Distinct influence of the anthracycline derivative doxorubicin on the differentiation efficacy of mESC-derived endothelial progenitor cells.

Cardiotoxicity is a highly relevant, because often life-threatening, adverse effect of doxorubicin (Doxo)-based anticancer therapy. Here, we investigated the Doxo-response of cardiovascular stem/progenitor cells employing a mouse embryonic stem cell (mESC)-based in vitro differentiation model. Endothelial progenitor cells revealed a pronounced Doxo sensitivity as compared to mESC, differentiated endothelial-like (EC) and cardiomyocyte-like cells (CM) and CM progenitors, which rests on the activation of senescence. Doxo treatment of EC progenitors altered protein expression of individual endothelial markers, actin cytoskeleton morphology, mRNA expression of genes related to mitochondrial functions, autophagy, apoptosis, and DNA repair as well as mitochondrial DNA content, respiration and ATP production in the surviving differentiated EC progeny. By contrast, LDL uptake, ATP-stimulated Ca2+ release, and cytokine-stimulated ICAM-1 expression remained unaffected by the anthracycline treatment. Thus, exposure of EC progenitors to Doxo elicits isolated and persistent dysfunctions in the surviving EC progeny. In conclusion, we suggest that Doxo-induced injury of EC progenitors adds to anthracycline-induced cardiotoxicity, making this cell-type a preferential target for pharmacoprotective and regenerative strategies.

Intrathecal B-cell activation in LGI1 antibody encephalitis.

OBJECTIVE: To study intrathecal B-cell activity in leucine-rich, glioma-inactivated 1 (LGI1) antibody encephalitis. In patients with LGI1 antibodies, the lack of CSF lymphocytosis or oligoclonal bands and serum-predominant LGI1 antibodies suggests a peripherally initiated immune response. However, it is unknown whether B cells within the CNS contribute to the ongoing pathogenesis of LGI1 antibody encephalitis. METHODS: Paired CSF and peripheral blood (PB) mononuclear cells were collected from 6 patients with LGI1 antibody encephalitis and 2 patients with other neurologic diseases. Deep B-cell immune repertoire sequencing was performed on immunoglobulin heavy chain transcripts from CSF B cells and sorted PB B-cell subsets. In addition, LGI1 antibody levels were determined in CSF and PB. RESULTS: Serum LGI1 antibody titers were on average 127-fold higher than CSF LGI1 antibody titers. Yet, deep B-cell repertoire analysis demonstrated a restricted CSF repertoire with frequent extensive clusters of clonally related B cells connected to mature PB B cells. These clusters showed intensive mutational activity of CSF B cells, providing strong evidence for an independent CNS-based antigen-driven response in patients with LGI1 antibody encephalitis but not in controls. CONCLUSIONS: Our results demonstrate that intrathecal immunoglobulin repertoire expansion is a feature of LGI1 antibody encephalitis and suggests a need for CNS-penetrant therapies.

Precise AuxPt1-x Alloy Nanoparticle Array of Tunable Composition for Catalytic Applications.

A 3-dimensional Block Copolymer Micellar nanoLithography (BCML) process was used to prepare AuxPt1-x alloy nanoparticles (NPs) monodisperse in size and composition, strongly anchored onto SiO2-particles (0.2 wt.% AuxPt1-x/SiO2). The particles possess a face-centered cubic (fcc) crystal structure and their size could be varied from 3-12 nm. We demonstrate the uniformity of the Au/Pt composition by analyzing individual NPs by energy-dispersive X-ray spectroscopy. The strongly bound AuxPt1-x NPs catalyzed the oxidation of CO with high activity. Thermal ageing experiments in pure CO2 as well as in ambient atmosphere demonstrated stability of the size distribution for times as long as 22 h.

The myelin oligodendrocyte glycoprotein directly binds nerve growth factor to modulate central axon circuitry.

