Marian Kampschulte

Vasa vasorum and atherosclerosis – Quid novi?

The role of vasa vasorum (VV) in atherosclerosis is hotly debated, and new experimental techniques have recently opened an opportunity to take a fresh look at this important topic. Although the proliferation of VV due to atherogenic stimuli is controversial, experimental and clinical evidence strongly suggest the potential of VV in vascular proliferative disorders. In the past, paradigms of atherosclerosis and restenosis have excluded the adventitia and VV in the artery wall due, in part, to a lack of i) appropriate animal models featuring adventitial VV neovascularization, ii) imaging technologies to quantitate adventitial VV and plaque neovascularization and iii) its consequences, concerning information on detectable plaque substrate in vulnerable lesions. VV proliferation is associated with increasing plaque burden and is linked to cellular processes which are critical during the development of atherosclerotic plaques such as inflammation, plaque perfusion and concomitant intraplaque hemorrhage - but the regulation and induction of VV based on pathological settings are poorly understood. This review discusses the current scientific status and its controversies and identifies open research questions.

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.

Macrophage and Cancer Cell Cross-talk via CCR2 and CX3CR1 Is a Fundamental Mechanism Driving Lung Cancer

RATIONALE Recent studies indicate that tumor-associated macrophages (MΦ) with an M2 phenotype can influence cancer progression and metastasis, but the regulatory pathways remain poorly characterized. OBJECTIVES This study investigated the role of tumor-associated MΦ in lung cancer. METHODS Coculturing of MΦ with mouse Lewis lung carcinoma (LLC1) and 10 different human lung cancer cell lines (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) caused up-regulation of CCR2/CCL2 and CX3CR1/CX3CL1 in both the cancer cells and the MΦ. MEASUREMENTS AND MAIN RESULTS In the MΦ-tumor cell system, IL-10 drove CCR2 and CX3CR1 up-regulation, whereas CCL1, granulocyte colony-stimulating factor, and MIP1α were required for the up-regulation of CCL2 and CX3CL1. Downstream phenotypic effects included enhanced LLC1 proliferation and migration and MΦ M2 polarization. In vivo, MΦ depletion (clodronate, MΦ Fas-induced apoptosis mice) and genetic ablation of CCR2 and CX3CR1 all inhibited LLC1 tumor growth and metastasis, shifted tumor-associated MΦ toward M1 polarization, suppressed tumor vessel growth, and enhanced survival (metastasis model). Furthermore, mice treated with CCR2 antagonist mimicked genetic ablation of CCR2, showing reduced tumor growth and metastasis. In human lung cancer samples, tumor MΦ infiltration and CCR2 expression correlated with tumor stage and metastasis. CONCLUSIONS Tumor-associated MΦ play a central role in lung cancer growth and metastasis, with bidirectional cross-talk between MΦ and cancer cells via CCR2 and CX3CR1 signaling as a central underlying mechanism. These findings suggest that the therapeutic strategy of blocking CCR2 and CX3CR1 may prove beneficial for halting lung cancer progression.

Effects of Multi-Deficiencies-Diet on Bone Parameters of Peripheral Bone in Ovariectomized Mature Rat

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.

Differences of bone healing in metaphyseal defect fractures between osteoporotic and physiological bone in rats

Discrepancies in bone healing between osteoporotic and non-osteoporotic bone remain uncertain. The focus of the current work is to evaluate potential healing discrepancies in a metaphyseal defect model in rat femora. Female Sprague-Dawley rats were either ovariectomized (OVX, n=14) and combined with a calcium-, phosphorus- and vitamin D3-, soy- and phytoestrogen-free diet or received SHAM operation with standard diet rat (SHAM, n=14). Three months post-ovariectomy, DEXA measurement showed a reduction of bone mineral density reflecting an osteoporotic bone status in OVX rats. Rats then underwent a 3 mm wedge-shaped osteotomy at the distal metaphyseal area of the left femur stabilized with a T-shaped mini-plate and allowed to heal for 6 weeks. Biomechanical competence by means of a non-destructive three-point bending test showed significant lower flexural rigidity in the OVX rats at 3 mm lever span compared to SHAM animals (p=0.048) but no differences at 10 mm lever span. Microcomputer tomography (μCT) showed bridging cortices and consolidation of the defect in both groups, however, no measurable differences were found in either total ossified tissue or vascular volume fraction. Furthermore, histology showed healing discrepancies that were characterized by cartilaginous remnant and more unmineralized tissue presence in the OVX rats compared to more mature consolidation appearance in the SHAM group. In summary, bone defect healing in metaphyseal bone slightly differs between osteoporotic and non-osteoporotic bone in the current 3 mm defect model in both 3mm lever span biomechanical testing and histology.

