeatedly proposed to represent a major cause of treatment-resistant hypertension (TRH); however, treatment decisions such as treating TRH with renal denervation depend on accurate judgment of adherence. We carefully analyzed adherence rates to medication before and after renal denervation and its effect on blood pressure (BP) control. METHODS AND RESULTS: Eighty patients with TRH were included in 2 prospective observational studies that assessed the difference of potential antihypertensive and nephroprotective effects of renal denervation. To compare prescribed with actual medication intake (representing a measure of adherence), we analyzed urine samples collected at baseline and at 6 months after renal denervation for antihypertensive compounds or metabolites (by liquid chromatography-mass spectrometry). In addition to office BP, 24-hour ambulatory BP and central hemodynamics (central systolic pressure, central pulse pressure) were assessed. Informed consent for analyses of urine metabolites was obtained from 79 of 80 patients. Actual intake of all antihypertensive drugs was detected at baseline and at 6 months after renal denervation in 44 (56%) and 52 (66%) patients, respectively; 1 drug was missing in 22 (28%) and 17 (22%) patients, respectively, and ≥2 drugs were missing in 13 (16%) and 10 (13%) patients, respectively. At baseline, 24-hour ambulatory BP (P=0.049) and central systolic BP (P=0.012) were higher in nonadherent patients. Adherence did not significantly change overall (McNemar-Bowker test, P=0.362). An increase in adherence was observed in 21 patients, and a decrease was observed in 11 patients. The decrease in 24-hour ambulatory BP was not different in those with stable adherence 6 months after renal denervation (n=41, -7±13 mm Hg) compared with those with increased adherence (n=21, -10±13 mm Hg) and decreased adherence (n=11, -7±14 mm Hg) (P>0.20). Our study is limited by the relatively small sample size and potentially by the specific health environment of our university center (Northern Bavaria, Germany). CONCLUSIONS: Nonadherence to medication among patients with TRH was relatively low: ≈1 of 6 patients with TRH did not take ≥2 of the prescribed drugs. Adherence pattern did not change significantly after renal denervation and had no impact on the overall observed BP changes, supporting the concept that renal denervation is an effective treatment in patients with TRH. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifiers: NCT00888433, NCT01442883 and NCT01687725.