Myelin oligodendrocyte glycoprotein (MOG) is a central nervous system myelin-specific molecule expressed on the outer lamellae of myelin. To date, the exact function of MOG has remained unknown, with MOG knockout mice displaying normal myelin ultrastructure and no apparent specific phenotype. In this paper, we identify nerve growth factor (NGF) as a binding partner for MOG and demonstrate that this interaction is capable of sequestering NGF from TrkA-expressing neurons to modulate axon growth and survival. Deletion of MOG results in aberrant sprouting of nociceptive neurons in the spinal cord. Binding of NGF to MOG may offer widespread implications into mechanisms that underlie pain pathways.

Rituximab efficiently depletes increased CD20-expressing T cells in multiple sclerosis patients.

In multiple sclerosis (MS), B cell-depleting therapy using monoclonal anti-CD20 Abs, including rituximab (RTX) and ocrelizumab, effectively reduces disease activity. Based on indirect evidence, it is generally believed that elimination of the Ag-presenting capabilities and Ag nonspecific immune functions of B cells underlie the therapeutic efficacy. However, a small subset of T lymphocytes (T cells) was shown to also express CD20, but controversy prevails surrounding the true existence of this T cell subpopulation. Using single-cell imaging flow cytometry and expression profiling of sorted lymphocyte subsets, we unequivocally demonstrate the existence of CD3(+)CD20(dim) T cells. We show that in MS patients, increased levels of CD3(+)CD20(dim) T cells are effectively depleted by RTX. The pathological relevance of this T cell subset in MS remains to be determined. However, given their potential proinflammatory functionality, depletion of CD20-expressing T cells may also contribute to the therapeutic effect of RTX and other mAbs targeting CD20.

Treatment of pulmonary arterial hypertension with circulating angiogenic cells.

Despite advances in the treatment of pulmonary arterial hypertension, a truly restorative therapy has not been achieved. Attention has been given to circulating angiogenic cells (CACs, also termed early endothelial progenitor cells) because of their ability to home to sites of vascular injury and regenerate blood vessels. We studied the efficacy of human CAC therapy in the treatment of pulmonary arterial hypertension at two different stages of disease severity. Cells were isolated from peripheral blood and administered to nude rats on day 14 ("early") or day 21 ("late") after monocrotaline injection. The control group received monocrotaline but no cell treatment. Disease progression was assessed using right heart catheterization and echocardiography at multiple time points. Survival differences, right ventricular hypertrophy (RVH), and vascular hypertrophy were analyzed at the study endpoint. Quantitative PCR was performed to evaluate cell engraftment. Treatment with human CACs either at the early or late time points did not result in increased survival, and therapy did not prevent or reduce the severity of disease compared with control. Histological analysis of RVH and vascular muscularization showed no benefit with therapy compared with control. No detectable signal was seen of human transcript in transplanted lungs at 14 or 21 days after cell transplant. In conclusion, CAC therapy was not associated with increased survival and did not result in either clinical or histological benefits. Future studies should be geared toward either earlier therapeutic time points with varying doses of unmodified CACs or genetically modified cells as a means of delivery of factors to the pulmonary arterial circulation.

Nitric oxide synthase expression and functional response to nitric oxide are both important modulators of circulating angiogenic cell response to angiogenic stimuli.

OBJECTIVE: Circulating angiogenic cells (CACs), also termed endothelial progenitor cells, play an integral role in vascular repair and are functionally impaired in coronary artery disease (CAD). The role of nitric oxide (NO) in CAC function is poorly understood. We hypothesized that CAC migration toward angiogenic signals is modulated by both NO synthase (NOS) expression and functional response to NO. METHODS AND RESULTS: Similar to endothelial cells, CAC chemotaxis to vascular endothelial growth factor (VEGF) was blocked by inhibition of NOS, phosphatidylinositol 3-kinase, or guanylyl cyclase or by treatment with an NO scavenger. Addition of an NO donor (S-nitroso-N-acetylpenicillamine) and the NOS substrate l-arginine increased random cell migration (chemokinesis) and enhanced VEGF-dependent chemotaxis. Healthy CACs expressed endothelial NOS, but endothelial NOS was not detected in CAD patient CACs. Both chemokinesis and chemotaxis to VEGF of patient CACs were decreased compared with healthy CACs but were restored to healthy values by S-nitroso-N-acetylpenicillamine. In parallel, CAD patients exhibited lower flow-mediated vasodilation and plasma NO source nitrite than young, healthy subjects, indicating endothelial dysfunction with reduced NO bioavailability. CONCLUSIONS: NOS activity is required for CAC chemotaxis. In CAD patients, impairment of NOS expression and NO bioavailability, rather than response to NO, may contribute to dysfunction of CACs and limit their regenerative capacity.