Assessment of angiogenesis in osseointegration of a silica–collagen biomaterial using 3D-nano-CT

Bony integration of biomaterials is a complex process in which angiogenesis plays a crucial role. We evaluated micro- and nano-CT imaging to demonstrate and quantify neovascularization in bony integration of a biomaterial and to give an image based estimation for the needed resolution for imaging angiogenesis in an animal model of femora defect healing. In 8 rats 5mm full-size defects were created at the left femur that was filled with silica-collagen bone substitute material and internally fixed with plate osteosynthesis. After 6 weeks the femora were infused in situ with Microfil, harvested and scanned for micro-CT (9 μm)(3) and nano-CT (3 μm)(3) imaging. Using those 3D images, the newly formed blood vessels in the area of the biomaterial were assessed and the total vascular volume fraction, the volume of the bone substitute material and the volume of the bone defect were quantitatively characterized. Results were complemented by histology. Differences were statistically assessed using (ANOVA). High-resolution nano-CT demonstrated new blood vessel formation surrounding the biomaterial in all animals at capillary level. Immunohistochemistry confirmed the newly formed blood vessels surrounding the bone substitute material. The mean vascular volume fraction (VVF) around the implant was calculated to be 3.01 ± 0.4%. The VVF was inversely correlated with the volume of the bone substitute material (r=0.8) but not with the dimension of the fracture zone (r=0.3). Nano-CT imaging is feasible for quantitative analysis of angiogenesis during bony integration of biomaterials and a promising tool in this context for the future.

3-Deazaadenosine inhibits vasa vasorum neovascularization in aortas of ApoE(-/-)/LDL(-/-) double knockout mice.

BACKGROUND Atherosclerosis and inflammation/angiogenesis are strongly associated including growth of vasa vasorum (VV) and plaque neovascularization, but a causative role for neovascularization has still not been established. Hence, we investigated the effect of 3-deazaadenosine (c(3)Ado), an anti-inflammatory and anti-proliferative drug, on plaque progression and VV neovascularization in apoE(-/-)/LDL(-/-) double knockout mice. METHODS The arterial trees from apoE(-/-)/LDL(-/-) mice with, or without c(3)Ado at the age of 16 weeks (n=10), 18 weeks (n=8) and 20 weeks (n=7) were infused in situ with Microfil, and the aortas harvested and scanned with micro-CT (12mum cubic voxel). We characterized plaque volume and VV luminal volume along the descending aorta using Analyze 6.0 software. Cellular effects of c(3)Ado on human endothelial and vascular smooth muscle cells were investigated in cell cultures and on nylon cDNA expression arrays. RESULTS Lesions spatially connected to VV increased from 16 to 20 weeks significantly (p<0.001). The volume of atherosclerotic lesions was significantly reduced in animals treated with c(3)Ado (p<0.01). This was accompanied by a significant decrease of vasa vasorum neovascularization along the descending aorta (p<0.01). Using nylon cDNA expression arrays, we identified the regulation of anti-proliferative, anti-inflammatory genes in human smooth muscle cells which might be involved in the anti-angiogenic effects of c(3)Ado. Moreover, c(3)Ado dose-dependently prevented the proliferation and migration of human coronary artery endothelial cells in vitro. CONCLUSION The smaller lesion volume in animals treated with c(3)Ado was closely associated with a reduced VV neovascularization, suggesting a direct relationship between lesion growth and VV development.

