The tyrosine kinase inhibitor nilotinib targets the discoidin domain receptor DDR2 in calcific aortic valve stenosis.

BACKGROUND AND PURPOSE: Tyrosine kinase inhibitors (TKI) used to treat chronic myeloid leukaemia (CML) have been associated with cardiovascular side effects, including reports of calcific aortic valve stenosis. The aim of this study was to establish the effects of first and second generation TKIs in aortic valve stenosis and to determine the associated molecular mechanisms. EXPERIMENTAL APPROACH: Hyperlipidemic APOE*3Leiden.CETP transgenic mice were treated with nilotinib, imatinib or vehicle. Human valvular interstitial cells (VICs) were isolated and studied in vitro. Gene expression analysis was perfromed in aortic valves from 64 patients undergoing aortic valve replacement surgery. KEY RESULTS: Nilotinib increased murine aortic valve thickness. Nilotinib, but not imatinib, promoted calcification and osteogenic activation and decreased autophagy in human VICs. Differential tyrosine kinase expression was detected between healthy and calcified valve tissue. Transcriptomic target identification revealed that the discoidin domain receptor DDR2, which is preferentially inhibited by nilotinib, was predominantly expressed in human aortic valves but markedly downregulated in calcified valve tissue. Nilotinib and selective DDR2 targeting in VICs induced a similar osteogenic activation, which was blunted by increasing the DDR2 ligand, collagen. CONCLUSIONS AND IMPLICATIONS: These findings suggest that inhibition of DDR2 by nilotinib promoted aortic valve thickening and VIC calcification, with possible translational implications for cardiovascular surveillance and possible personalized medicine in CML patients.

Plaque Evaluation by Ultrasound and Transcriptomics Reveals BCLAF1 as a Regulator of Smooth Muscle Cell Lipid Transdifferentiation in Atherosclerosis.

BACKGROUND: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. METHODS: Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). RESULTS: Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. CONCLUSIONS: Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

Upregulated Autophagy in Calcific Aortic Valve Stenosis Confers Protection of Valvular Interstitial Cells.

Autophagy serves as a cell survival mechanism which becomes dysregulated under pathological conditions and aging. Aortic valve thickening and calcification causing left ventricular outflow obstruction is known as calcific aortic valve stenosis (CAVS). CAVS is a chronic and progressive disease which increases in incidence and severity with age. Currently, no medical treatment exists for CAVS, and the role of autophagy in the disease remains largely unexplored. To further understand the role of autophagy in the progression of CAVS, we analyzed expression of key autophagy genes in healthy, thickened, and calcified valve tissue from 55 patients, and compared them with nine patients without significant CAVS, undergoing surgery for aortic regurgitation (AR). This revealed a upregulation in autophagy exclusively in the calcified tissue of CAVS patients. This difference in autophagy between CAVS and AR was explored by LC3 lipidation in valvular interstitial cells (VICs), revealing an upregulation in autophagic flux in CAVS patients. Inhibition of autophagy by bafilomycin-A1 led to a decrease in VIC survival. Finally, treatment of VICs with high phosphate led to an increase in autophagic activity. In conclusion, our data suggests that autophagy is upregulated in the calcified tissue of CAVS, serving as a compensatory and pro-survival mechanism.

Identification and management of alcohol use and illicit substance use in outpatient psychiatric clinics in Sweden: a national survey of clinic directors and staff.

BACKGROUND: Swedish national guidelines recommend that all health care settings systematically screen patients for alcohol use and illicit substance use. When hazardous use is identified, it should immediately be addressed, preferably through brief interventions (BI). It is well known that the prevalence of alcohol use and illicit substance use among psychiatric patients is high, but it is not known to what extent screening and BI are routinely carried out in such clinics. METHODS: Two online surveys investigating the use of screening and BI for alcohol and illicit substances were constructed; one for psychiatric outpatient clinic directors and one for staff at these clinics. The main analyses were calculated as simple frequencies. In secondary analyses, we investigated the associations between substance abuse training, type of clinic and screening/BI delivery. For these analyses, the Chi square test was used. RESULTS: Most clinic directors reported that they have guidelines to screen for alcohol (93.1%) and illicit substance use (78.9%) at initial assessment. Fifty percent reported having guidelines for delivering BI when identifying hazardous alcohol use (35.9% for hazardous illicit substance use). Among staff, 66.6% reported always screening for alcohol use and 57.8% reported always screening for illicit substance use at initial assessment. Further, 36.7% reported that they usually deliver BI when identifying hazardous alcohol use (35.7% for hazardous illicit substance use). Secondary analyses indicated that staff with substance abuse training were significantly more likely to screen for alcohol use than staff without such training. Further, staff at psychosis clinics were significantly less likely to screen for both alcohol and substance use than staff at both general and specialist psychiatric clinics. CONCLUSIONS: Most clinic directors reported having clear guidelines for staff to screen for alcohol use and illicit substance use, but fewer staff members than expected indicated that these guidelines were adhered to. Providing training about substance use disorders for staff may increase use of screening for alcohol use, and psychosis clinics may need to improve their screening routines.