Improvement of endothelial function with dietary flavanols is associated with mobilization of circulating angiogenic cells in patients with coronary artery disease.

OBJECTIVES: In patients with coronary artery disease (CAD) medically managed according to currently accepted guidelines, we tested whether a 1-month dietary intervention with flavanol-containing cocoa leads to an improvement of endothelial dysfunction and whether this is associated with an enhanced number and function of circulating angiogenic cells (CACs). BACKGROUND: Dietary flavanols can improve endothelial dysfunction. The CACs, also termed endothelial progenitor cells, are critical for vascular repair and maintenance of endothelial function. METHODS: In a randomized, controlled, double-masked, cross-over trial, 16 CAD patients (64+/-3 years of age) received a dietary high-flavanol intervention (HiFI [375 mg]) and a macronutrient- and micronutrient-matched low-flavanol intervention (LoFI [9 mg]) twice daily in random order over 30 days. RESULTS: Endothelium-dependent vasomotor function, as measured by flow-mediated vasodilation of the brachial artery, improved by 47% in the HiFI period compared with the LoFI period. After HiFI, the number of CD34+/KDR+-CACs, as measured by flow cytometry, increased 2.2-fold as compared with after LoFI. The CAC functions, as measured by the capacity to survive, differentiate, proliferate, and to migrate were not different between the groups. The HiFI led to a decrease in systolic blood pressure (mean change over LoFI: -4.2+/-2.7 mm Hg), and increase in plasma nitrite level (mean change over LoFI: 74+/-32 nM). Applying a mixed-effects linear regression model, the results demonstrated a significant increase in flow-mediated vasodilation and a decrease in systolic blood pressure with increasing levels of CD34+/KDR+-CACs. CONCLUSIONS: Sustained improvements in endothelial dysfunction by regular dietary intake of flavanols are associated with mobilization of functional CACs. (Effect of Cocoa Flavanols on Vascular Function in Optimally Treated Coronary Artery Disease Patients: Interaction Between Endothelial Progenitor Cells, Reactivity of Micro- and Macrocirculation; NCT00553774).

Cytokine combination therapy with long-acting erythropoietin and granulocyte colony stimulating factor improves cardiac function but is not superior than monotherapy in a mouse model of acute myocardial infarction.

BACKGROUND: Erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF) are potential novel therapies after myocardial infarction (MI). We first established the optimal and clinically applicable dosages of these drugs in mobilizing hematopoietic stem cells (HSC), and then tested the efficacy of monotherapy and combination therapy post-MI. METHODS AND RESULTS: Optimal doses were established in enhanced green fluorescent protein (eGFP) + chimeric mice (n = 30). Next, mice underwent MI and randomized into 4 groups (n = 18/group): 1) GCSF; 2) EPO; 3) EPO+GCSF; and 4) control. Left ventricular (LV) function was analyzed pre-MI, at 4 hours and at 28 days post-MI. Histological assessment of infarct size, blood vessels, apoptotic cardiomyocytes, and engraftment of eGFP+ mobilized cells were analyzed at day 28. LV function in the control group continued to deteriorate, whereas all treatments showed stabilization. The treatment groups resulted in less scarring, increased numbers of mobilized cells to the infarct border zone (BZ), and a reduction in the number of apoptotic cardiomyocytes. Both EPO groups had significantly more capillaries and arterioles at the BZ. CONCLUSION: We have established the optimal doses for EPO and GCSF in mobilizing HSC from the bone marrow and demonstrated that therapy with these agents, either as monotherapy or combination therapy, led to improvement of cardiac function post-MI. Combination therapy does not seem to have additive benefit over monotherapy in this model.