Implications of combined ovariectomy and glucocorticoid (dexamethasone) treatment on mineral, microarchitectural, biomechanical and matrix properties of rat bone

Osteoporosis is one of the deleterious side effects of long‐term glucocorticoid therapy. Since the condition is particularly aggressive in postmenopausal women who are on steroid therapy, in this study we have attempted to analyse the combined effect of glucocorticoid (dexamethasone) treatment and cessation of oestrogen on rat bone. The dual aim was to generate osteoporotic bone status in a short time scale and to characterise the combination of glucocorticoid–postmenopausal osteoporotic conditions. Sprague Dawley rats (N = 42) were grouped randomly into three groups: untreated control, sham‐operated and ovariectomized–steroid (OVX‐Steroid) rats. Control animals were euthanized with no treatment [Month 0 (M0)], while sham and OVX‐Steroid rats were monitored up to 1 month (M1) and 3 months (M3) post laparotomy/post OVX‐Steroid treatment. Histology, dual‐energy X‐ray absorptiometry (DXA), micro‐computed tomography (micro‐CT), and biomechanical and mRNA expression analysis of collagenous, non‐collagenous matrix proteins and osteoclast markers were examined. The study indicated enhanced osteoclastogenesis and significantly lower bone mineral density (BMD) in the OVX‐Steroid rats with Z‐scores below −2.5, reduced torsional strength, reduced bone volume (BV/TV%), significantly enhanced trabecular separation (Tb.S), and less trabecular number (Tb.N) compared with sham rats. Osteoclast markers, cathepsin K and MMP 9 were upregulated along with Col1α1 and biglycan with no significant expression variation in fibronectin, MMP 14, LRP‐5, Car II and TNC. These results show higher bone turnover with enhanced bone resorption accompanied with reduced torsional strength in OVX‐Steroid rats; and these changes were attained within a short timeframe. This could be a useful model which mimics human postmenopausal osteoporosis that is associated with steroid therapy and could prove of value both in disease diagnosis and for testing generating and testing biological agents which could be used in treatment.

Role of Computed Tomography Voxel Size in Detection and Discrimination of Calcium and Iron Deposits in Atherosclerotic Human Coronary Artery Specimens

Objective: This study evaluated the influence of voxel size on its ability to discriminate calcium from iron deposits in ex vivo coronary arteries. Methods: Postmortem human coronary arteries underwent multislice computed tomographic scan at (600-&mgr;m)3 voxel size to provide an index of computed tomography (CT) image noise and synchrotron-based micro-CT at (4-&mgr;m)3 voxel size to provide data for generating a range of voxel sizes 4 to (600-&mgr;m)3 after grayscale noise was added to the projection images before reconstruction so as to mimic the effect of retaining the same radiation exposure involved in the multislice computed tomographic scan. Results: At voxel sizes of (20-&mgr;m)3 or smaller, iron deposits could be identified based on CT grayscale value. Voxels of (100-&mgr;m)3 or larger cannot resolve nor distinguish iron deposits from calcifications by virtue of CT grayscale value. Conclusions: Clinical CT scanners cannot be expected to discriminate iron deposits from calcifications by their CT value alone in the arterial wall.

Quantitative analyses of bone composition in acetylcholine receptor M3R and alpha7 knockout mice

AIMS Increasing collagen synthesis was observed in lung after stimulation of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) on fibroblasts. Since collagen synthesis is an important process during fracture healing and bone remodelling, we asked whether cholinergic receptors are involved in bone collagen production. MAIN METHODS In the present study we analysed 16 week old male knockout mice for nAChRα7 (α7-KO) and mAChR M3R (M3R-KO) in correlation to their corresponding wild types (WT). Microarchitecture of right femora, vertebrae Th13 and L1 were analysed by 3D Micro-CT, left femora by a three-point bending test and humeri by real-time RT-PCR. KEY FINDINGS A significant decrease in relative bone volume, trabecular thickness, trabecular number, bone surface density, and a significant increase in trabecular separation and structure model index were measured for the M3R-KO using Micro-CT analysis. Bending stiffness of M3R-KO was significantly reduced in comparison to WT as well as the collagen 1α1 and 1α2 mRNA expression was down-regulated. No changes were detected for α7-KO using Micro-CT, biomechanical testing, and collagen mRNA expression. SIGNIFICANCE Our results indicate that nAChRα7 are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis. Since the M3R-KO exhibit bone structures similar to systemically diseased bone it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities.