Immune Response to Molecular Radiotherapy with 177Lu-DOTATOC: Predictive Value of Blood Cell Counts for Therapy Outcome.

Purpose: In a prior, retrospective study, 76% of patients with advanced neuroendocrine tumors undergoing 177Lu-DOTATOC molecular radiotherapy (MRT) showed their best response within 8 months from the first MRT cycle. In 24% of patients, latency was much greater up to >22 months after the first cycle, and long after near-complete decay of 177Lu from the last cycle. An immune response induced by MRT seems a likely explanation. As a crude measure of immunocompetence, the authors investigated whether blood cell counts (BCCs) may have predictive value for MRT outcome with 177Lu-DOTATOC. Methods: 56 Patients with neuroendocrine tumors (NET) were administered 177Lu-DOTATOC (mean 2.1 cycles; range 1-4) with median radioactivity of 7.0 GBq/cycle at 3-month intervals. Patients' BCCs were evaluated for four responder categories: CR, PR, SD, and PD (RECIST 1.1). Furthermore, baseline BCCs were correlated with progression-free survival (PFS). Finally, BCCs of patients with (PMT+) and without prior medical therapy (PMT-) were compared. Results: Significant differences between responder categories were found for baseline hemoglobin (Hb), erythrocytes, neutrophils, lymphocytes, neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and LEHN-score, integrating lymphocyte, erythrocyte, and neutrophil counts, and Hb level, but not for leukocytes and platelets. LEHN-score yielded an almost complete separation between CR and PD groups. In analogy, PFS times showed significant correlations with baseline Hb, erythrocytes, neutrophils, lymphocytes, NLR, PLR, and LEHN-score, the LEHN-score showing the strongest correlation, but not with leukocytes and platelets. For PMT- patients, median PFS was 34.5 months, compared with 20.8 months in PMT+ patients, with corresponding baseline lymphocyte (32.1 ± 9.6% vs. 24.5 ± 11.6%, p = 0.028) and neutrophil (54.9 ± 11.6% vs. 63.5 ± 13.7%, p = 0.039) counts. Conclusion: These findings emphasize the significance of an immune response to MRT for obtaining optimal therapy efficacy and support concepts to enhance the immune response of less immunocompetent patients before MRT. It seems advisable to avoid prior or concomitant immunosuppressant medical therapy.

Safety and Efficacy of Para-Aminohippurate Coinfusion for Renal Protection During Peptide Receptor Radiotherapy in Patients with Neuroendocrine Tumors.

Para-aminohippurate, also known as p-aminohippuric acid (PAH), is used clinically to measure effective renal plasma flow. Preclinically, it was shown to reduce 177Lu-DOTATOC uptake in the kidneys while improving bioavailability compared with amino acid (AA) coinfusion. We report the safety and efficacy of PAH coinfusion during peptide receptor radiotherapy in patients with neuroendocrine tumors. Methods: Twelve patients with metastatic or unresectable gastroenteropancreatic neuroendocrine tumors received 177Lu-DOTATOC in 33 treatment cycles. Either 8 g of PAH or a mixture of 25 g of arginine and 25 g of lysine were coinfused. Safety was assessed by monitoring laboratory data, including hematologic and renal data, as well as electrolytes obtained before and 24 h after treatment. For radiation dosimetry, whole-body scans were performed at 1, 24, and 48 h and a SPECT/CT scan was performed at 48 h, along with blood sampling at 5 min and 0.5, 2, 4, 24, and 48 h after administration. Absorbed dose estimations for the kidneys and bone marrow were performed according to the MIRD concept. Results: In 15 treatment cycles, PAH was coinfused. No changes in mean creatinine level, glomerular filtration rate, and serum electrolytes were observed before or 24 h after treatment when using PAH protection (P ≥ 0.20), whereas serum chloride and serum phosphate increased significantly under AA (both P < 0.01). Kidney-absorbed dose coefficients were 0.60 ± 0.14 Gy/GBq with PAH and 0.53 ± 0.16 Gy/GBq with AA. Based on extrapolated cumulative kidney-absorbed doses for 4 cycles, 1 patient with PAH protection and 1 patient with AA protection in our patient group would exceed the 23-Gy conservative threshold. The bone marrow-absorbed dose coefficient was 0.012 ± 0.004 Gy/GBq with PAH and 0.012 ± 0.003 Gy/GBq with AA. Conclusion: PAH is a promising alternative to AA for renal protection during peptide receptor radiotherapy. Further research is required to systematically investigate the safety profile and radiation dosimetry at varying PAH plasma concentrations.

[(177)Lu-DOTA](0)-D-Phe(1)-Tyr(3)-Octreotide ((177)Lu-DOTATOC) For Peptide Receptor Radiotherapy in Patients with Advanced Neuroendocrine Tumours: A Phase-II Study.

PURPOSE: To characterise efficacy and safety of (177)Lu-DOTATOC as agent for peptide receptor radiotherapy (PRRT) of advanced neuroendocrine tumours (NET). PATIENTS AND METHODS: Fifty-six subjects with metastasized and progressive NET (50% gastroenteral, 26.8% pancreatic, 23.2% other primary sites) treated consecutively with (177)Lu-DOTATOC were analysed retrospectively. Subjects were administered (177)Lu-DOTATOC (mean 2.1 cycles; range 1-4) as 7.0GBq (median) doses at three-monthly intervals. Efficacy was analysed using CT and/or MRI according to RECIST 1.1 criteria and results were stratified for the number of administered cycles and the primary tumour origin. RESULTS: In the total NET population (A), median progression-free (PFS) and overall survival (OS) were 17.4 and 34.2 months, respectively, assessed in a follow-up time (mean ± SD) of 16.1 ± 12.4 months. In patients receiving more than one cycle, mean follow-up time was 22.4 ± 11.0 months for all NETs (B) and PFS was 32.0 months for all NETs (B), 34.5 months for GEP-NET (C), and 11.9 months for other NETs (D). Objective response rates (Complete/Partial Responses) were 33.9%, 40.6%, 54.2%, and 0% for A, B, C, and D groups, respectively, while disease control rates in the same were 66.1%, 93.8%, 100%, and 75%. Complete responses (16.1%, 18.8% and 25.0% for groups A, B and C) were high, 78% of which were maintained throughout the follow up. There were no serious adverse events. One case of self-limiting grade 3 myelotoxicity was reported. Although 20% of patients had mild renal insufficiency at baseline, there was no evidence of exacerbated or de novo renal toxicity after treatment. CONCLUSION: (177)Lu-DOTATOC is a novel agent for PRRT with major potential to induce objective tumour responses and sustained disease control in progressive neuroendocrine tumours, even when administered in moderate activities. The observed safety profile suggests a particularly favourable therapeutic index, including in patients with impaired bone marrow or renal function, which reflects a uniquely low uptake of (177)Lu-DOTATOC by normal organs.

23Na chemical shift imaging and Gd enhancement of myocardial edema.

Myocardial edema can arise in several disease states. MRI contrast agent can accumulate in edematous tissue, which complicates differential diagnosis with contrast-enhanced (CE)-MRI and might lead to overestimation of infarct size. Sodium Chemical Shift Imaging ((23)Na-CSI) may provide an alternative for edema imaging. We have developed a non-infarct, isolated rat heart model with two levels of edema, which was studied with (23)Na-CSI and CE-MRI. In edematous, but viable tissue the extracellular sodium (Na (e) (+)) signal is hypothesized to increase, but not the intracellular sodium (Na (i) (+)) signal. Isolated hearts were perfused at 60 (n = 6) and 140 mmHg (n = 5). Dimethyl methylphosphonate (DMMP) and phenylphosphonate (PPA) were used to follow edema formation by (31)P-MR Spectroscopy. In separate groups, Thulium(III)1,4,7,10 tetraazacyclododecane-N,N',N″,N'''-tetra(methylenephosphonate) (TmDOTP(5-)) and Gadovist were used for (23)Na-CSI (n = 8) and CE-MRI (n = 6), respectively. PPA normalized signal intensity (SI) was higher at 140 versus 60 mmHg, with a ratio of 1.27 ± 0.12 (p < 0.05). The (DMMP-PPA)/dry weight ratio, as a marker of intracellular volume, remained unchanged. The mid-heart cross sectional area (CSA) of the left ventricle (LV) was significantly increased at 140 mmHg. In addition, at 140 mmHg, the LV Na (e) (+) SI increased with a 140 mmHg/60 mmHg ratio of 1.24 ± 0.18 (p < 0.05). Na (i) (+) SI remained essentially unchanged. With CE-MRI, a subendocardially enhanced CSA was identified, increasing from 0.20 ± 0.02 cm(2) at 60 mmHg to 0.31 ± 0.02 cm(2) at 140 mmHg (p < 0.05). Edema shows up in both CE-MRI and Na (e) (+) . High perfusion pressure causes more edema subendocardially than subepicardially. (23)Na-CSI is an attractive alternative for imaging of edema and is a promising tool to discriminate between edema, acute and chronic MI.

Quantitative T 2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice.

OBJECT: Imaging of myocardial infarct composition is essential to assess efficacy of emerging therapeutics. T (2) (*) mapping has the potential to image myocardial hemorrhage and fibrosis by virtue of its short T (2) (*) . We aimed to quantify T (2) (*) in acute and chronic myocardial ischemia/reperfusion (I/R) injury in mice. MATERIALS AND METHODS: I/R-injury was induced in C57BL/6 mice (n = 9). Sham-operated mice (n = 8) served as controls. MRI was performed at baseline, and 1, 7 and 28 days after surgery. MRI at 9.4 T consisted of Cine, T (2) (*) mapping and late-gadolinium-enhancement (LGE). Mice (n = 6) were histologically assessed for hemorrhage and collagen in the fibrotic scar. RESULTS: Baseline T (2) (*) values were 17.1 ± 2.0 ms. At day 1, LGE displayed a homogeneous infarct enhancement. T (2) (*) in infarct (12.0 ± 1.1 ms) and remote myocardium (13.9 ± 0.8 ms) was lower than at baseline. On days 7 and 28, LGE was heterogeneous. T (2) (*) in the infarct decreased to 7.9 ± 0.7 and 6.4 ± 0.7 ms, whereas T (2) (*) values in the remote myocardium were 14.2 ± 1.1 and 15.6 ± 1.0 ms. Histology revealed deposition of iron and collagen in parallel with decreased T (2) (*) . CONCLUSION: T (2) (*) values are dynamic during infarct development and decrease significantly during scar maturation. In the acute phase, T (2) (*) values in infarcted myocardium differ significantly from those in the chronic phase. T (2) (*) mapping was able to confirm the presence of a chronic infarction in cases where LGE was inconclusive. Hence, T (2) (*) may be used to discriminate between acute and chronic infarctions.

A view on imaging in drug research and development for respiratory diseases.

With the incidence of respiratory diseases increasing throughout the world, new therapies are needed. This review provides a short overview of different imaging techniques of interest for drug discovery and development within the pulmonary disease area. The focus is on studies performed in both animals and humans, which are of importance for understanding pathophysiological aspects and evaluating new drugs. Rather than emphasizing particular lung diseases, the noninvasive diagnosis and quantification of a number of characteristics related to several pathological conditions of the lung are addressed: inflammation, mucus secretion and clearance, emphysema, ventilation, perfusion, fibrosis, airway remodeling, and pulmonary arterial hypertension. Techniques are discussed based on their present use or potential future utilization in the context of drug studies.

Evaluation of infarcted murine heart function: comparison of prospectively triggered with self-gated MRI.

Measurement of cardiac function is often performed in mice after, for example, a myocardial infarction. Cardiac MRI is often used because it is noninvasive and provides high temporal and spatial resolution for the left and right ventricle. In animal cardiac MRI, the quality of the required electrocardiogram signal is variable and sometimes deteriorates over time, especially with infarcted hearts or cardiac hypertrophy. Therefore, we compared the self-gated IntraGateFLASH method with a prospectively triggered FLASH (fast low-angle shot) method in mice with myocardial infarcts (n = 16) and in control mice (n = 21). Mice with a myocardial infarct and control mice were imaged in a vertical 9.4-T MR system. Images of contiguous 1-mm slices were acquired from apex to base with prospective and self-gated methods. Data were processed to calculate cardiac function parameters for the left and right ventricle. The signal-to-noise and contrast-to-noise ratios were calculated in mid-ventricular slices. The signal-to-noise and contrast-to-noise ratios of the self-gated data were higher than those of the prospectively gated data. Differences between the two gating methods in the cardiac function parameters for both left and right ventricle (e.g. end-diastolic volumes) did not exceed the inter-observer variability in control or myocardial infarcted mice. Both methods gave comparable results with regard to the cardiac function parameters in both healthy control mice and mice with myocardial infarcts. Moreover, the self-gated method provided better signal-to-noise and contrast-to-noise ratios when the acquisition time was equal. In conclusion, the self-gated method is suitable for routine use in cardiac MRI in mice with myocardial infarcts as well as in control mice, and obviates the need for electrocardiogram triggering and respiratory gating. In both gating methods, more than 10 frames per cardiac cycle are recommended.

Characterization and in vitro and in vivo testing of CB2-receptor- and NGAL-targeted paramagnetic micelles for molecular MRI of vulnerable atherosclerotic plaque.

PURPOSE: Atherosclerotic plaque macrophages express the peripheral cannabinoid receptor (CB2-R) and promote fibrous cap degradation by secretion of neutrophil gelatinase-associated lipocalin 2 (NGAL). In this study, we report the preparation, characterization, and in vitro and in vivo testing of double-labeled (MR and fluorescent) CB2-R- and NGAL-targeted micelles. PROCEDURES/RESULTS: Specific CB2-R agonists or antibodies directed to 24p3 (mouse homolog of NGAL) were incorporated into di-oleoyl-polyethylene glycol-phosphatidylethanolamine 1000 (DOPE-PEG1000) micelles or di-stearoyl-polyethylene glycol-phosphatidylethanolamine 2000 (DSPE-PEG2000) micelles. The hydrodynamic diameter, determined by dynamic light scattering, was 16.5 and 19.0 nm for CB2-R-targeted DOPE-PEG1000 and DSPE-PEG2000 micelles, respectively, and 23.0 nm for Ab-conjugated DSPE-PEG2000 micelles. In vitro and in vivo MRI and fluorescence microscopy showed specific binding of CB2-R-targeted and 24p3-targeted micelles to in vitro systems and to aortic plaque in apoE(-/-)/eNOS(-/-) mice, respectively. CONCLUSIONS: CB2-R- and NGAL-targeted micelles show promise as tools for in vivo characterization of vulnerable plaque.

Antioxidant treatment attenuates pulmonary arterial hypertension-induced heart failure.

ROS have been implicated in the development of pathological ventricular hypertrophy and the ensuing contractile dysfunction. Using the rat monocrotaline (MCT) model of pulmonary arterial hypertension (PAH), we recently reported oxidative stress in the failing right ventricle (RV) with no such stress in the left ventricle of the same hearts. We used the antioxidant EUK-134 to assess the role of ROS in the pathological remodeling and dysfunction of the RV. PAH was induced by an injection of MCT (80 mg/kg, day 0), treatment with EUK-134 (25 mg/kg, once every 2 days) of control and MCT-injected animals [congestive heart failure (CHF) group] was started on day 10, and animals were analyzed on day 22. EUK-134 treatment of the CHF group attenuated cardiomyocyte hypertrophy and associated changes in mRNA expression (myosin heavy chain-beta and deiodinase type 3). It also reduced RV oxidative stress and proapoptotic signaling and prevented interstitial fibrosis. Cardiac MRI showed that ROS scavenging did not affect the 37% increase in end-diastolic volume of the RV in the CHF relative to the control group, but the threefold increase in end-systolic volume was reduced by 42% in the EUK-134-treated CHF group. The improved systolic function was confirmed using echocardiography by an assessment of tricuspid annular plane systolic excursion. These data indicate an important role of ROS in RV cardiomyocyte hypertrophy and contractile dysfunction due to PAH and show the potential of EUK-class antioxidants as complementary therapeutics in the treatment of RV dysfunction in PAH.

Recent developments and new perspectives on imaging of atherosclerotic plaque: role of anatomical, cellular and molecular MRI Part I and II.