A comparison of echocardiography to invasive measurement in the evaluation of pulmonary arterial hypertension in a rat model.

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by progressive elevation in pulmonary artery pressure (PAP) and total pulmonary vascular resistance (TPVR). Recent advances in imaging techniques have allowed the development of new echocardiographic parameters to evaluate disease progression. However, there are no reports comparing the diagnostic performance of these non-invasive parameters to each other and to invasive measurements. Therefore, we investigated the diagnostic yield of echocardiographically derived TPVR and Doppler parameters of PAP in screening and measuring the severity of PAH in a rat model. Serial echocardiographic and invasive measurements were performed at baseline, 21 and 35 days after monocrotaline-induction of PAH. The most challenging echocardiographic derived TPVR measurement had good correlation with the invasive measurement (r = 0.92, P < 0.001) but also more simple and novel parameters of TPVR were found to be useful although the non-invasive TPVR measurement was feasible in only 29% of the studies due to lack of sufficient tricuspid valve regurgitation. However, echocardiographic measures of PAP, pulmonary artery flow acceleration time (PAAT) and deceleration (PAD), were measurable in all animals, and correlated with invasive PAP (r = -0.74 and r = 0.75, P < 0.001 for both). Right ventricular thickness and area correlated with invasive PAP (r = 0.59 and r = 0.64, P < 0.001 for both). Observer variability of the invasive and non-invasive parameters was low except in tissue-Doppler derived isovolumetric relaxation time. These non-invasive parameters may be used to replace invasive measurements in detecting successful disease induction and to complement invasive data in the evaluation of PAH severity in a rat model.

Brief secondhand smoke exposure depresses endothelial progenitor cells activity and endothelial function: sustained vascular injury and blunted nitric oxide production.

OBJECTIVES: This study sought to analyze the effects of acute secondhand smoke (SHS) exposure on the number and function of endothelial progenitor cells (EPCs) over 24 h. BACKGROUND: Secondhand smoke increases the risk of vascular disease and is a major public health concern, but the mechanism(s) of action are not fully understood. METHODS: Healthy nonsmokers (age SEM 30.3 +/- 1.3 years, n = 10) were exposed to 30 min of SHS yielding cotinine levels commonly observed in passive smokers and to smokefree air on 2 separate days. Measurements were taken before exposure (baseline), immediately after (0 h), and at 1 h, 2.5 h, and 24 h after. The EPCs (CD133(+)/KDR(+), CD34(+)/KDR(+)) and endothelial microparticles (EMPs: CD31(+)/CD41(-), CD144(+), CD62e(+)) were determined in blood using flow cytometry. The EPC chemotaxis toward vascular endothelial growth factor was measured. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound. RESULTS: Secondhand smoke exposure increased EPCs and plasma vascular endothelial growth factor and completely abolished EPC chemotaxis during 24 h after exposure. Secondhand smoke increased EMPs and decreased FMD. Although FMD returned to baseline at 2.5 h, EMPs and vascular endothelial growth factor levels remained elevated at 24 h, suggesting endothelial activation and injury with functional impairment of the vascular endothelium. Exposure to smokefree air had no effect. Incubation of EPCs from nonexposed subjects with plasma isolated from SHS-exposed subjects in vitro decreased chemotaxis by blockade of vascular endothelial growth factor-stimulated nitric oxide production. CONCLUSIONS: Brief exposure to real-world levels of SHS leads to sustained vascular injury characterized by mobilization of dysfunctional EPCs with blocked nitric oxide production. Our results suggest that SHS not only affects the vascular endothelium, but also the function of EPCs.