Irina Berger

Vascular Endothelial Growth Factor Gene‐Activated Matrix (VEGF165‐GAM) Enhances Osteogenesis and Angiogenesis in Large Segmental Bone Defects

Healing of fractures is dependent on vascularization of bone, which is in turn promoted by VEGF. It was shown that 0.1 and 1 mg of pVEGF165‐GAM led to a significant increase in vascularization and bone regeneration in defects that would otherwise have led to atrophic nonunions.

Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis

Genetic and pharmacological evidence suggests that overexpression of cyclooxygenase-2 (COX-2) is critical for epithelial carcinogenesis and provides a major target for cancer chemoprevention by nonsteroidal antiinflammatory drugs. Transgenic mouse lines with keratin 5 promoter-driven COX-2 overexpression in basal epidermal cells exhibit a preneoplastic skin phenotype. As shown here, this phenotype depends on the level of COX-2 expression and COX-2-mediated prostaglandin accumulation. The transgenics did not develop skin tumors spontaneously but did so after a single application of an initiating dose of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Long-term treatment with the tumor promoter phorbol 12-myristate 13-acetate, as required for tumorigenesis in wild-type mice, was not necessary for transgenics. The ratios of squamous cell carcinomas to papillomas and of sebaceous gland adenomas to papillomas plus squamous cell carcinomas were increased markedly in transgenic mice treated with DMBA alone compared with DMBA/phorbol 12-myristate 13-acetate-treated transgenic and wild-type mice. Thus, COX-2 overexpression, which leads to high levels of epidermal prostaglandin E2, prostaglandin F2α, and 15-deoxyΔ12,14-PGJ2, is insufficient for tumor induction but transforms epidermis into an “autopromoted” state, i.e., dramatically sensitizes the tissue for genotoxic carcinogens.

Calcification of Coronary Intima and Media: Immunohistochemistry, Backscatter Imaging, and X-Ray Analysis in Renal and Nonrenal Patients

Coronary calcification is a potent predictor of cardiac events. In patients with chronic renal disease, both prevalence and intensity of coronary calcification are increased. It has remained uncertain whether it is the intima of the coronaries or the media that is calcified and whether the morphologic details of calcified plaques differ between renal and nonrenal patients. Autopsy samples of coronaries were obtained from standard sites in 23 renal and 23 age- and gender-matched nonuremic patients. Specimens were examined using light and electron microscopy, immunohistochemistry, backscatter imaging, and x-ray analysis. In coronaries, calcified plaques occupied a similar proportion of the intima area in renal versus nonrenal patients (17.3 +/- 11.9 versus 18.1 +/- 11.9%) but occupied a significantly higher proportion of the media (16.6 +/- 10.6 versus 3.8 +/- 2.31%). Expression of the proteins osteocalcin, C-reactive protein, TGF-beta, and collagen IV was significantly more intensive around coronary plaques of renal compared with nonrenal patients. The non-plaque-bearing intima of renal patients showed minimal staining for fetuin, but fetuin staining was seen surrounding calcified plaques. In addition, more pronounced deposition of C5b-9 was found around coronary plaques of renal patients, and glycophorin deposition pointed to more past intraplaque hemorrhage in renal patients. Calcification by electron backscatter analysis is more intense in the coronary media, but not if the intima is more intense in renal compared with nonrenal patients. A more marked inflammatory response in renal patients is suggested by more frequent presence and greater intensity of markers of inflammation.

Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn's disease

Abstract Abstract. To define mediator profiles in inflamed and noninflamed areas in inflammatory bowel disease (IBD) we analyzed the expression of 35 messenger-RNAs (mRNAs) encoding cytokines, chemokines, and some related molecules in transmural gut tissues (n=138) from patients with ulcerative colitis (UC), Crohns disease (CD), and inflammatory and normal controls by real-time quantitative reverse transcription polymerase chain reaction. Using sample collectives with a comparable degree of inflammation, most parameters investigated showed similarly increased mRNA expression levels in both active UC and CD. This included proinflammatory cytokines, but also interferon (IFN) γ and several IFN-γ inducible chemokines. Only macrophage inflammatory protein (MIP)-2α mRNA was expressed at higher levels in inflamed UC vs. CD. IH revealed that MIP-2α protein was produced mainly by intestinal epithelial cells. Importantly, in histologically noninflamed/inactive IBD samples mRNAs for several mediators were significantly enhanced, accompanied by elevated levels of migration-inhibition factor related protein (MRP) 14 transcripts. CD14 positive macrophages were found especially in noninflamed/inactive UC, many of which coexpressed the RFD-7 antigen. Our results indicate a substantial overlap in cytokine/chemokine mRNA expression in UC and CD. Elevated mediator expression is evident in noninflamed/inactive areas in both diseases. Local recruitment of MRP-14 positive leukocytes might contribute to this phenomenon. In inactive UC a phenotypically altered population of macrophages expressing CD14 might play an additional role.

