Shear stress modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/l drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress downregulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

Differential gene and protein expression in abluminal sprouting and intraluminal splitting forms of angiogenesis

In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.

A brief update on lung stereology

Lung stereology has a long and successful tradition. From mice to men, the application of new stereological methods at several levels (alveoli, parenchymal cells, organelles, proteins) has led to new insights into normal lung architecture, parenchymal remodelling in emphysema-like pathology, alveolar type II cell hyperplasia and hypertrophy and intracellular surfactant alterations as well as distribution of surfactant proteins. The Euler number of the network of alveolar openings, estimated using physical disectors at the light microscopic level, is an unbiased and direct estimate of alveolar number. Surfactant-producing alveolar type II cells can be counted and sampled for local size estimation with physical disectors at a high magnification light microscopic level. The number of their surfactant storage organelles, lamellar bodies, can be estimated using physical disectors at the EM level. By immunoelectron microscopy, surfactant protein distribution can be analysed with the relative labelling index. Together with the well-established classical stereological methods, these design-based methods now allow for a complete quantitative phenotype analysis in lung development and disease, including the structural characterization of gene-manipulated mice, at the light and electron microscopic level.

Zoledronic acid efficacy and safety over five years in postmenopausal osteoporosis

In a 5-year study involving 119 postmenopausal women, zoledronic acid 4 mg given once-yearly for 2, 3 or 5 years was well tolerated with no evidence of excessive bone turnover reduction or any safety signals. BMD increased significantly. Bone turnover markers decreased from baseline and were maintained within premenopausal reference ranges. INTRODUCTION: After completion of the core study, two consecutive, 2-year, open-label extensions investigated the efficacy and safety of zoledronic acid 4 mg over 5 years in postmenopausal osteoporosis. METHODS: In the core study, patients received 1 to 4 mg zoledronic acid or placebo. In the first extension, most patients received 4 mg per year and then patients entered the second extension and received 4 mg per year or calcium only. Patients were divided into three subgroups according to years of active treatment received (2, 3 or 5 years). Changes in BMD and bone turnover markers (bone ALP and CTX-I) were assessed. RESULTS: All subgroups showed substantial increases in BMD and decreases in bone markers. By the end of the core study, 37.5% of patients revealed a suboptimal reduction (< 30%) of bone ALP levels. After subsequent study drug administration during the extensions, there was no evidence of progressive reduction of bone turnover markers. Furthermore, increased marker levels after treatment discontinuation demonstrates preservation of bone remodelling capacity. CONCLUSIONS: This study showed that zoledronic acid 4 mg once-yearly was well tolerated and effective in reducing biomarkers over 5 years. Detailed analysis of bone marker changes, however, suggests that this drug regimen causes insufficient reduction of remodelling activity in one third of patients.

Coronary plaque composition of culprit/target lesions according to the clinical presentation: a virtual histology intravascular ultrasound analysis

AIMS: To evaluate the plaque composition obtained by virtual histology (VH) IVUS according to the clinical presentation and to compare those data to previously published histopathology data. METHODS AND RESULTS: VH was performed on 95 de novo significant lesions (>75% stenosis) in 85 patients [28 acute coronary syndrome (ACS) patients, 30 lesions; 57 stable angina pectoris (SAP) patients, 65 lesions]. There were a higher prevalence of positive remodelling (47 vs. 22%, P=0.013), thrombus (20 vs. 1.5%, P=0.0037), and echo-lucent area (23.3 vs. 7.7%, P=0.047) in ACS patients. At the minimal lumen site, fibrous plaque area was significantly larger in ACS lesions than in SAP lesions (66.0+/-10.7 vs. 61.4+/-8.9%, P=0.034), whereas necrotic core and dense calcium plaque area were smaller in ACS lesions (Necrotic core: 6.8+/-6.0 vs. 11.0+/-8.3%, P=0.02; Dense calcium: 2.6+/-3.0 vs. 4.9+/-5.8%, P=0.03). No differences in rate of thin cap fibroatheroma, thick fibrotheroma, or for the presence of multiple necrotic core layers were observed between both groups. CONCLUSION: Plaque composition obtained by VH-IVUS shows less necrotic core and more fibrous tissue in ACS compared to SAP lesions, which is in contradiction with previously published histopathologic data.

