Lars Stangenberg

The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions

Healing of myocardial infarction (MI) requires monocytes/macrophages. These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling. The mechanisms that orchestrate such divergent functions remain unknown. In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI. We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing. Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6Chi and -6Clo monocytes via CCR2 and CX3CR1, respectively. Ly-6Chi monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions. Ly-6Clo monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular–endothelial growth factor. Consequently, Ly-6Chi monocytes digest damaged tissue, whereas Ly-6Clo monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen. MI in atherosclerotic mice with chronic Ly-6Chi monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response. These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI.

Tracking the inflammatory response in stroke in vivo by sensing the enzyme myeloperoxidase

Inflammation can extend ischemic brain injury and adversely affect outcome in experimental animal models. A key difficulty in translating animal studies to humans is the lack of a definitive method to confirm and track inflammation in the brain in vivo. Myeloperoxidase (MPO), a key inflammatory enzyme secreted by activated neutrophils and macrophages/microglia, can generate highly reactive oxygen species to cause additional damage in cerebral ischemia. We report here that a functional, enzyme-activatable MRI agent can accurately track the oxidative activity of MPO noninvasively in stroke in living animals. We found that MPO is widely distributed in ischemic tissues, correlates positively with infarct size, and is detected even 3 weeks postinfarction. The peak level of MPO activity, determined by activation of the MPO-sensing agent in vivo and confirmed by MPO activity and quantitative RT-PCR assays, occurred on day 3 after ischemia. Both neutrophils and macrophages/microglia contribute to secrete MPO in the ischemic brain, although neutrophils peak earlier (days 1–3) whereas macrophages/microglia are most abundant later (days 3–7). In contrast to the conventional MRI agent diethylenetriamine-pentatacetate gadolinium, which reports blood–brain barrier disruption, MPO imaging is able to additionally track MPO activity and confirm inflammation on the molecular level in vivo, information that was previously only possible to obtain on ex vivo brain sections and impossible to assess in living human patients. Our findings could allow efficient noninvasive serial screening of therapies targeting inflammation and the use of MPO imaging as an imaging biomarker to risk-stratify patients.

Hypoxia-Dependent Modification of Collagen Networks Promotes Sarcoma Metastasis

UNLABELLED Intratumoral hypoxia and expression of hypoxia-inducible factor-1α (HIF-1α) correlate with metastasis and poor survival in patients with sarcoma. We show here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF-1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF-1α or PLOD2 expression disrupts collagen modification, cell migration, and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSL-Kras(G12D/+); Trp53(fl/fl) murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF-1α-deficient tumors, and analysis of human sarcomas reveals elevated HIF1A and PLOD2 expression in metastatic primary lesions. Pharmacologic inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF-1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination. SIGNIFICANCE Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, which frequently and fatally metastasizes to the lung. Here, we show the potential use of a novel therapeutic target for the treatment of metastatic UPS, specifi cally the collagen-modifying enzyme PLOD2.

Transglutaminase activity in acute infarcts predicts healing outcome and left ventricular remodelling: implications for FXIII therapy and antithrombin use in myocardial infarction

AIMS The transglutaminase factor XIII (FXIII) emerges as a key enzyme in healing after myocardial infarction (MI). Here we assess the impact of transglutaminase-modulating therapies on healing and evolution of heart failure using a novel, non-invasive molecular imaging technique. METHODS AND RESULTS Immunoblotting revealed lower FXIII levels in the myocardium of nine patients with infarct rupture when compared to MI patients without rupture (P < 0.0045). In a murine model of MI, we assessed healing while modulating local FXIII activity. Infarct tissue activity was monitored with molecular in vivo single photon emission computed tomography-computed tomography (SPECT-CT) imaging, and activity was found to be increased by 80% in FXIII-treated mice (400 IU FXIII/kg iv.), and decreased by 65% in dalteparin (DP)-treated mice (600 IU/kg DP sc., P < 0.05). DP-treated mice exhibited increased mortality due to infarct rupture (64% by day 7, P < 0.018). Serial Magnetic Resonance Imaging (MRI) showed that left ventricular dilation post-MI was attenuated by FXIII treatment when compared to saline control-treated mice with MI (P = 0.04). Quantitative histological and reverse transcription-polymerase chain reaction analyses revealed that FXIII treatment induced a faster resolution of the neutrophil response, enhanced macrophage recruitment, increased collagen content and augmented angiogenesis in the healing infarct (P < 0.05 vs. control-treated mice with MI). CONCLUSION FXIII tissue levels are decreased in patients with insufficient healing. Therapeutic strategies that modulate FXIII activity impact murine myocardial healing. Molecular imaging of FXIII activity predicts prognosis in mice with experimental MI.