The intravertebral vacuum phenomen as specific sign of osteonecrosis in vertebral compression fractures: results from a radiological and histological study

This study investigated the prevalence of the intravertebral vacuum phenomenon (IVP) and osteonecroses in vertebral compression fractures (VCFs). We therefore performed an histological analysis of biopsies obtained from VCFs prior to balloon kyphoplasty. Computed tomography (CT) scans were reviewed regarding the presence of an IVP (i.e. cleft sign, Kümmell disease). We reviewed the data of 266 consecutive patients treated by balloon kyphoplasty in 501 procedures from 2002 to 2004. From 180 patients (68%) we obtained adequate bone tissue for histological evaluation. Biopsy specimens were analysed regarding the presence of osteoporosis, infection, malignancy and osteonecrosis. CT scans of all 180 patients were reviewed for presence of an IVP. Histological examination revealed 135 (75%) osteoporoses, 20 (11%) neoplasms, 12 (7%) trauma cases and 13 (7%) osteonecroses. An IVP was present in 12 (7%) patients. There was a significant association of osteonecrosis and IVP (P < 0.0001). Eleven of 12 patients with a vacuum phenomenon showed an osteonecrosis on histology, wheras 11 of 13 patients with osteonecrosis showed an IVP on CT. The IVP is a specific sign of osteonecrosis in vertebral compression fractures (sensitivity 85%, specificity 99%, positive predictive value 91%). Our findings strongly support the thesis that an IVP indicates local bone ischemia associated with a non-healing vertebral collapse and pseudarthrosis.

Circulating fibronectin affects bone matrix, whereas osteoblast fibronectin modulates osteoblast function

The bone matrix is composed mostly of collagen, but the initial and continuous presence of fibronectin was found to be crucial for collagen matrix integrity in vitro. It has been assumed that osteoblasts produce the fibronectin required for bone matrix formation. Using transgenic mice, we conditionally deleted fibronectin in the osteoblasts and in the liver using the cre‐loxP system. We also used mice with mutated fibronectin and conditionally deleted β1‐integrin in osteoblasts to identify the receptor involved in fibronectin effects on osteoblasts. Conditional deletion of fibronectin in the differentiating osteoblasts [using the 2.3 kb collagen‐α1(I) promoter] failed to show a decrease in fibronectin amount in the bone matrix despite evidence of successful deletion. Using these mice we established that osteoblast‐derived fibronectin solely affects osteoblast function. This effect was not mediated by integrins that bind to the RGD motif. Conditional deletion of fibronectin in the liver showed a marked decrease in fibronectin content in the matrix associated with decreased mineral‐to‐matrix ratio and changed biomechanical properties but had no effect on osteoblasts or osteoclasts. In conclusion, osteoblast fibronectin affects osteoblasts function. This does not seem to be mediated by the RGD motif on fibronectin. In contrast, liver‐derived fibronectin affects bone matrix properties without affecting osteoblast or osteoclast function. A novel role for liver‐derived circulating fibronectin thus was defined and delineated from that of locally produced fibronectin.

The peroxynitrite decomposition catalyst FP15 improves ageing-associated cardiac and vascular dysfunction

Overproduction of oxidants and free radicals in ageing tissues induces nitro-oxidative stress, which has recently been implicated in the pathogenesis of cardiovascular dysfunction associated with ageing. Peroxynitrite, a strong cytotoxic oxidant damages proteins and DNA and activates several pathways causing tissue injury, including the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway. In this study, we investigated the effectiveness of the peroxynitrite decomposition catalyst FP15 on ageing-associated cardiac and vascular dysfunction. Young and ageing rats were treated with vehicle or FP15 intraperitoneally. Using a microtip Millar pressure catheter we performed left ventricular blood pressure analysis to assess systolic and diastolic function. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Ageing animals showed a marked reduction of systolic and diastolic cardiac function and loss of endothelium-dependent relaxant responsiveness of aortic rings. FP15-treatment significantly improved cardiac performance and endothelial function. Immunohistochemical staining confirmed that FP15 effectively reduced nitrosative stress and prevented the activation of PARP in the aortic wall of ageing rats. Our results demonstrate the importance of endogenous peroxynitrite-overproduction in the pathogenesis of ageing-associated cardiovascular dysfunction. Pharmacological decomposition of peroxynitrite by FP15 may represent a novel therapeutic utility to improve cardiac and vascular dysfunction associated with ageing.