Temporary scaffolding of coronary arteries with bioabsorbable magnesium stents: a prospective, non-randomised multicentre trial

BACKGROUND: Coronary stents improve immediate and late results of balloon angioplasty by tacking up dissections and preventing wall recoil. These goals are achieved within weeks after angioplasty, but with current technology stents permanently remain in the artery, with many limitations including the need for long-term antiplatelet treatment to avoid thrombosis. We report a prospective multicentre clinical trial of coronary implantations of absorbable magnesium stents. METHODS: We enrolled 63 patients (44 men; mean age 61.3 [SD 9.5 years]) in eight centres with single de novo lesions in a native coronary artery in a multicentre, non-randomised prospective study. Follow-up included coronary angiography and intravascular ultrasound at 4 months and clinical assessment at 6 months and 12 months. The primary endpoint was cardiac death, non-fatal myocardial infarction, or clinically driven target lesion revascularisation at 4 months FINDINGS: 71 stents, 10-15 mm in length and 3.0-3.5 mm in diameter, were successfully implanted after pre-dilatation in 63 patients. Diameter stenosis was reduced from 61.5 (SD 13.1%) to 12.6 (5.6%) with an acute gain of 1.41 mm (0.46 mm) and in-stent late loss of 1.08 mm (0.49 mm). The ischaemia-driven target lesion revascularisation rate was 23.8% after 4 months, and the overall target lesion revascularisation rate was 45% after 1 year. No myocardial infarction, subacute or late thrombosis, or death occurred. Angiography at 4 months showed an increased diameter stenosis of 48.4 (17.0%). After serial intravascular ultrasound examinations, only small remnants of the original struts were visible, well embedded into the intima. Neointimal growth and negative remodelling were the main operating mechanisms of restenosis. INTERPRETATION: This study shows that biodegradable magnesium stents can achieve an immediate angiographic result similar to the result of other metal stents and can be safely degraded after 4 months. Modifications of stent characteristics with prolonged degradation and drug elution are currently in development.

Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

BACKGROUND: Despite a large body of clinical and histological data demonstrating beneficial effects of enamel matrix proteins (EMPs) for regenerative periodontal therapy, it is less clear how the available biological data can explain the mechanisms underlying the supportive effects of EMPs. OBJECTIVE: To analyse all available biological data of EMPs at the cellular and molecular levels that are relevant in the context of periodontal wound healing and tissue formation. METHODS: A stringent systematic approach was applied using the key words "enamel matrix proteins" OR "enamel matrix derivative" OR "emdogain" OR "amelogenin". The literature search was performed separately for epithelial cells, gingival fibroblasts, periodontal ligament cells, cementoblasts, osteogenic/chondrogenic/bone marrow cells, wound healing, and bacteria. RESULTS: A total of 103 papers met the inclusion criteria. EMPs affect many different cell types. Overall, the available data show that EMPs have effects on: (1) cell attachment, spreading, and chemotaxis; (2) cell proliferation and survival; (3) expression of transcription factors; (4) expression of growth factors, cytokines, extracellular matrix constituents, and other macromolecules; and (5) expression of molecules involved in the regulation of bone remodelling. CONCLUSION: All together, the data analysis provides strong evidence for EMPs to support wound healing and new periodontal tissue formation.

Stability of the hard and soft tissue profile after mandibular advancement in sagittal split osteotomies: a longitudinal and long-term follow-up study

The aim of the study was to conduct a long-term follow-up investigation of the stability of hard and soft tissues after bilateral sagittal split osteotomy (BSSO) with rigid internal (RIF) fixation to advance the mandible. Sixteen consecutive patients (12 females and 4 males, mean age 21.4 years) were available for re-examination 12.7 years (T5) after surgery. The preceding follow-ups were before (T1), and 5 days (T2), 7.3 months (T3), and 13.9 months (T4) after surgery. Lateral cephalograms were traced by hand, digitized, and evaluated with the Dentofacial Planner program. The x-axis for the system of co-ordinates ran through sella (point zero) and the line NSL -7 degrees. Thus, the program determined the x- and y-values of each variable and the usual angles and distances. Statistical analysis was carried out using Wilcoxon's matched-pair signed-ranks test with Bonferroni adjustments. The relationships between the examined variables were analysed by Spearman rank correlation coefficients. The backward relapse at point B (T5) was 2.42 mm, or 50 per cent, and at pogonion 3.21 mm, or 60 per cent of the initial advancement. The mean net effect at T5 on the labial fold (soft tissue point B) was 94 per cent of the advancement at point B. For the soft tissue chin (soft tissue pogonion), it was 119 per cent of the advancement at pogonion. The net effect on the lower lip (labrale inferior) was 55 per cent of the advancement at incision inferior. The amount of the surgical advancement of the mandible was correlated with the long-term relapse in point B. Among possible reasons for this relapse are the initial soft tissue profile, the initial growth direction, and the remodelling processes of the hard tissue.