Atherosclerotic plaque disruption accounts for the major part of cardiovascular mortality and the risk of disruption appears to depend on plaque composition. Carotid plaques in patients, scheduled for endarterectomy, have been successfully characterised with MRI. MRI has the advantage of combining information about morphology and function. Unfortunately, the tortuosity and size of the coronary arteries, and the respiratory and cardiac motion hinder the in vivo characterisation of human coronary plaque. In addition to plaque composition several molecular markers of the different processes involved in atherosclerosis, such as integrins, matrix metalloproteinases and fibrin seem to correlate with risk of plaque rupture and clinical outcome. These molecular markers can be targeted with antibodies coupled to carriers, which are loaded with gadolinium for detection (molecular MRI). Several cellular/molecular MRI studies in animal models and some in human patients have been conducted with varying levels of success. The advent of clinical high field magnets, the development of contrast agent carriers with high relaxivity and the development of relatively new MR contrast techniques are promising in the field of plaque imaging. Future MRI studies will have to focus on the molecular target of the atherosclerotic process, which has the highest prognostic value with regard to acute coronary syndromes and on the most suitable contrast agent to visualize that target.

Recent developments and new perspectives on imaging of atherosclerotic plaque: role of anatomical, cellular and molecular MRI part III.

Atherosclerotic plaque disruption accounts for the major part of cardiovascular mortality and the risk of disruption appears to depend on plaque composition. Carotid plaques in patients, scheduled for endarterectomy, have been successfully characterised with MRI. MRI has the advantage of combining information about morphology and function. Unfortunately, the tortuosity and size of the coronary arteries, and the respiratory and cardiac motion hinder the in vivo characterisation of human coronary plaque. In addition to plaque composition several molecular markers of the different processes involved in atherosclerosis, such as integrins, matrix metalloproteinases and fibrin seem to correlate with risk of plaque rupture and clinical outcome. These molecular markers can be targeted with antibodies coupled to carriers, which are loaded with gadolinium for detection (molecular MRI). Several cellular/molecular MRI studies in animal models and some in human patients have been conducted with varying levels of success. The advent of clinical high field magnets, the development of contrast agent carriers with high relaxivity and the development of relatively new MR contrast techniques appear to be promising in the field of plaque imaging. Future MRI studies will have to focus on the molecular target of the atherosclerotic process, which has the highest prognostic value with regard to acute coronary syndromes and on the most suitable contrast agent to visualize that target.

Negative MR contrast caused by USPIO uptake in lymph nodes may lead to false positive observations with in vivo visualization of murine atherosclerotic plaque.

OBJECTIVE: USPIOs are used clinically as contrast agent for magnetic resonance imaging (MRI) of lymph nodes, and in research settings for MRI of macrophages in atherosclerotic lesions. However, T2* weighted (T2*w) imaging can lead to "blooming" with overestimation of the area occupied by USPIOs. In this study, plaque uptake of USPIOs in atherosclerotic mice was investigated in the presence and absence of circulating monocytes. The influence of peri-aortic lymph node uptake on the interpretation of T2*w images of the aortic wall was studied. METHODS: Atherosclerotic mice were fed an atherogenic diet and were randomized to total body irradiation or non-irradiation. After 2 days, T2*w MRI of the abdominal aorta was performed, followed by intravenous administration of 100mumol/kg USPIOs (t=0). At t=3 and 5 days MRI of the abdominal aorta was repeated. Animals were sacrificed and histological evidence for iron uptake by aortic wall and lymph nodes was compared with the degree of focal signal loss on in vivo MR images. RESULTS: Aortic walls in irradiated and non-irradiated mice, but also in healthy wild-type mice, showed signal loss on T2*w MRI. Signal loss however did not correspond with histological evidence of USPIO uptake by aortic wall but by peri-aortic lymph nodes. CONCLUSIONS: The versatility of USPIOs as a negative MR contrast agent for both lymph node staging and atherosclerosis may limit the use for detection of atherosclerotic lesions in vessels where lymph nodes are highly prevalent.

Targeted deletion of nuclear factor kappaB p50 enhances cardiac remodeling and dysfunction following myocardial infarction.