Improved detection of ovarian cancer metastases by intraoperative quantitative fluorescence protease imaging in a pre-clinical model

OBJECTIVES Cytoreductive surgery is a cornerstone of therapy in metastatic ovarian cancer. While conventional white light (WL) inspection detects many obvious tumor foci, careful histologic comparison has shown considerable miss rates for smaller foci. The goal of this study was to compare tumor detection using WL versus near infrared (NIR) imaging with a protease activatable probe, as well as to evaluate the ability to quantify NIR fluorescence using a novel quantitative optical imaging system. METHODS A murine model for peritoneal carcinomatosis was generated and metastatic foci were imaged using WL and NIR imaging following the i.v. administration of the protease activatable probe ProSense750. The presence of tumor was confirmed by histology. Additionally, the ability to account for variations in fluorescence signal intensity due to changes in distance between the catheter and target lesion during laparoscopic procedures was evaluated. RESULTS NIR imaging with a ProSense750 significantly improved upon the target-to-background ratios (TBRs) of tumor foci in comparison to WL imaging (minimum improvement was approximately 3.5 fold). Based on 52 histologically validated samples, the sensitivity for WL imaging was 69%, while the sensitivity for NIR imaging was 100%. The effects of intraoperative distance changes upon fluorescence intensity were corrected in realtime, resulting in a decrease from 89% to 5% in signal variance during fluorescence laparoscopy. CONCLUSIONS With its molecular specificity, low background autofluorescence, high TBRs, and quantitative signal, optical imaging with NIR protease activatable probes greatly improves upon the intraoperative detection of ovarian cancer metastases.

Cross Species Genomic Analysis Identifies a Mouse Model as Undifferentiated Pleomorphic Sarcoma/Malignant Fibrous Histiocytoma

Undifferentiated pleomorphic sarcoma/Malignant Fibrous Histiocytoma (MFH) is one of the most common subtypes of human soft tissue sarcoma. Using cross species genomic analysis, we define a geneset from the LSL-KrasG12D; Trp53Flox/Flox mouse model of soft tissue sarcoma that is highly enriched in human MFH. With this mouse geneset as a filter, we identify expression of the RAS target FOXM1 in human MFH. Expression of Foxm1 is elevated in mouse sarcomas that metastasize to the lung and tissue microarray analysis of human MFH correlates overexpression of FOXM1 with metastasis. These results suggest that genomic alterations present in human MFH are conserved in the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue sarcoma and demonstrate the utility of this pre-clinical model.

Efficacy of sunitinib and radiotherapy in genetically engineered mouse model of soft-tissue sarcoma.

PURPOSE Sunitinib (SU) is a multitargeted receptor tyrosine kinase inhibitor of the vascular endothelial growth factor and platelet-derived growth factor receptors. The present study examined SU and radiotherapy (RT) in a genetically engineered mouse model of soft tissue sarcoma (STS). METHODS AND MATERIALS Primary extremity STSs were generated in genetically engineered mice. The mice were randomized to treatment with SU, RT (10 Gy x 2), or both (SU+RT). Changes in the tumor vasculature before and after treatment were assessed in vivo using fluorescence-mediated tomography. The control and treated tumors were harvested and extensively analyzed. RESULTS The mean fluorescence in the tumors was not decreased by RT but decreased 38-44% in tumors treated with SU or SU+RT. The control tumors grew to a mean of 1378 mm(3) after 12 days. SU alone or RT alone delayed tumor growth by 56% and 41%, respectively, but maximal growth inhibition (71%) was observed with the combination therapy. SU target effects were confirmed by loss of target receptor phosphorylation and alterations in SU-related gene expression. Cancer cell proliferation was decreased and apoptosis increased in the SU and RT groups, with a synergistic effect on apoptosis observed in the SU+RT group. RT had a minimal effect on the tumor microvessel density and endothelial cell-specific apoptosis, but SU alone or SU+RT decreased the microvessel density by >66% and induced significant endothelial cell apoptosis. CONCLUSION SU inhibited STS growth by effects on both cancer cells and tumor vasculature. SU also augmented the efficacy of RT, suggesting that this combination strategy could improve local control of STS.

Denervation protects limbs from inflammatory arthritis via an impact on the microvasculature.