Stability of hard tissue profile after mandibular setback in sagittal split osteotomies: a longitudinal and long-term follow-up study

The aim of the study was to conduct a long-term follow-up on the stability of the hard tissues after bilateral sagittal split osteotomy (BSSO) with rigid internal fixation (RIF)to set back the mandible and to compare it with that of mandibular advancement performed by the same team of surgeons and with the same examination protocol. Seventeen consecutive patients (6 females and 11 males) could be re-examined 12.7 years (T5) after surgery. The previous examinations were before surgery (T1), 5 days (T2), and 6.6 (T3) and 14.4 (T4) months after surgery. Lateral cephalograms were traced by hand, digitized, and evaluated with the Dentofacial Planner software program. The x-axis for the system of co-ordinates ran through sella (point zero) and the line nasion-sella-line minus 7 degrees. The program determined the x- and y-values of each variable and the usual angles and distances. The effects of treatment were determined with Wilcoxon matched pairs, signed ranks test, with Bonferroni adjustment, and the relationship between variables with Spearman rank correlation coefficient. Relapse at point B was 0.94 mm or 15 per cent and at pogonion 1.46 mm or 21 per cent of the initial setback at T5. Relapse was mainly short-term (T4-T2), 13 per cent for point B and 17 per cent for pogonion. Gender correlated significantly with relapse (T5-T2) at point B (P = 0.002) and pogonion (P = 0.021), i.e. females in contrast to males showed further distalization of the mandible instead of relapse. No correlations were seen for age or the amount of surgical setback. The long-term results in mandibular setback patients were more stable when compared with the mandibular advancement patients examined previously. The initial soft tissue profile, the initial growth direction, and the remodelling processes of the hard tissues must be considered as reasons for long-term relapse. Growth direction positively influenced the long-term results in females: further distalization of the mandible occurred.

Gene and stem cell therapy in peripheral arterial occlusive disease

Peripheral arterial occlusive disease (PAOD) is a manifestation of systemic atherosclerosis strongly associated with a high risk of cardiovascular morbidity and mortality. In a considerable proportion of patients with PAOD, revascularization either by endovascular means or by open surgery combined with best possible risk factor modification does not achieve limb salvage or relief of ischaemic rest pain. As a consequence, novel therapeutic strategies have been developed over the last two decades aiming to promote neovascularization and remodelling of collaterals. Gene and stem cell therapy are the main directions for clinical investigation concepts. For both, preclinical studies have shown promising results using a wide variety of genes encoding for growth factors and populations of adult stem cells, respectively. As a consequence, clinical trials have been performed applying gene and stem cell-based concepts. However, it has become apparent that a straightforward translation into humans is not possible. While several trials reported relief of symptoms and functional improvement, other trials did not confirm this early promise of efficacy. Ongoing clinical trials with an improved study design are needed to confirm the potential that gene and cell therapy may have and to prevent the gaps in our scientific knowledge that will jeopardize the establishment of angiogenic therapy as an additional medical treatment of PAOD. This review summarizes the experimental background and presents the current status of clinical applications and future perspectives of the therapeutic use of gene and cell therapy strategies for PAOD.

Cardiovascular risk factors impair native collateral development and may impair efficacy of therapeutic interventions

Animal and early clinical studies of gene therapy for tissue ischaemia suggested that this approach might provide benefit to patients with coronary artery disease not amenable to traditional revascularization. This enthusiasm was then tempered by the subsequent disappointing results of randomized clinical trials and led researchers to develop strategies using progenitor cells as an alternative to improve collateral function. However, the recent publication of several randomized clinical trials reporting either negative or weakly positive results using this approach have led to questions regarding its effectiveness. There are several factors that need to be considered in explaining the discordance between the positive studies of such treatments in animals and the disappointing results seen in randomized patient trials. Aside from the practical issues of arteriogenic therapies, such as effective delivery, vascular remodelling is an extraordinarily complex process, and the administration of a single agent or cell in the hope that it would lead to lasting physiological effects may be far too simplistic an approach. In addition, however, evidence now suggests that many of the traditional cardiovascular risk factors-such as age and hypercholesterolemia-may impair the host response not only to ischaemia but, critically, also to treatment as well. This review discusses the evidence and mechanisms for these observations and highlights future directions that might be taken in an effort to provide more effective therapies.

