Vascular remodeling in mice lacking the cytoplasmic domain of tissue factor

Tissue factor (TF), the cell surface receptor for the serine protease FVIIa supports cell migration by interaction with the cytoskeleton. Intracellular signaling pathways dependent on the cytoplasmic domain of TF modify cell migration and may alter vascular remodeling. Vascular remodeling was analyzed in a femoral artery injury and a blood flow cessation model in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids of TF (TF(Deltact/Deltact)) and compared with TF wild-type mice (TF(wt/wt)). Morphometric analysis revealed a decrease in the intima/media ratio after vascular injury in arteries from TF(Deltact/Deltact) compared with TF(wt/wt) mice (femoral artery injury: 2.4+/-0.3 TF(wt/wt) versus 0.6+/-0.3 TF(Deltact/Deltact), n=9 to 10, P=0.002; carotis ligation: 0.45+0.11 TF(wt/wt) versus 0.22+0.03 TF(Deltact/Deltact), n=12 to 14, P=0.09). This was caused by an increase in the media by 54% (P=0.04) in the femoral artery model and by 32% (P=0.03) after carotis ligation and was associated with an increased number of proliferating cells. Isolated aortic smooth muscle cells (SMCs) of TF(wt/wt) mice showed an increased migratory response toward the TF ligand active site-inhibited FVIIa that was abolished in TF(Deltact/Deltact) SMC. In contrast, the unstimulated proliferation rate was increased in TF(Deltact/Deltact) SMC compared with TF(wt/wt) SMCs. Thus, retention of SMCs attributable to a migratory defect and increased proliferation results in thickening of the media and in decrease in neointima formation after arterial injury. TF cytoplasmic domain signaling alters vascular remodeling and, thereby, may play a role in the development of restenosis, atherosclerotic disease, and neovascularization.     

Critical role for p47phox in renin-angiotensin system activation and blood pressure regulation

OBJECTIVE: Renin-angiotensin system (RAS) activation leads to increased production of NAD(P)H oxidase-derived reactive oxygen species (ROS), and both have been implicated in the initiation and progression of arterial hypertension, atherosclerosis, and cardiac hypertrophy. The cytosolic subunit p47phox is critically involved in agonist-induced NAD(P)H oxidase activation. Here, we investigated the role of p47phox in blood pressure control, endothelium-dependent relaxation, cardiac hypertrophy, RAS activation, and renal oxidative stress under resting conditions. METHODS AND RESULTS: Mice deficient in p47phox (on C57BL/6 background) developed significantly higher systolic blood pressure levels compared to C57BL/6 wild-type animals (136.0+/-3.0 mmHg vs. 112.2+/-2.6, P<0.01, n=16) as measured by the tail cuff method from week 6 up to week 12 post partum. The increase in blood pressure in p47phox-/- mice was associated with an impaired endothelium-dependent relaxation (P<0.005 vs. wild-type, n=11). At the age of 12 weeks p47phox-/- mice showed increased plasma renin activity as analyzed by radioimmunoassay (14.5+/-1.8 ng/mL/h vs. 9.6+/-1.7 ng/mL/h, P<0.05, n=10) and enhanced angiotensin converting enzyme (ACE) activity in the kidney and aorta as measured by Hip-His-Leu cleavage (7.6+/-0.8 vs. 4.8+/-0.9 nmol/L His-Leu/mg protein, P<0.05, n=5) compared to wild-type mice. No differences in oxygen radical formation was determined in kidney samples by lucigenin- and luminol-enhanced chemiluminescence or by electron spin resonance spectroscopy. Consistently, treatment with the radical scavenger tempol did not lower blood pressure in p47phox-/- mice, whereas ACE and angiotensin II type I receptor inhibition normalized blood pressure. CONCLUSION: Deficiency of the NAD(P)H oxidase subunit p47phox leads to RAS activation, which subsequently contributes to blood pressure increase in a ROS-independent manner.     

Elevated IgM anti-IgE in sera from allergic patients.

The role of anti-IgE autoantibodies in IgE-related allergic diseases has not been elucidated sufficiently. For example, anti-IgE antibodies have been reported to cause both proallergic and antiallergic blocking reactions. Contrary to other authors, some authors revealed a positive correlation between total IgE and the amount of IgE/IgG complexes detected. By comparing the IgE levels of allergic patients with those of control persons and rheumatoid arthritis patients the present study contributes to our understanding of the role of anti-IgE autoantibodies. The sera were tested by means of ELISA concerning their content of free and complexed IgG and IgM anti-IgE. In addition, the amounts of IgE/IgG and IgE/IgM complexes were determined in the sera of allergic and control persons after Superose 6 column separation. We found that the allergic patients revealed significantly higher values of free IgM anti-IgE than patients with rheumatoid arthritis and than control persons (mean 0.470, p = 0.0164 and p = 0.0061, respectively). The corresponding values for IgE/IgM complexes also tend to be higher (mean 0.431, p = 0.0784 and p = 0.0601, respectively). However, the corresponding contents of IgG anti-IgE autoantibodies and IgE/IgG complexes did not differ significantly. After Superose 6 column separation, we detected IgE/IgG in fractions corresponding to MW of 150 to 180 kDa for the sera of both allergic and control persons. In contrast, the IgE/IgM complexes were found in fractions corresponding to MW 330 kDa. We conclude that the increased IgM anti-IgE autoantibody titer in the sera of allergic patients is not correlated with the high total IgE level. Moreover, we suggest that the IgE/IgG complexes form de nova during ELISA. Unlike the IgE/IgG complexes, the IgE/IgM complexes are assumed to occur already in circulating blood.     

Comparison of glucose and lipid metabolism and bone mineralization in patients with growth hormone deficiency with and without long-term growth hormone replacement

The effects of long-term growth hormone (GH) substitution in pituitary-insufficient patients with GH deficiency (GHD-pats) on glucose and lipid metabolism and bone mineral density (BMD) have yet to be ascertained. We performed this cross-sectional study comparing GHD-pats with and without long-term GH substitution. We measured lipid parameters at baseline and glucose and insulin concentrations for 3 hours during oral glucose tolerance test in 52 GHD-pats (21 female and 31 male; median age, 51.5 years [27-82]). Twenty-two GHD-pats were on constant GH substitution (GH-Subs) for a median of 10 years (2-42 years). Thirty GHD-pats had not been substituted for at least 2 years (non-Subs). For analyses of β-cell function, insulin resistance (IR), and sensitivity, homeostatic model assessment (HOMA)-β , HOMA-IR, and insulin sensitivity index were used, respectively. Body composition and BMD were measured by dual-energy x-ray absorptiometry. Age and body mass index did not differ significantly between groups. Fasting glucose was significantly lower for GH-Subs than non-Subs (87 mg/dL [71-103] vs non-Subs 89 mg/dL [71-113], P < .05), whereas basal insulin did not differ significantly (10 μU/mL (4-42) vs non-Subs 10μU/mL [4-63]). Glucose and insulin levels at 120 minutes as well as patients' area under the curve, C-peptide, hemoglobin A_1c, waist-hip ratio, HOMA-β, HOMA-IR, insulin sensitivity index, lipid parameters, and BMD did not differ significantly; but total fat mass was significantly higher in non-Subs (37% [20%-52%] vs GH-sub 31% [13%-54%], P < .01). More non-Subs had abnormal glucose tolerance (19 [63%] vs GH-Subs 9 [41%]). Long-term GH substitution trends to beneficially influence fasting glucose and glucose tolerance, although differences are sparse. Growth hormone substitution alone does not seem to significantly impact on insulin sensitivity, lipid metabolism, and BMD in patients with pituitary insufficiency.     

l-Carnitine l-tartrate supplementation favorably affects biochemical markers of recovery from physical exertion in middle-aged men and women