Local Delivery of miR-21 Stabilizes Fibrous Caps in Vulnerable Atherosclerotic Lesions.

miRNAs are potential regulators of carotid artery stenosis and concordant vulnerable atherosclerotic plaques. Hence, we analyzed miRNA expression in laser captured micro-dissected fibrous caps of either ruptured or stable plaques (n = 10 each), discovering that miR-21 was significantly downregulated in unstable lesions. To functionally evaluate miR-21 in plaque vulnerability, miR-21 and miR-21/apolipoprotein-E double-deficient mice (Apoe-/-miR-21-/-) were assessed. miR-21-/- mice lacked sufficient smooth muscle cell proliferation in response to carotid ligation injury. When exposing Apoe-/-miR-21-/- mice to an inducible plaque rupture model, they presented with more atherothrombotic events (93%) compared with miR-21+/+Apoe-/- mice (57%). We discovered that smooth muscle cell fate in experimentally induced advanced lesions is steered via a REST-miR-21-REST feedback signaling pathway. Furthermore, Apoe-/-miR-21-/- mice presented with more pronounced atherosclerotic lesions, greater foam cell formation, and substantially higher levels of arterial macrophage infiltration. Local delivery of a miR-21 mimic using ultrasound-targeted microbubbles into carotid plaques rescued the vulnerable plaque rupture phenotype. In the present study, we identify miR-21 as a key modulator of pathologic processes in advanced atherosclerosis. Targeted, lesion site-specific overexpression of miR-21 can stabilize vulnerable plaques.

Short-term Results of a Magnetic Resonance Imaging-Based Swedish Screening Program for Individuals at Risk for Pancreatic Cancer.

IMPORTANCE: Pancreatic cancer is the fourth leading cause of cancer-related death in Western countries. In approximately 10% of all patients with pancreatic cancer, it is possible to define a positive family history for pancreatic cancer or for one of the other related genetic syndromes. A screening program for individuals at risk is recommended; however, surveillance modalities have not been defined yet. OBJECTIVE: To analyze the short-term results of a prospective clinical surveillance program for individuals at risk for pancreatic cancer using a noninvasive magnetic resonance imaging (MRI)-based screening protocol. DESIGN, SETTING AND PARTICIPANTS: A prospective observational study of all patients with a genetic risk for developing pancreatic cancer who were referred to Karolinska University Hospital between January 1, 2010, and January 31, 2013, using an MRI-based surveillance program. All patients were investigated for the most common genetic mutations associated with pancreatic cancer. EXPOSURE: A noninvasive MRI-based screening protocol. MAIN OUTCOMES AND MEASURES: The ability of MRI to identify potential precancerous or early cancers in individuals at risk for pancreatic cancer. RESULTS: Forty patients (24 women and 16 men) were enrolled. The mean age was 49.9 years. The mean length of follow-up was 12.9 months. The numbers of relatives affected by pancreatic cancer were 5 in 2 patients (5%), 4 in 5 patients (12.5%), 3 in 17 patients (42.5%), 2 in 14 patients (35%), and 1 in 2 patients (5%). In 4 patients (10%), a p16 mutation was found; in 3, a BRCA2 mutation (7.5%); and in 1, a BRCA1 mutation (2.5%). In 16 patients (40%), MRI revealed a pancreatic lesion: intraductal papillary mucinous neoplasia (14 patients, 35%) and pancreatic ductal adenocarcinoma (2 patients, 5%). One patient had a synchronous intraductal papillary mucinous neoplasia and pancreatic ductal adenocarcinoma. Five patients (12.5%) required surgery (3 for pancreatic ductal adenocarcinoma and 2 for intraductal papillary mucinous neoplasia), while the remaining 35 are under continued surveillance. CONCLUSIONS AND RELEVANCE: During a median follow-up of approximately 1 year, pancreatic lesions were detected in 40% of the patients, of whom 5 underwent surgery. Although the study time was relatively short, the surveillance program in individuals at risk seems to be effective.