The Gup1 homologue of Trypanosoma brucei is a GPI glycosylphosphatidylinositol remodelase

Glycosylphosphatidylinositol (GPI) lipids of Trypanosoma brucei undergo lipid remodelling, whereby longer fatty acids on the glycerol are replaced by myristate (C14:0). A similar process occurs on GPI proteins of Saccharomyces cerevisiae where Per1p first deacylates, Gup1p subsequently reacylates the anchor lipid, thus replacing a shorter fatty acid by C26:0. Heterologous expression of the GUP1 homologue of T. brucei in gup1Delta yeast cells partially normalizes the gup1Delta phenotype and restores the transfer of labelled fatty acids from Coenzyme A to lyso-GPI proteins in a newly developed microsomal assay. In this assay, the Gup1p from T. brucei (tbGup1p) strongly prefers C14:0 and C12:0 over C16:0 and C18:0, whereas yeast Gup1p strongly prefers C16:0 and C18:0. This acyl specificity of tbGup1p closely matches the reported specificity of the reacylation of free lyso-GPI lipids in microsomes of T. brucei. Depletion of tbGup1p in trypanosomes by RNAi drastically reduces the rate of myristate incorporation into the sn-2 position of lyso-GPI lipids. Thus, tbGup1p is involved in the addition of myristate to sn-2 during GPI remodelling in T. brucei and can account for the fatty acid specificity of this process. tbGup1p can act on GPI proteins as well as on GPI lipids.

Sirolimus ameliorates the enhanced expression of metalloproteinases in a rat model of autosomal dominant polycystic kidney disease

BACKGROUND: Remodelling of matrix and tubular basement membranes (TBM) is a characteristic of polycystic kidney disease. We hypothesized that matrix and TBM degradation by metalloproteinases (MMPs) could promote cyst formation. We therefore investigated the renal expression of MMPs in the Han:SPRD rat model of autosomal dominant polycystic kidney disease (ADPKD) and examined the effect of sirolimus treatment on MMPs. METHODS: 5-week-old male heterozygous (Cy/+) and wild-type normal (+/+) rats were treated with sirolimus (2 mg/kg/day) through drinking water for 3 months. RESULTS: The mRNA and protein levels of MMP-2 and MMP-14 were markedly increased in the kidneys of heterozygous Cy/+ animals compared to wild-type +/+ as shown by RT-PCR and Western blot analyses for MMP-2 and MMP-14, and by zymography for MMP-2. Strong MMP-2 expression was detected by immunoperoxidase staining in cystic epithelial cells that also displayed an altered, thickened TBM. Tissue inhibitor of metalloproteinases-2 (TIMP-2) expression was not changed in Cy/+ kidneys. Sirolimus treatment leads to decreased protein expression of MMP-2 and MMP-14 in Cy/+, whereas MMP-2 and MMP-14 mRNA levels and TIMP-2 protein levels were not affected by sirolimus. CONCLUSION: In summary, in kidneys of the Han:SPRD rat model of ADPKD, there is a marked upregulation of MMP-2 and MMP-14. Sirolimus treatment was associated with a marked improvement of MMP-2 and MMP-14 overexpression, and this correlated also with less matrix and TBM alterations and milder cystic disease.

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.

Orbitale Fettgewebsatrophie bei lokaler Bimatoprost-Therapie - Kann Bimatoprost einen Enophthalmus verursachen?

PURPOSE: The aim of this communication is to report enophthalmos as a possible new adverse effect of topical bimatoprost treatment. PATIENTS AND METHODS: A retrospective case series of five glaucoma patients under long-term topical bimatoprost treatment was evaluated. Documentation with photo and Hertel exophthalmometry was reviewed. RESULTS: In all five patients a deep lid sulcus, reduced infraocular fat pads and enophthalmos-suspicious Hertel values were found (mean 11.9 mm; SD 2.4). Other aetiologies for enophthalmos were excluded anamnestically and by clinical examination. CONCLUSION: Bimatoprost may lead to an alteration of the eyelid with deepening of the lid sulcus and may also be responsible for an iatrogenic orbital fat atrophy. A possible mechanism of action might be the induction of apoptosis of orbital fibroblasts with a remodelling of the extracellular matrix. Prospective studies are necessary to confirm this cross-sectional observation.