Significance Individuals who suffer paralysis on one side of the body and then develop rheumatoid arthritis show joint inflammation only on the neurologically intact side. We successfully modeled hemiplegia-induced protection from arthritis by transferring arthritogenic serum from K/BxN mice into recipients that had undergone unilateral sciatic and femoral nerve transection. Protection from serum-transferred arthritis could not be achieved by inhibiting the sympathetic, parasympathetic, or sensory arms of the nervous system. However, nerve transection did inhibit the joint-localized, inflammation-enhancing vascular leak rapidly induced by arthritogenic immune complexes and suggestively altered the transcriptome of the ankle microvasculature. Two-way communication between the mammalian nervous and immune systems is increasingly recognized and appreciated. An intriguing example of such crosstalk comes from clinical observations dating from the 1930s: Patients who suffer a stroke and then develop rheumatoid arthritis atypically present with arthritis on only one side, the one not afflicted with paralysis. Here we successfully modeled hemiplegia-induced protection from arthritis using the K/BxN serum-transfer system, focused on the effector phase of inflammatory arthritis. Experiments entailing pharmacological inhibitors, genetically deficient mouse strains, and global transcriptome analyses failed to associate the protective effect with a single nerve quality (i.e., with the sympathetic, parasympathetic, or sensory nerves). Instead, there was clear evidence that denervation had a long-term effect on the limb microvasculature: The rapid and joint-localized vascular leak that typically accompanies and promotes serum-transferred arthritis was compromised in denervated limbs. This defect was reflected in the transcriptome of endothelial cells, the expression of several genes impacting vascular leakage or transendothelial cell transmigration being altered in denervated limbs. These findings highlight a previously unappreciated pathway to dissect and eventually target in inflammatory arthritis.

In Vivo Optical Molecular Imaging of Matrix Metalloproteinase Activity following Celecoxib Therapy for Colorectal Cancer

We present an optical molecular imaging approach to measure the efficacy of the cyclooxygenase-2 (COX-2) inhibitor celecoxib on tumor growth rate through its effect on matrix metalloproteinase (MMP) activity. A xenograft model of colorectal cancer was generated in nude mice, which were then randomized to receive celecoxib versus vehicle. MMP activity was measured by an enzyme-activatable optical molecular probe. A novel genetically engineered mouse (GEM) model of colorectal cancer was also used to assess celecoxibs effect on MMP activity, which was measured by quantitative fluorescence colonoscopy. Subcutaneously implanted xenograft tumors were 84% (SD 20.2%) smaller in volume in the treatment group versus the control group. Moreover, treated animals exhibited only a 7.6% (SEM 9%) increase in MMP activity versus 106% (SEM 8%) for untreated animals. There was an apparent linear relationship (r = .91) between measured MMP activity and tumor growth rate. Finally, in the GEM model experiment, treated murine tumors remained relatively unchanged in volume and MMP activity; however, untreated tumors grew significantly and showed an increase in MMP activity. This method may provide for the improved identification of patients for whom COX-2 inhibition therapy is indicated by allowing one to balance the patients cardiovascular risk with the cancers responsiveness to celecoxib.

Low degree of formal education and musical experience predict degree of music-induced stress reduction in relatives and friends of patients: a single-center, randomized controlled trial.

Objective:To determine the factors that may predict music-induced relaxation in friends and family of patients in the emergency department. Background:It remains unclear to date which demographic and experiential factors predict the effectiveness of music-induced relaxation. Furthermore, in-hospital stressors for friends and family of patients rather than patients themselves are underresearched and deserve in-depth investigation to improve this groups experience in health care environments. Methods:A total of 169 relatives and friends of patients in the emergency department–waiting area completed a series of questionnaires, including the Spielberger State-Trait Anxiety Inventory (STAI), the Music Experience Questionnaire (MEQ), and a demographic survey. They were then randomly assigned to either Case Group (1 hour in the waiting area with classical music in the background) or Control Group (1 hour with no music) before completing a second, identical copy of the STAI to measure change from baseline. Data were analyzed for associations between music intervention, change in STAI scores, MEQ scores, and demographic characteristics. Results:Participants who underwent the music intervention experienced a 9.8% decrease in overall mean State Anxiety, whereas those in the Control Group experienced no change over time (P = 0.001). Higher education significantly inversely correlated with the effectiveness of music intervention: participants with no formal education beyond high school showed a greater overall mean decrease in State Anxiety than those with a college education or beyond in response to classical music (P = 0.006). Furthermore, MEQ scores indicated that the Social Uplift scale (a measure of ones tendency to be uplifted in a group-oriented manner by music) was highly predictive of the effectiveness of music intervention. Conclusions:Music is an effective and inexpensive means of reducing anxiety in friends and family of patients, who are underresearched in medicine. Moreover, low educational attainment and tendency to respond positively to music in a group setting can predict the effectiveness of music-induced relaxation.