Calcimimetic R-568 or calcitriol: equally beneficial on progression of renal damage in subtotally nephrectomized rats

Patients with renal insufficiency develop secondary hyperparathyroidism. Monotherapy with active vitamin D or calcimimetics ameliorates secondary hyperparathyroidism. We compared kidney damage in subtotally nephrectomized (SNX) rats treated with active vitamin D (calcitriol) or the calcimimetic R-568. Male Sprague-Dawley SNX and sham-operated (sham-op) rats were randomized into the following treatment groups: SNX + R-568, SNX + calcitriol, SNX + vehicle, sham-op + R-568, sham-op + calcitriol, and sham-op + vehicle. Albuminuria and blood pressure were monitored and kidneys were examined using morphometry, immunohistochemistry, quantitative RT-PCR, and in situ hybridization. Parathyroid hormone concentrations were lowered to the same extent by the two interventions, although phosphorus and the calcium-phosphorus product were reduced only by R-568 treatment. SNX rats developed marked albuminuria, which was significantly reduced in ad libitum- and pair-fed animals treated with R-568 and animals treated with calcitriol. Mean glomerular volume (6.05 +/- 1.46 vs. 2.70 +/- 0.91 mm(3)), podocyte volume (831 +/- 127 vs. 397 +/- 67 microm(3)), the degree of foot process fusion (mean width of foot processes = 958 +/- 364 vs. 272 +/- 35 nm), and glomerular basement membrane thickness (244 +/- 6 vs. 267 +/- 23 nm), as well as desmin staining, were significantly higher in vehicle-treated SNX than sham-operated animals. These changes were ameliorated with R-568 and calcitriol. In SNX, as well as sham-operated, animals, expression of the calcium-sensing receptor (protein and mRNA) was upregulated by treatment with the calcimimetic, but not calcitriol. Calcitriol and R-568 were similarly effective in ameliorating kidney damage.

First histological observations on the incorporation of a novel nanocrystalline hydroxyapatite paste OSTIM® in human cancellous bone

Background:A commercially available nanocrystalline hydroxyapatite paste Ostim® has been reported in few recent studies to surpass other synthetic bone substitutes with respect to the observed clinical results. However, the integration of this implantable material has been histologically evaluated only in animal experimental models up to now. This study aimed to evaluate the tissue incorporation of Ostim® in human cancellous bone after reconstructive bone surgery for trauma.Methods:Biopsy specimens from 6 adult patients with a total of 7 tibial, calcaneal or distal radial fractures were obtained at the time of osteosynthesis removal. The median interval from initial operation to tissue sampling was 13 (range 3–15) months. Samples were stained with Masson-Goldner, von Kossa, and toluidine blue. Osteoid volume, trabecular width and bone volume, and cortical porosity were analyzed. Samples were immunolabeled with antibodies against CD68, CD56 and human prolyl 4-hydroxylase to detect macrophages, osteoblasts, and fibroblasts, respectively. TRAP stainings were used to identify osteoclasts.Results:Histomorphometric data indicated good regeneration with normal bone turnover: mean osteoid volume was 1.93% of the trabecular bone mass, trabecular bone volume – 28.4%, trabecular width – 225.12 μm, and porosity index – 2.6%. Cortical and spongious bone tissue were well structured. Neither inflammatory reaction, nor osteofibrosis or osteonecrosis were observed. The implanted material was widely absorbed.Conclusion:The studied nanocrystalline hydroxyapatite paste showed good tissue incorporation. It is highly biocompatible and appears to be a suitable bone substitute for juxtaarticular comminuted fractures in combination with a stable screw-plate osteosynthesis.

Arterial Distribution Characteristics of Embozene Particles and Comparison with Other Spherical Embolic Agents in the Porcine Acute Embolization Model

PURPOSE To determine the arterial distribution pattern of the embolic agent Embozene within the porcine kidney and compare it with those of other spherical embolic agents. MATERIALS AND METHODS Embozene, Embosphere, Bead Block, and Contour SE in size classes of 100-300 microm, 500-700 microm, and 700-900 microm and Embozene and Embosphere in the size class of 40-120 microm were used for total arterial occlusion in minipig kidneys. Organs were evaluated microscopically regarding vascular distribution of the different embolic agents and particle sizes. RESULTS The following variations of arterial distribution were identified. In the 40-120-microm size class, Embosphere particles penetrated significantly deeper compared with Embozene (P = .04). In the 100-300-microm size class, Bead Block showed a significantly deeper distribution as microscopy identified particles in arteries much smaller than their nominal size. In the 500-700-microm size class, Embosphere and Contour SE showed a deeper distribution. The most uniform arterial distribution was observed in the 700-900 microm size class,. However, few Embosphere and Contour SE particles were found in arcuate arteries, also indicating a distal distribution. CONCLUSIONS Throughout the four most-used size classes, from very small (40-120 microm) to large (700-900 microm), the distribution characteristics of the four tested materials vary substantially. Particularly, small Embosphere particles and small Bead Block particles showed a more distal distribution, as did medium-sized Embosphere and Contour SE particles. In the largest investigated size class, the distribution was more uniform. In general, the Embozene particles are very uniform in size, and they seem to reach vessels closely corresponding to their nominal size.