Myocardial infarction is commonly complicated by left ventricular remodeling, a process that leads to cardiac dilatation, congestive heart failure and death. The innate immune system plays a pivotal role in the remodeling process via nuclear factor (NF)-kappaB activation. The NF-kappaB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. The function of NF-kappaB p50, however, is controversial in this process. To clarify the role of NF-kappaB p50 in postinfarct left ventricular remodeling, myocardial infarction was induced in wild-type 129Bl6 mice and NF-kappaB p50-deficient mice. Without affecting infarct size, deletion of NF-kappaB p50 markedly increased the extent of expansive remodeling (end-diastolic volume: 176+/-13 microL versus 107+/-11 microL; P=0.003) and aggravated systolic dysfunction (left ventricular ejection fraction: 16.1+/-1.5% versus 24.7+/-3.7%; P=0.029) in a 28-day time period. Interstitial fibrosis and hypertrophy in the noninfarcted myocardium was increased in NF-kappaB p50 knockout mice. In the infarct area, a lower collagen density was observed, which was accompanied by an increased number of macrophages, higher gelatinase activity and increased inflammatory cytokine expression. In conclusion, targeted deletion of NF-kappaB p50 results in enhanced cardiac remodeling and functional deterioration following myocardial infarction by increasing matrix remodeling and inflammation.

Modulation of serotonin transporter function during fetal development causes dilated heart cardiomyopathy and lifelong behavioral abnormalities.

BACKGROUND: Women are at great risk for mood and anxiety disorders during their childbearing years and may become pregnant while taking antidepressant drugs. In the treatment of depression and anxiety disorders, selective serotonin reuptake inhibitors (SSRIs) are the most frequently prescribed drugs, while it is largely unknown whether this medication affects the development of the central nervous system of the fetus. The possible effects are the product of placental transfer efficiency, time of administration and dose of the respective SSRI. METHODOLOGY/PRINCIPAL FINDINGS: In order to attain this information we have setup a study in which these parameters were measured and the consequences in terms of physiology and behavior are mapped. The placental transfer of fluoxetine and fluvoxamine, two commonly used SSRIs, was similar between mouse and human, indicating that the fetal exposure of these SSRIs in mice is comparable with the human situation. Fluvoxamine displayed a relatively low placental transfer, while fluoxetine showed a relatively high placental transfer. Using clinical doses of fluoxetine the mortality of the offspring increased dramatically, whereas the mortality was unaffected after fluvoxamine exposure. The majority of the fluoxetine-exposed offspring died postnatally of severe heart failure caused by dilated cardiomyopathy. Molecular analysis of fluoxetine-exposed offspring showed long-term alterations in serotonin transporter levels in the raphe nucleus. Furthermore, prenatal fluoxetine exposure resulted in depressive- and anxiety-related behavior in adult mice. In contrast, fluvoxamine-exposed mice did not show alterations in behavior and serotonin transporter levels. Decreasing the dose of fluoxetine resulted in higher survival rates and less dramatic effects on the long-term behavior in the offspring. CONCLUSIONS: These results indicate that prenatal fluoxetine exposure affects fetal development, resulting in cardiomyopathy and a higher vulnerability to affective disorders in a dose-dependent manner.

Toll-like receptor 4 mediates maladaptive left ventricular remodeling and impairs cardiac function after myocardial infarction.

Left ventricular (LV) remodeling leads to congestive heart failure and is a main determinant of morbidity and mortality following myocardial infarction. Therapeutic options to prevent LV remodeling are limited, which necessitates the exploration of alternative therapeutic targets. Toll-like receptors (TLRs) serve as pattern recognition receptors within the innate immune system. Activation of TLR4 results in an inflammatory response and is involved in extracellular matrix degradation, both key processes of LV remodeling following myocardial infarction. To establish the role of TLR4 in postinfarct LV remodeling, myocardial infarction was induced in wild-type BALB/c mice and TLR4-defective C3H-Tlr4(LPS-d) mice. Without affecting infarct size, TLR4 defectiveness reduced the extent of LV remodeling (end-diastolic volume: 103.7+/-6.8 microL versus 128.5+/-5.7 microL; P<0.01) and preserved systolic function (ejection fraction: 28.2+/-3.1% versus 16.6+/-1.3%; P<0.01), as assessed by MRI. In the noninfarcted area, interstitial fibrosis, and myocardial hypertrophy were reduced in C3H-Tlr4(LPS-d) mice. In the infarcted area, however, collagen density was increased, which was accompanied by fewer macrophages, reduced inflammation regulating cytokine expression levels (interleukin [IL]-1alpha, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, tumor necrosis factor-alpha, interferon-gamma, granulocyte/macrophage colony-stimulating factor), and reduced matrix metalloproteinase-2 (4684+/-515 versus 7573+/-611; P=0.002) and matrix metalloproteinase-9 activity (76.0+/-14.3 versus 168.0+/-36.2; P=0.027). These data provide direct evidence for a causal role of TLR4 in postinfarct maladaptive LV remodeling, probably via inflammatory cytokine production and matrix degradation. TLR4 may therefore constitute a novel target in the treatment of ischemic heart failure.