The purpose of this study was to examine the effects of Carnipure tartrate (Lonza, Allendale, NJ) supplementation (total dose of 2 g/d of l-carnitine) on markers of performance and recovery from physical exertion in middle-aged men and women. Normally active and healthy men (n = 9, 45.4 ± 5.3 years old) and women (n = 9, 51.9 ± 5.0 years old) volunteered to participate in the investigation. Double-blind, placebo, balanced treatment presentation and crossover design were used with 3 weeks and 3 days of supplementation followed by a 1-week washout period before the other counterbalanced treatment was initiated. After 3 weeks of each supplementation protocol, each participant then performed an acute resistance exercise challenge of 4 sets of 15 repetitions of squat/leg press at 50% 1-repetition maximum and continued supplementation over the recovery period that was evaluated. Blood samples were obtained at preexercise and at 0, 15, 30, and 120 minutes postexercise during the acute resistance exercise challenge and during 4 recovery days as well. Two grams of l-carnitine supplementation had positive effects and significantly (P ≤ .05) attenuated biochemical markers of purine metabolism (ie, hypoxanthine, xanthine oxidase), free radical formation (malondialdehyde), muscle tissue disruption (myoglobin, creatine kinase), and muscle soreness after physical exertion. However, markers of physical performance (ie, strength, power, get up and go) were not affected by supplementation. These findings support our previous findings of l-carnitine in younger people that such supplementation can reduce chemical damage to tissues after exercise and optimize the processes of muscle tissue repair and remodeling.     

HLA–B27–restricted antigen presentation by human chondrocytes to CD8+ T cells: Potential contribution to local immunopathologic processes in ankylosing spondylitis

Objective

Analysis of the histopathologic features of hip arthritis in patients with ankylosing spondylitis (AS) has revealed accumulation of infiltrating mononuclear cells in the bone end plate and presence of hyaline articular cartilage that is not found in areas of total cartilage destruction. This study was undertaken to assess whether chondrocytes attract lymphocytes and whether cartilage chondrocytes from patients with AS have the potential to directly stimulate T cells in an HLA‐restricted manner.

Methods

Human HLA–B27+ T cell lines, specific for the Epstein‐Barr virus–derived peptide EBNA_258–266, and autologous chondrocytes, serving as nonprofessional antigen‐presenting cells (APCs), were available for use in a model system to study chondrocyte functions in femoral head joint cartilage of patients with AS. Peptide functionality of cytotoxic T cells was assessed by flow cytometry, and cellular interactions were detected by fluorescence confocal microscopy.

Results

When maintained in an alginate matrix, chondrocytes isolated from the femoral heads of patients with AS constitutively expressed type II collagen and CD80. When pulsed with the EBNA_258–266 peptide, autologous chondrocytes functioned as APCs and, specifically, induced interferon‐γ production in CD8+ T cells. In mixed chondrocyte–T cell cultures, cell–cell contacts were dependent on the presence of the EBNA_258–266 peptide. T cells adjacent to chondrocytes produced perforin and granzyme B; both molecules were found in focal aggregates, a prerequisite for antigen‐specific lysis of target cells.

Conclusion

Antigen presentation through human chondrocytes allows the stimulation of peptide‐specific CD8+ T cells. These results indicate that human chondrocytes can act as nonprofessional APCs, and suggest that there is an interferon‐γ–triggered autocrine loop of immune cell–mediated chondrocyte activation in the already inflamed environment. Thus, local HLA‐dependent activation of peptide‐specific cytotoxic CD8+ T cells by chondrocytes might contribute to inflammatory processes in the spondylarthritides.

     

Serum concentrations of DKK‐1 decrease in patients with multiple myeloma responding to anti‐myeloma treatment

Lytic bone destruction is a hallmark of multiple myeloma (MM) and is because of an uncoupling of bone remodeling. Secretion of Dickkopf (DKK)‐1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. In this study, the effect of different treatment regimens for MM on serum DKK‐1 was evaluated and correlated with the response to treatment in 101 myeloma patients receiving bortezomib, thalidomide, lenalidomide, adriamycin and dexamethasone (AD) or high‐dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT). At baseline, myeloma patients had increased serum DKK‐1 as compared with patients with MGUS (mean 3786 pg/mL vs. 1993 pg/mL). There was no difference between previously untreated MM patients and patients at relapse. A significant decrease of DKK‐1 after therapy was seen in the following groups: Bortezomib (4059 pg/mL vs. 1862 pg/mL, P = 0.016), lenalidomide (11837 pg/mL vs. 4374 pg/mL, P = 0.039), AD (1668 pg/mL vs. 1241 pg/mL, P = 0.016), and AD + HDCT + ASCT (2446 pg/mL vs. 1082 pg/mL, P = 0.001). Thalidomide led to a non‐significant decrease in DKK‐1 (1705 pg/mL vs. 1269 pg/mL, P = 0.081). Within all groups, a significant decrease of DKK‐1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non‐responders. We show for the first time that serum DKK‐1 levels decrease in myeloma patients responding to treatment, irrespective of the regimen chosen. These data suggest that myeloma cells are the main source of circulating DKK‐1 protein and provide a framework for clinical trials on anti‐DKK‐1 treatment in MM.     