Combined blockade of the Na+ channel and the Na+/H+ exchanger virtually prevents ischemic Na+ overload in rat hearts.

Blocking either the Na(+) channel or the Na(+)/H(+) exchanger (NHE) has been shown to reduce Na(+) and Ca(2+) overload during myocardial ischemia and reperfusion, respectively, and to improve post-ischemic contractile recovery. The effect of combined blockade of both Na(+) influx routes on ionic homeostasis is unknown and was tested in this study. [Na(+)](i), pH(i) and energy-related phosphates were measured using simultaneous (23)Na- and (31)P-NMR spectroscopy in isolated rat hearts. Eniporide (3 muM) and/or lidocaine (200 muM) were administered during 5 min prior to 40 min of global ischemia and 40 min of drug free reperfusion to block the NHE and the Na(+) channel, respectively. Lidocaine reduced the rise in [Na(+)](i) during the first 10 min of ischemia, followed by a rise with a rate similar to the one found in untreated hearts. Eniporide reduced the ischemic Na(+) influx during the entire ischemic period. Administration of both drugs resulted in a summation of the effects found in the lidocaine and eniporide groups. Contractile recovery and infarct size were significantly improved in hearts treated with both drugs, although not significantly different from hearts treated with either one of them.

Endoglin has a crucial role in blood cell-mediated vascular repair.

BACKGROUND: Endoglin, an accessory receptor for transforming growth factor-beta in vascular endothelial cells, is essential for angiogenesis during mouse development. Mutations in the human gene cause hereditary hemorrhagic telangiectasia type 1 (HHT1), a disease characterized by vascular malformations that increase with age. Although haploinsufficiency is the underlying cause of the disease, HHT1 individuals show great heterogeneity in age of onset, clinical manifestations, and severity. METHODS AND RESULTS: In situ hybridization and immunohistochemical analysis of mouse and human hearts revealed that endoglin is upregulated in neoangiogenic vessels formed after myocardial infarction. Microvascularity within the infarct zone was strikingly lower in mice with reduced levels of endoglin (Eng+/-) compared with wild-type mice, which resulted in a greater deterioration in cardiac function as measured by magnetic resonance imaging. This did not appear to be because of defects in host inflammatory cell numbers in the infarct zone, which accumulated to a similar extent in wild-type and heterozygous mice. However, defects in vessel formation and heart function in Eng+/- mice were rescued by injection of mononuclear cells from healthy human donors but not by mononuclear cells from HHT1 patients. CONCLUSIONS: These results establish defective vascular repair as a significant component of the origin of HHT1. Because vascular damage or inflammation occurs randomly, it may also explain disease heterogeneity. More generally, the efficiency of vascular repair may vary between individuals because of intrinsic differences in their mononuclear cells.

MEF2 activates a genetic program promoting chamber dilation and contractile dysfunction in calcineurin-induced heart failure.

BACKGROUND: Hypertrophic growth, a risk factor for mortality in heart disease, is driven by reprogramming of cardiac gene expression. Although the transcription factor myocyte enhancer factor-2 (MEF2) is a common end point for several hypertrophic pathways, its precise cardiac gene targets and function in cardiac remodeling remain to be elucidated. METHODS AND RESULTS: We report the existence of synergistic interactions between the nuclear factor of activated T cells and MEF2 transcription factors triggered by calcineurin signaling. To circumvent the embryonic lethality and mitochondrial deficiency associated with germ-line null mutations for MEF2C and MEF2A respectively, we used conditional transgenesis to express a dominant-negative form of MEF2 in the murine postnatal heart and combined this with magnetic resonance imaging to assess MEF2 transcriptional function in Ca2+/calcineurin-induced cardiac remodeling. Surprisingly, end-diastolic and end-systolic ventricular dimensions and contractility were normalized in the presence of severely hypertrophied left ventricular walls on MEF2 inhibition in calcineurin transgenic mice. In line, we generated lines of transgenic mice expressing MEF2A in the heart, which displayed primarily chamber dilation. Microarray profiling indicated that MEF2 promotes a gene profile functioning primarily to or at the nucleus, cytoskeletal and microtubular networks, and mitochondria. CONCLUSIONS: These findings assign a novel function to MEF2 transcription factors in the postnatal heart, where they activate a genetic program that minimally affects cardiac growth yet promotes chamber dilation, mechanical dysfunction, and dilated cardiomyopathy.