Persistent symptoms in patients with Crohn’s disease in remission: An exploratory study on the role of diet

Objectives: Patients with Crohn’s disease (CD) often report food hypersensitivities with gastrointestinal (GI) symptoms despite being in clinical remission. We aimed to identify the most frequent symptoms and dietary triggers in such patients, and also explored whether a strict elimination diet may reduce their GI symptoms.

Methods: We assessed GI symptoms and dietary triggers in 16 patients with CD in clinical remission. Of these, 12 patients subsequently participated in a dietary intervention trial: two weeks on a habitual diet including wheat and dairy products followed by two weeks of a strict elimination diet. The severity of seven symptoms (overall symptoms, abdominal pain, bloating, abnormal feces, wind, fatigue, and musculoskeletal pain) was measured by using visual analog scales throughout the four weeks intervention period.

Main results: The most common symptoms were abdominal pain, wind, bloating, odorous wind/feces, and diarrhea. Dairy and wheat products were reported as the most frequent dietary symptom triggers. All symptoms improved (p?Conclusion: Our exploratory study suggests that dietary interventions such as an elimination diet may reduce GI symptoms in patients with CD in remission.     

Precision Medicine for CRC Patients in the Veteran Population: State-of-the-Art,Challenges and Research Directions

Colorectal cancer (CRC) accounts for ~9% of all cancers in the Veteran population, a fact which has focused a great deal of the attention of the VA’s research and development efforts. A field-based meeting of CRC experts was convened to discuss both challenges and opportunities in precision medicine for CRC. This group, designated as the VA Colorectal Cancer Cell-genomics Consortium (VA4C), discussed advances in CRC biology, biomarkers, and imaging for early detection and prevention. There was also a discussion of precision treatment involving fluorescence-guided surgery, targeted chemotherapies and immunotherapies, and personalized cancer treatment approaches. The overarching goal was to identify modalities that might ultimately lead to personalized cancer diagnosis and treatment. This review summarizes the findings of this VA field-based meeting, in which much of the current knowledge on CRC prescreening and treatment was discussed. It was concluded that there is a need and an opportunity to identify new targets for both the prevention of CRC and the development of effective therapies for advanced disease. Also, developing methods integrating genomic testing with tumoroid-based clinical drug response might lead to more accurate diagnosis and prognostication and more effective personalized treatment of CRC.     

The proteasome inhibitor bortezomib stimulates osteoblastic differentiation of human osteoblast precursors via upregulation of vitamin D receptor signalling

Interactions of myeloma cells with the bone marrow microenvironment lead to enhanced osteoclast recruitment and impaired osteoblast activity. Recent evidence revealed that the proteasome inhibitor bortezomib stimulates osteoblast differentiation, but the mechanisms are not fully elucidated. We hypothesised that bortezomib could influence osteoblastic differentiation via alteration of vitamin D signalling by blocking the proteasomal degradation of the vitamin D receptor (VDR). This is of clinical importance, as a high rate of vitamin D deficiency was reported in patients with myeloma. We performed cocultures of primary human mesenchymal stem cells (hMSCs) and human osteoblasts (hOBs) with myeloma cells, which resulted in an inhibition of the vitamin D‐dependent differentiation of osteoblast precursors. Treatment with bortezomib led to a moderate increase in osteoblastic differentiation markers in hMSCs and hOBs. Importantly, this effect could be strikingly increased when vitamin D was added. Bortezomib led to enhanced nuclear VDR protein levels in hMSCs. Primary hMSCs transfected with a VDR luciferase reporter construct showed a strong increase in VDR signalling with bortezomib. In summary, stimulation of VDR signalling is a mechanism for the bortezomib‐induced stimulation of osteoblastic differentiation. The data suggest that supplementation of vitamin D in patients with myeloma treated with bortezomib is crucial for optimal bone formation.