Relative contributions of Na+/H+ exchange and Na+/HCO3- cotransport to ischemic Nai+ overload in isolated rat hearts.

The Na(+)/H(+) exchanger (NHE) and/or the Na(+)/HCO(3)(-) cotransporter (NBC) were blocked during ischemia in isolated rat hearts. Intracellular Na(+) concentration ([Na(+)](i)), intracellular pH (pH(i)), and energy-related phosphates were measured by using simultaneous (23)Na and (31)P NMR spectroscopy. Hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. Cariporide (3 microM) or HCO(3)(-)-free HEPES buffer was used, respectively, to block NHE, NBC, or both. End-ischemic [Na(+)](i) was 320 +/- 18% of baseline in HCO(3)(-)-perfused, untreated hearts, 184 +/- 6% of baseline when NHE was blocked, 253 +/- 19% of baseline when NBC was blocked, and 154 +/- 6% of baseline when both NHE and NBC were blocked. End-ischemic pH(i) was 6.09 +/- 0.06 in HCO(3)(-)-perfused, untreated hearts, 5.85 +/- 0.02 when NHE was blocked, 5.81 +/- 0.05 when NBC was blocked, and 5.70 +/- 0.01 when both NHE and NBC were blocked. NHE blockade was cardioprotective, but NBC blockade and combined blockade were not, the latter likely due to a reduction in coronary flow, because omission of HCO(3)(-) under conditions of NHE blockade severely impaired coronary flow. Combined blockade of NHE and NBC conserved intracellular H(+) load during reperfusion and led to massive Na(+) influx when blockades were lifted. Without blockade, both NHE and NBC mediate acid-equivalent efflux in exchange for Na(+) influx during ischemia, NHE much more than NBC. Blockade of either one does not affect the other.

Assessment of myocardial viability by intracellular 23Na magnetic resonance imaging.

BACKGROUND: Because of rapid changes in myocardial intracellular Na+ (Na+(i)) during ischemia and reperfusion (R), 23Na magnetic resonance imaging (MRI) appears to be an ideal diagnostic modality for early detection of myocardial ischemia and viability. So far, cardiac 23Na MRI data are limited and mostly concerned with imaging of total Na+. For proper interpretation, imaging of both Na+(i) and extracellular Na+ is essential. In this study, we tested whether Na+(i) imaging can be used to assess viability after low-flow (LF) ischemia. METHODS AND RESULTS: Isolated rat hearts were subjected to LF (1%, 2%, or 3% of control coronary flow) and R. A shift reagent was used to separate Na+(i) and extracellular Na+ resonances. Acquisition-weighted 23Na chemical shift imaging (CSI) was alternated with 23Na MR spectroscopy. Already during control perfusion, Na+(i) could be clearly seen on the images. Na+(i) image intensity increased with increasing severity of ischemia. During R, Na+(i) image intensity remained highest in 1% LF hearts. Not only did we find very good correlations between Na+(i) image intensity at end-R and end-diastolic pressure (R=0.85, P<0.001) and recovery of the rate-pressure product (R=-0.88, P<0.001) at end-R, but most interestingly, also Na+(i) image intensity at end-LF was well correlated with end-diastolic pressure (R=0.78, P<0.01) and with recovery of the rate-pressure product (R=-0.81, P<0.01) at end-R. Furthermore, Na+(i) image intensity at end-LF was well correlated with creatine kinase release during R (R=0.79, P<0.05) as well as with infarct size (R=0.77, P<0.05). CONCLUSIONS: These data indicate that 23Na CSI is a promising tool for the assessment of myocardial viability.